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ESCG-O-12

Disclosures: No disclosures to report. Increased physical exercise has been reported to improve the clinical symptoms of chronic enteropathies, such as inflammatory bowel disease, in human patients. The aim of this investigation was to evaluate the impact of an intervention to increase physical exercise in dogs with chronic enteropathies.
Twenty-two dogs (11 each in the exercise and control groups) with chronic enteropathies and no response to an elimination diet were included. Routine diagnostic work-up (haematology, plasma biochemistry profile, urinalysis, faecal parasitology, abdominal radiographs, and ultrasound) was conducted in all dogs to eliminate underlying causes. All dogs were given oral prednisolone (1 mg/kg/day) for 14 days, followed by a tapering dosage over 10 weeks. After 4 weeks of prednisolone treatment, a certified canine rehabilitation therapist instructed the owners of dogs in the exercise group on how to increase their dogs' physical exercise. The exercise protocol combined aerobic and resistance exercises in low-to moderate-intensity interval training. Owners of dogs in the control group were asked to maintain the dogs' routine lifestyles. Modified canine inflammatory bowel disease activity scores (CIB-DAIs), based on the parameters of activity level, appetite, vomiting, stool consistency, stool frequency, bloating, and weight loss, were calculated pre-treatment and 4 and 10 weeks post-treatment for all dogs. CIBDAI scores were compared among timepoints (pre-treatment and 2 post-treatment assessments) and between groups (exercise and control) using multivariate repeated-measures models for multiple comparisons.
An increased physical activity intervention had positive effects on clinical symptoms in dogs with chronic enteropathies.

ESVNU

Disclosures: No disclosures to report. Chronic kidney disease (CKD) produces progressive reduction in the number of functional nephrons and directly affects the homeostasis of the solutes excreted in the urine, including phosphorus. Hyperphosphatemia is considered a factor directly related to the increased mortality in humans, cats and dogs. In order to provide data from controlled clinical studies to examine the effects of hyperphosphatemia on the progression and survival of naturally occurring canine CKD, the following study was conducted.
In conclusion, plasma phosphate concentration in dogs increases as chronic kidney disease develops. And an inverse relationship to survival in dogs with phosphorus concentrations above 5 mg/dl, and as it progresses the IRIS scale was observed.
Disclosures: No disclosures to report. High blood pressure causes an increase in vascular endothelial growth factor (VEGF) secretion. Feline hypertension is commonly associated with chronic kidney disease (CKD) Amlodipine is the first choice antihypertensive treatment in cats but could have a negative effect on the kidney by increasing glomerular pressure through afferent arteriolar dilatation. The aims of this study were to: (1) validate a method for the quantification of VEGF in feline serum samples; (2) assess the association between urinary VEGF, serum VEGF (sVEGF) and biochemical and clinical variables in hypertensive cats and (3) investigate changes in urinary VEGF with amlodipine treatment.

ISCAID

Disclosures: This study was financially supported by the ECVIM clinical studies fund. The authors declare no further conflict of interest. The evaluation of liver fibrosis is of major importance for the management of chronic liver disease and the prediction of prognosis. Although liver biopsy is the gold standard for evaluation of fibrosis, non-invasive tests enable the clinician to stage and monitor a variety of liver diseases in human medicine. As such transforming growth factor b (TGF-b) and hyaluronic acid (HA) are biomarkers of hepatic fibrogenesis, that reflect the activity of the fibrogenic and fibrinolytic process, their use has not been validated in dogs.

ESCG-P-4 ASSESSMENT OF IMAGE QUALITY PRODUCED BY

Disclosures: Dr Sharman has shares in Optiscan Imaging Pty Ltd. Chronic enteropathies (CE) and exocrine pancreatic insufficiency (EPI) can both cause hypocobalaminemia in cats. Current supplementation protocols for cobalamin in cats call for repeated parenteral injections. In humans, several studies have reported equal efficacy of oral administration of cobalamin. There is also evidence that oral supplementation is effective in dogs with hypocobalaminemia. Recently, it has also been reported that oral cobalamin substitution restores normocobalaminemia in healthy elderly cats. The purpose of this retrospective case series was to evaluate whether oral cobalamin supplementation can restore normocobalaminemia in hypocobalaminemic cats with chronic enteropathies.
The study group consisted of 11 dogs with IBD, including 4 with PLE. Diagnosis was based on compatible clinical and histopathology and exclusion of other causes of chronic gastrointestinal disease. PLE was defined as IBD plus hypoproteinaemia (serum total protein <58 g/l) and hypoalbuminaemia (serum albumin <26 g/l). PTE was diagnosed in one dog with PLE, and suspected in a second.
Disclosures: No disclosures to report. The intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (CE) in dogs. While imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with CE involving the ileum and colon. The aim of the present study was to use fluorescence in situ hybridization (FISH) techniques to investigate the composition and spatial organization of mucosal microbiota in endoscopic biopsies obtained from dogs with CE and controls. Tissue sections from the ileum and colon from 19 dogs with inflammatory bowel disease (IBD), 6 dogs with granulomatous colitis (GC), 12 dogs with intestinal neoplasia, and 15 controls were studied by FISH targeting the 16S rRNA genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human IBD. The numbers of mucosal bacteria were analyzed using generalized linear models for each of the colon and ileum tissues, with Spearman's rank correlation coefficients used to test the correlation between mucosal microbiota and inflammatory (CIB-DAI score, histopathology) indices. The ileal and colonic mucosa of healthy dogs and dogs with CE was predominantly colonized by bacteria localized to free and adherent mucus compartments. Dogs with CE harbored more (P < 0.05) mucosal bacteria belonging to the Clostridium-coccoides/Eubacterium rectale group, Bacteroides, Enterobacteriaceae, and Escherichia coli versus controls. Within the CE group, IBD dogs had increased (P < 0.05) Enterobacteriaceae and E. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. Bacterial invasion with E. coli was present in the ileal and colonic mucosa of dogs with GC (P < 0.05). Dogs with intestinal neoplasia had increased (P < 0.05) adherent (total bacteria, Enterobacteriaceae, E. coli) and invasive (Enterobacteriaceae, E. coli, and Bacteroides) bacteria in biopsy specimens versus all other groups. Increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity (CIBDAI score) in IBD dogs (P < 0.05). These results indicate that histopathologic lesions of canine CE are associated with different populations in ileal and colonic mucosal microbiota. These spatial, segment-specific structure and differential response of select bacterial groups to intestinal inflammation may be pivotal regarding the functional consequences of these alterations in the pathogenesis of canine CE.

ESVC

Disclosures: No disclosures to report. Chronic mitral valve disease is by far the most common cardiovascular disease in dogs. The disease is caused by myxomatous degeneration of the mitral valve leaflets and, in approximately 30% of cases, it's accompanied by degeneration of the tricuspid valve. It is also described in previous studies that approximately 14% of affected dogs also have evidence of associated pulmonary arterial hypertension.
The present study aims to characterize the disease in a population of dogs in Portugal. We retrospectively reviewed the medical records of dogs presented to Hospital Veterin ario do Porto, with an echocardiographic diagnosis of canine chronic mitral valve disease, during a period of 13 years.

ESVIM-P-2 PET OWNERS USE OF THE INTERNET FOR THEIR PETS' HEALTH. A. Chatard, M. Hugonnard. VetAgro Sup, Marcy l'etoile, France

Disclosures: Johnson: Feline advisory board, speaker honoraria. Chronic respiratory disease, often characterized by a chronic cough, is common in dogs.

ESVIM-P-10

The purpose of this study was to determine the etiology of chronic respiratory disease in dogs that were presented with persistent and chronic coughing.
A retrospective study of 126 client-owned dogs with signs of persistent and chronic lower respiratory disease, that underwent bronchoscopy together with either an endotracheal wash (ETW) or broncho-alveolar lavage (BAL), was performed.
A definitive diagnosis was made in 118/126 cases (93.6%). Chronic bronchitis was the most common diagnosis (37.3%), median age 8 years; followed by airway tracheal collapse or bronchomalacia (23%), median age 11 years,; and primary bacterial infections (15.8%), median age 3 years. Less common etiologies identified included neoplasia (5.5%), median age 14 years; parasitic infections (4.8%), median age 7 years; and eosinophilic bronchopneumopathy (3.2%), median age 6 years. Rare etiologies identified included primary pulmonary hypertension, primary ciliary dyskinesia, excitement-induced cough, and obesity. Myxodematous mitral valve disease was found concurrently in 12/126 (9.5%) dogs.
This study concluded that by using a structured combination of survey thoracic radiography, bronchoscopy and ETW or BAL with cytology and culture, a diagnosis could be made in the majority of dogs with chronic respiratory disease.

ESVIM-P-15 RETROSPECTIVE CHARACTERIZATION OF MESENTE-RIAL PURULENT LYMPHADENITIS AND LYMPH NODE ABSCESSES IN DOGS REVEALS AN INCREASED RISK FOR THE SMALL MUNSTERLANDER. S. Schmitz. Small Animal Hospital Justus Liebig University Giessen, Giessen, Germany

Disclosures: No disclosures to report. Feline Chronic Kidney Disease (CKD) is a common feature of ageing cats. Angiotensin-converting enzyme inhibitors (ACEi) are recommended to treat hypertension associated with CKD to limit target-organ damage and especially glomerular hypertension. In addition, the International Renal Interest Society (IRIS) guidelines recommends the prescription of an ACEi in patients with CKD and proteinuria. To our knowledge no study has demonstrated the effects of long term administration of ACEI in a client owned population of cats suffering from CKD on glomerular filtration rate (GFR).
The aim of the study was to evaluate the effect of an ACEi (imidapril, Prilium Ò , V etoquinol SA) on the GFR of cats suffering from naturally occurring chronic kidney disease (CKD) over 12 months.
Daily long term imidapril treatment compared to placebo, may be an effective treatment to slow the progression of renal failure in cats with naturally occurring chronic kidney disease.

ISCAID-P-4 DETECTION OF FELINE CORONAVIRUS SPIKE GENE MUTATIONS AS

The present study describes early xanthinuria, renal mineralization and urolithiasis as adverse effects due to chronic allopurinol treatment in dogs with leishmaniosis. Neither mineral analysis nor renal biopsy was performed to confirm the origin of these lesions, but no urinary abnormality was present before allopurinol treatment was instituted. A thorough monitoring of dogs treated against leishmaniosis combined with urinalysis and abdominal ultrasound should be performed to evaluate urinary adverse effects and to help in the clinical management of these adverse effects.

ESCG-P-4 ASSESSMENT OF IMAGE QUALITY PRODUCED BY

Disclosures: Dr Sharman has shares in Optiscan Imaging Pty Ltd. Chronic enteropathies (CE) and exocrine pancreatic insufficiency (EPI) can both cause hypocobalaminemia in cats. Current supplementation protocols for cobalamin in cats call for repeated parenteral injections. In humans, several studies have reported equal efficacy of oral administration of cobalamin. There is also evidence that oral supplementation is effective in dogs with hypocobalaminemia. Recently, it has also been reported that oral cobalamin substitution restores normocobalaminemia in healthy elderly cats. The purpose of this retrospective case series was to evaluate whether oral cobalamin supplementation can restore normocobalaminemia in hypocobalaminemic cats with chronic enteropathies.

ESCG

The study group consisted of 11 dogs with IBD, including 4 with PLE. Diagnosis was based on compatible clinical and histopathology and exclusion of other causes of chronic gastrointestinal disease. PLE was defined as IBD plus hypoproteinaemia (serum total protein <58 g/L) and hypoalbuminaemia (serum albumin <26 g/L). PTE was diagnosed in one dog with PLE, and suspected in a second.
Disclosures: No disclosures to report. The intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (CE) in dogs. While imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with CE involving the ileum and colon. The aim of the present study was to use fluorescence in situ hybridization (FISH) techniques to investigate the composition and spatial organization of mucosal microbiota in endoscopic biopsies obtained from dogs with CE and controls. Tissue sections from the ileum and colon from 19 dogs with inflammatory bowel disease (IBD), 6 dogs with granulomatous colitis (GC), 12 dogs with intestinal neoplasia, and 15 controls were studied by FISH targeting the 16S rRNA genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human IBD. The numbers of mucosal bacteria were analyzed using generalized linear models for each of the colon and ileum tissues, with Spearman's rank correlation coefficients used to test the correlation between mucosal microbiota and inflammatory (CIB-DAI score, histopathology) indices. The ileal and colonic mucosa of healthy dogs and dogs with CE was predominantly colonized by bacteria localized to free and adherent mucus compartments. Dogs with CE harbored more (P < 0.05) mucosal bacteria belonging to the Clostridium-coccoides/Eubacterium rectale group, Bacteroides, Enterobacteriaceae, and Escherichia coli versus controls. Within the CE group, IBD dogs had increased (P < 0.05) Enterobacteriaceae and E. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. Bacterial invasion with E. coli was present in the ileal and colonic mucosa of dogs with GC (P < 0.05). Dogs with intestinal neoplasia had increased (P < 0.05) adherent (total bacteria, Enterobacteriaceae, E. coli) and invasive (Enterobacteriaceae, E. coli, and Bacteroides) bacteria in biopsy specimens versus all other groups. Increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity (CIBDAI score) in IBD dogs (P < 0.05). These results indicate that histopathologic lesions of canine CE are associated with different populations in ileal and colonic mucosal microbiota. These spatial, segment-specific structure and differential response of select bacterial groups to intestinal inflammation may be pivotal regarding the functional consequences of these alterations in the pathogenesis of canine CE.

ESVC-P-8 QUANTIFICATION OF SYSTOLIC AND DIASTOLIC RIGHT

Disclosures: No disclosures to report. Chronic mitral valve disease is by far the most common cardiovascular disease in dogs. The disease is caused by myxomatous degeneration of the mitral valve leaflets and, in approximately 30% of cases, it's accompanied by degeneration of the tricuspid valve. It is also described in previous studies that approximately 14% of affected dogs also have evidence of associated pulmonary arterial hypertension.
The present study aims to characterize the disease in a population of dogs in Portugal. We retrospectively reviewed the medical records of dogs presented to Hospital Veterin ario do Porto, with an echocardiographic diagnosis of canine chronic mitral valve disease, during a period of 13 years.

ESVE-P-4 VALIDATION OF AN ENZYME FLUORESCENCE ASSAY (ELFA) TO MEASURE TOTAL THYROXINE IN DOGS AND CATS.

Disclosures: Johnson: Feline advisory board, speaker honoraria. Chronic respiratory disease, often characterized by a chronic cough, is common in dogs.

ESVIM-P-10

The purpose of this study was to determine the etiology of chronic respiratory disease in dogs that were presented with persistent and chronic coughing.
A retrospective study of 126 client-owned dogs with signs of persistent and chronic lower respiratory disease, that underwent bronchoscopy together with either an endotracheal wash (ETW) or broncho-alveolar lavage (BAL), was performed.
A definitive diagnosis was made in 118/126 cases (93.6%). Chronic bronchitis was the most common diagnosis (37.3%), median age 8 years; followed by airway tracheal collapse or bronchomalacia (23%), median age 11 years,; and primary bacterial infections (15.8%), median age 3 years. Less common etiologies identified included neoplasia (5.5%), median age 14 years; parasitic infections (4.8%), median age 7 years; and eosinophilic bronchopneumopathy (3.2%), median age 6 years. Rare etiologies identified included primary pulmonary hypertension, primary ciliary dyskinesia, excitement-induced cough, and obesity. Myxodematous mitral valve disease was found concurrently in 12/126 (9.5%) dogs.
This study concluded that by using a structured combination of survey thoracic radiography, bronchoscopy and ETW or BAL with cytology and culture, a diagnosis could be made in the majority of dogs with chronic respiratory disease.

ESVIM

Disclosures: No disclosures to report. Feline Chronic Kidney Disease (CKD) is a common feature of ageing cats. Angiotensin-converting enzyme inhibitors (ACEi) are recommended to treat hypertension associated with CKD to limit target-organ damage and especially glomerular hypertension. In addition, the International Renal Interest Society (IRIS) guidelines recommends the prescription of an ACEi in patients with CKD and proteinuria. To our knowledge no study has demonstrated the effects of long term administration of ACEI in a client owned population of cats suffering from CKD on glomerular filtration rate (GFR).
The aim of the study was to evaluate the effect of an ACEi (imidapril, Prilium Ò , V etoquinol SA) on the GFR of cats suffering from naturally occurring chronic kidney disease (CKD) over 12 months.
Daily long term imidapril treatment compared to placebo, may be an effective treatment to slow the progression of renal failure in cats with naturally occurring chronic kidney disease.

ISCAID

The present study describes early xanthinuria, renal mineralization and urolithiasis as adverse effects due to chronic allopurinol treatment in dogs with leishmaniosis. Neither mineral analysis nor renal biopsy was performed to confirm the origin of these lesions, but no urinary abnormality was present before allopurinol treatment was instituted. A thorough monitoring of dogs treated against leishmaniosis combined with urinalysis and abdominal ultrasound should be performed to evaluate urinary adverse effects and to help in the clinical management of these adverse effects.

259 (17.3%) cats presented for gastrointestinal signs had an hypocobalaminemia: the majority were Domestic Short Hair, 47% were males (84% castrated) and 53% females (94% castrated), aged from 3 months to 18 years. The main clinical signs included chronic diarrhea (93%), weight loss (71%), polyuropolydypsia (48%), vomiting (43%), polyphagia (38%), fatigability (36%) and dysorexia (19%) with a median duration of 4 months before diagnosis. Cobalamin values ranged from 44 to 400 ng/L (median: 296 ng/L). 60% of the hypocobalaminemic cats had also a hyperfolatemia (folate > 12 ng/L) at diagnosis. fT4 was measured in the 64 older cats (> 12 years) and revealed an hyperthyroidism (fT4 > 40 pmol/L) in 20% of the cases. 67/259 hypocobalaminemic cats had a known clinical and biological follow-up (median time follow-up = 44 days): cobalamin significantly improved 1 month after treatment (50 lg/kg IM cyanocobalamin in a single dose) for 87% of the cats, even if 16% remained hypocobalaminemic. 62% of the followed cats were clinically improved, of which 85% with an associated higher cobalamin value. Clinical and biological improvement after cobalamin supplementation was significantly associated with an increase in folate concentration (P-value = 0.02). However, 33% of the cats with an improved cobalamin value did not show any clinical improvement.
Disclosures: No disclosures to report. Cobalamin malabsorption is common in old cats with weight loss, macronutrient malabsorption and enteric protein loss due to idiopathic chronic enteropathy, ICE (Patil AP and Cupp CJ. Proc. Nestle-Purina Compan Anim Nutr Summit, 55-61, 2010, Williams and Czarnecki-Maulden, Proc 23rd ECVIM-CA Congress, 2013) . High dose oral cobalamin supplementation reverses subnormal serum concentration within 1 week but serum cobalamin can become undetectable within as little as 1 month following cessation of supplementation (Williams and Czarnecki-Maulden, Proc 23rd ECVIM-CA Congress, 2014). The objectives of this study were to determine if serum cobalamin concentrations in cats with ICE and the response to oral supplementation and withdrawal are associated with differences in the intestinal microbiome.
David Williams is a consultant and adviser for Nestle-Purina, Idexx Laboratories, and the Gastrointestinal Laboratory at Texas A&M University. He receives royalties from Idexx Laboratories and has given continuing education lectures sponsored by Nestle-Purina. Inflammatory bowel disease (IBD) is a common cause of chronic gastrointestinal signs in cats. Typically, lymphoplasmacytic inflammation is found in biopsies, but a subset of cats with IBD has neutrophilic inflammation. The clinical significance of neutrophilic infiltration is unclear.

ESCG-O-3 INVESTIGATION INTO THE ROLE OF CAMPYLOBACTER

The role of the intestinal virome in health and disease is gaining increased attention in human medicine. The use of next generation sequencing (NGS) technologies has allowed identification of diversity and distribution of the virome. These approaches can equally be applied to dogs.This study aimed to identify and characterise the virome present in faeces of dogs with chronic enteropathy (CE) compared to the virome of healthy dogs (HD). Faecal samples were evaluated from 8 HD and 8 dogs with CE (4 Food, 3 Antibiotic and 1 Steroid Responsive) using a NGS approach. A viral enrichment protocol, using a series of centrifugation, endonuclease treatments and bacterial filtration were performed. The enriched viral DNA and RNA were extracted and amplified using sequence-independent single-primer amplification (SISPA) protocol, and subsequently sequenced by NGS using the Illumina MiSeq platform at the AGRF. Two bioinformatic pipelines were used to analyse the viral population. After selecting high quality reads and removing dog and bacterial sequences, sequence information was compared against 2 reference databases. We identified a total of 15,358 viral contigs, with 14,241 DNA viral sequences and 1,041 RNA sequences across all 16 dog samples. The majority of viral hits from both groups of faecal samples were bacteriophage (73.8% HD and 99.7% CE), from several families mainly from the Caudovirales order. After all analyses, only 6 viral eukaryotic families were identified across all samples. Two groups of sequences similar to known virus families, Reoviridae and Papillomaviridae, were identified in both groups (HD 1/8 and 1/8 and CE 2/8 and 2/8, respectively). Sequences similar to Picornaviridae were identified only in one dog with CE and sequences similar to Adenoviridae, Parvoviridae and Coronaviridae were identified only in healthy dogs (1/8 each).Further genomic characterisation and phylogenetic analysis was undertaken on 2 viruses. The 11 genome segments of a Rotavirus (Reoviridae) isolate were determined. Similarly, the sequence of the entire coding region of a kobuvirus (Picornaviridae) isolate was determined. Preliminary analyses indicated that all rotavirus gene segments exhibited between 55% -98% nt homology to previously reported canine rotaviruses. The kobuvirus sequence exhibited moderate nt homology (55%) to previously described genomes and clustered with other canine kobuvirus sequences available in GenBank. In conclusion, viral sequences from a range of different virus families, including both RNA and DNA families, and known pathogens were identified and characterised, and the largest proportion of viral contigs identified belonged to bacteriophages.
Disclosures: No disclosures to report. Canine S100A12 has potential as a biomarker of inflammation in dogs. Fecal S100A12 concentrations were increased in dogs with chronic gastroenteropathy (CE), and correlated with the severity of clinical and endoscopic disease. A negative outcome was associated with higher fecal S100A12 concentrations in CE dogs, but the response to different forms of treatment and fecal S100A12 has not been reported, and this information will be important to further evaluate the utility of fecal S100A12 as a biomarker for gastrointestinal disease. Aim of this study was to evaluate the association between responses to various treatments (i.e., elimination diet, antimicrobial drugs, or corticosteroids/other immunosuppressants) and fecal S100A12 in dogs with CE. Fecal samples were collected from dogs diagnosed with CE, and fecal S100A12 was measured in all specimens using an established in-house ELISA. Based on the response to treatment, dogs were classified as having antibiotic-responsive diarrhea (ARD), food-responsive diarrhea (FRD), or steroid-responsive/therapy-resistant idiopathic inflammatory bowel disease (IBD). Statistical analysis was performed using non-parametric 2-or multiple-group comparisons, the likelihood ratio to evaluate the association between groups of dogs and response to treatment, and a receiver operating characteristic curve to calculate sensitivity and specificity at the optimum cut-off concentration. A total of 64 dogs with CE (median age: 6.3 years; 33 males/ 31 females) were included in the study, the final diagnosis of which were ARD (n = 9), FRD (n = 30), or IBD (n = 25). Response to treatment was complete remission (n = 35), partial response (n = 25), or no response (n = 4). Fecal S100A12 concentrations ranged from 1 to 34,500 ng/g, and higher S100A12 levels were seen in dogs with IBD than in dogs with FRD (P = 0.010) or ARD (P = 0.025). Dogs that did not respond to treatment had significantly higher S100A12 levels than dogs with partial (P = 0.005) or complete (P = 0.003) remission, but response to treatment was associated with disease classification (P = 0.020). Despite a small number of patients, fecal S100A12 levels of >2,700 ng/g at the time of diagnosis distinguished dogs that failed responding to treatment from those with at least partial remission with a sensitivity of 100% and specificity of 87%. We conclude that, in line with our previous finding that fecal S100A12 may be a useful biomarker of disease severity in dogs with IBD, fecal S100A12 may also have utility in predicting the lack of response to treatment in dogs with CE. The utility of serial fecal S100A12 concentrations to monitor treatment response in dogs with CE warrants further research.

ESCG-O-12

Disclosures: The Eukanuba diet used in this study is manufactured by Spectrum Brands whilst DMM is employed by Spectrum Brands. VJA is an independent epidemiologist who helped analyze the data and was financially supported by Spectrum Brands for this work. PJW has participated in veterinary seminars organised by Spectrum Brands and has received an honorarium for this work. Introduction: In healthy animals, the phosphate (P) in combination with calcium homeostasis is regulated in comparatively narrow limits: excessively ingested P is excreted via urine. Common knowledge is, however, that P is a progressive factor in chronic renal failure (CRF) wherefore typically a P restricted diet is prescribed for affected patients. In 1995, Pastoor demonstrated that a P excess (significantly at~890 mg P/MJ ME; Ca/P 0.4/1 for 28 days) impairs renal function even in healthy cats, diagnosed mainly by reduced endogenous creatinine clearance. Dietary P originates from meat and other protein sources, bones and cartilages, mineral supplements and technical additives (water binding, palatability a/o texture enhancer etc.). The daily amount ingested with complete diets from the European market often exceeds the recommended daily allowance (RDA; NRC 2006 , FEDIAF 2014 considerably (up to 10times, Anonymus 2014).

ESVC-O-4 SERUM AND URINE CARDIAC TROPONIN I IN CATS

Disclosures: This study was funded by grants provided by the Faculty of Veterinary Science at the University of Sydney. M. Yata received financial support from Luoda Pharma, the Australian Postgraduate Awards Scholarship, and the Eric Horatio Maclean Scholarship whilst undertaking this project. None of the authors involved in this study have current affiliations with the drug company that manufactured the product used in this study (Boehringer Ingelheim Pimobendan has positive inotropic, positive lusitropic and vasodilator effects and is licensed for use in dogs with cardiac disease in many countries. Numerous studies have shown benefit with the use of pimobendan in canine dilated cardiomyopathy and chronic degenerative mitral valve disease, and whilst not licensed for use in cats, recent studies have reported benefits with the use of oral pimobendan in a variety of cardiac diseases including dilated and hypertrophic cardiomyopathies. An intravenous formulation has been available in the UK since January 2013. The use of intravenous pimobendan in cats in the clinical setting has not previously been described. The aim of this study was to describe the use of intravenous pimobendan in cats with naturally occurring heart failure and report tolerability and side effects/adverse reactions.

ESVC-O-

Disclosures: No disclosures to report. Within the distal nephron, the enzyme 11-beta hydroxysteroid dehydrogenase 2 (11bHSD2) protects the mineralocorticoid receptor (MR) from activation by cortisol, allowing it to interact with aldosterone. In humans, mutations of 11bHSD2 cause apparent mineralocorticoid excess, characterised by sodium and water retention with resultant hypertension. Sodium and water retention is also a hallmark of canine congestive heart failure (CHF). This could partly be explained by dysregulation of renal 11bHSD2 activity, exposing MR to activation by cortisol. The aim of this study was to investigate the activity of renal 11bHSD2 in canine CHF by measuring the concentration of cortisol and its metabolites in the urine from affected dogs. Owners collected urine in a home environment from healthy adult dogs (n = 7), and from dogs prior to presentation with non-cardiac chronic disease (n = 6), and dogs with cardiac disease (ISACHC Ib, n = 4; ISACHCII or III, n = 5). Levels of cortisol (F) and cortisone (E) excreted in urine were measured by mass spectrometry. Urinary cortisol was normalised to creatinine to account for variations in glomerular filtration rate. Cortisol was also measured in plasma obtained from all unhealthy dogs. Plasma cortisol levels (P = 0.75)and urinary cortisol:creatinine ratio (P = 0.22) did not differ between groups. However, the F/E ratio, was increased in dogs with class II-III heart failure (P = 0.048). An increased F/E ratio, in the presence of unchanged plasma cortisol, implies decreased renal 11bHSD2 activity and enhanced MR activation by cortisol in canine CHF. This data suggests that changes in renal cortisol metabolism in canine CHF cannot be explained by chronicity of disease, that the urinary F/E ratio has potential as a biomarker for canine CHF and that renal 11bHSD2 could offer a therapeutic target in its management. Further studies investigating 11bHSD2 expression and bioactivity in canine CHF are ongoing.

ESVNU

Disclosures: The author and co-authors work for Royal Canin, the company commercializing one of the diets evaluated. Serum cystatin C (sCysC) and urinary cystatin C (uCysC) are potential markers for detection of feline chronic kidney disease (CKD). Our aims were twofold. Firstly, we evaluated CysC as marker for CKD. We compared sCysC and uCysC between CKD and healthy cats, correlated sCysC and sCr with glomerular filtration rate (GFR) and calculated sensitivity, specificity for detecting decreased GFR. Secondly, we compared assay performance of the turbidimetric assay (PETIA) with the previously validated nephelometric assay (PENIA).
Disclosures: No disclosures to report. A broad range of clinical manifestations and immune responses have been described in canine leishmaniosis. Canine L. infantum infection can manifest as a chronic subclinical infection, self-limiting disease, or non-self-limiting illness. A protective CD4+ T-cellmediated immune response characterized by production of inter-feron-gamma, IL-2 and TNF-alpha is believed to be present in resistant subclinical dogs while this response seems to be diminished or absent in sick dogs. However, there are few and poorly standardized assays to evaluate this response in the dog. In addition, cellular mediated immunity has been mainly investigated in subclinical or vaccinated dogs but limited information is available in sick dogs with different degrees of disease severity. The aim of this study was to investigate L. infantum-specific cellular immunity in dogs with clinical leishmaniosis at the time of diagnosis. Twenty-six dogs were diagnosed based on physical examination, routine laboratory tests (CBC, biochemistry profile, serum electrophoresis, urinalysis and urinary protein/creatinine ratio) and L. infantum-specific antibody levels measured by quantitative ELISA. Heparin whole blood was stimulated with L. infantum soluble antigen (LSA) and the mitogen concavalin A (ConA) and incubated during 5 days. Unstimulated whole blood from each dog was used as control. Supernatants were collected and IFNgamma concentration was measured with a commercial sandwich ELISA. The majority of dogs (n = 16) were classified as LeishVet stage II (moderate disease). The rest of dogs were classified as stage I (n = 3), stage III (n = 4) and stage IV (n = 2). Twelve dogs (stage I, n = 2; stage IIa: n = 9, stage III: n = 1) produced IFNgamma after stimulation with LSA (mean AE SD: 2137AE1526 pg/ mL) and ConA (meanAESD: 6596 AE 4448 pg/mL). In contrast, 13 dogs (stage IIa: n = 5; stage IIb: n = 3; stage III = n = 3; stage IV, n = 2) did not produce detectable IFN-gamma after stimulation with LSA but 12 dogs produced IFN-gamma after stimulation with ConA (mean AE SD: 5665AE4923 pg/mL) while one dog was unresponsiveness to ConA. No differences in IFN-gamma concentration were found between IFN-gamma producer and nonproducer dogs when blood was stimulated with ConA (P = 0.6). Interestingly, IFN-gamma producer sick dogs presented with a lower antibody levels (meanAESD: 1068AE1217 ELISA units (EU)) when compared with IFN-gamma non-producer sick dogs (mean AE SD: 11448AE17602 EU) but differences were not statistically significant (P = 0.1). The results of this study suggest that sick dogs with a more exaggerated humoral response and a more severe disease lack L. infantum specific IFN-gamma production in stimulated blood.

ISCAID

Echosens covers a part of the biological measurements expenses in the clinical trial associated to this abstract. Primary hepatitis is a common disorder in dogs. Treatments for primary hepatitis are typically symptomatic and importantly, predicting prognosis at point of diagnosis remains challenging. In contrast to human medicine, where the type of hepatitis is defined by the inciting cause, few causes of chronic hepatitis have been identified in the dog, and the majority of cases are idiopathic. Systemic inflammation is well recognised in humans with liver disease. Systemic Inflammatory Response Syndrome (SIRS) is the clinical expression of the action of complex intrinsic mediators of the acute phase reaction. The presence of SIRS has been linked to a poor outcome in various liver diseases. The prevalence and predictive value of a SIRS in dogs with primary hepatitis has not been examined in dogs with liver disease. This is surprising given the accumulating evidence that SIRS is linked to the development of hepatic encephalopathy (HE) in dogs with liver disease. Although the pathogenesis of HE is not completely understood, it has been indicated that ammonia and inflammatory cytokines play a crucial role in the development of HE. HE is an important cause of morbidity and mortality in patients with liver disease.

ESCG-P-4 ASSESSMENT OF IMAGE QUALITY PRODUCED BY

ALI was successfully performed in 5/5 patients, with no adverse effects. Average study duration was 15.7AE4.1 hours and mean image acquisition count was 22,572AE17,315. Gastric and small intestinal transit times were 79.2AE39.8 minutes and 119.4AE43.7 minutes, respectively. Median (range) image quality scores were 9 (8-10), 8 (6-10) and 6 (5-9), for the stomach, SI and colon, respectively. Image quality scores were significantly higher in the stomach and SI than in the colon (P < 0.001). Visualized lesions were consistent with GI ulcers (2 dogs), inflammatory bowel disease (1 dog), and bilious vomiting syndrome (1 dog). One dog receiving chronic NSAIDs had a normal study.

ESVC

In conclusion, STE provides a rapid and non-invasive evaluation of RV function that may be used for clinical investigations in canine cardiology. Doppler-derived +dP/dt and -dP/dt from mitral regurgitation are considered indexes for assessment of systolic and diastolicfunction respectively, that have less load dependence than the ejection phase indexes. This study aimed to determine correlation between Doppler-deriveddP/dt and other systolic and diastolic echocardiographic indexes, and if they can be used to identify dogs with and without remodeling, with or without congestive heart failure (CHF) and for evaluation of chronic mitral valve disease (CMVD) severity. Fifty-seven dogs with CMVD (stages B1, B2, C+D) were included prospectively in an observational cross-sectional clinical study and distributed in groups regarding the presence of remodeling and CHF, to evaluate+dP/dt and -dP/dt, and distributed according to TDI-diastolic pattern tocompare -dP/dt. Group C+D (2142 mmHg/s, P 25 -P 75 = 2023-2456)had +dP/dt significantly lower compared to B1 (2865 mmHg/s, P 25 -P 75 = 2383-3308)and B2 (2721 mmHg/s, P 25 -P 75 = 2241-3186) (P = 0.0023). GroupC+D also had lower -dP/dt, compared to B1 (968.5 mmHg/sAE266.8 and 1198 mmHg/sAE165.7; P = 0.0115). Dogs with CHF compared to those without CHF, presented lower +dP/dt(2142 mmHg/s, P 25 -P 75 = 2023-2456; 2858 mmHg/s, P 25 -P 75 = 2299-3241; P = 0.0007) and
Disclosures: No disclosures to report. In people anemia is frequent in patients with heart failure (HF) and it is associated with poor outcomes. The most likely pathogenic factors include iron deficiency, chronic kidney disease (CKD), and cytokine production, although other factors may contribute. Little is known about the prevalence of anemia in dog with cardiovascular disease.
Disclosures: No disclosures to report. The objective of this study was to evaluate left atrial (LA) function by left atrial total fractional area change (LA-FACtotal) and left atrial ejection fraction (LAEF) in dogs affected with chronic mitral valve disease (CMVD) naturally acquired with and without congestive heart failure (CHF). Our hypothesis was that LA-FACtotal and LAEF decrease with severity of CMVD. Eighty dogs were included in a prospective observational cross-section clinical study, grouped according to CMVD severity based on echocardiographic evaluation and clinical signs. The dogs were equally distributed in each group: A, B1, B2 and C, according to American College of Veterinary Internal Medicine staging system. Indicators of LA function were calculated with the following equations: LA-FACtotal = 100 9 (LAmaximum area -LAminimum area)/LAmaximum area, measured by apical 4 view; and LAEF = 100 x (LAmaximum volume -LAminimum volume)/LAmaximum volume, by biplane area-length method from the left apical 4 and 2chamber views. LA-FACtotal showed lower values (P < 0.0001) in group C (31.88%, P25-75% = 26.47-41.12) compared with groups A (52.75%, P25-75% = 48.08-56.07), B1 (48.38%, P25-75% = 42.57-51.91) and B2 (46.15%, P25-75% = 41.17-50). Group C had lower LAEF (40.69%, P25-75% = 34.89-52.09) than groups A (68.12%, P25-75% = 64.96-69.91), B1 (58.72%, P25-75% = 52.25-64.60) and B2 (56.98%, P25-75% = 52.08-61) (P < 0.0001). Left atrial function, assessed by LA-FACtotal and LAEF, was reduced in dogs with CMVD and CHF compared with healthy and asymptomatic CMVD groups.

ESVC-P-

Twenty-one dogs affected by MVD (cases) were included and 20 healthy dogs (controls) were randomly selected among the eligible population. The 33% of cases experienced at least one episode of congestive heart failure (CHF), but none of these patients developed chronic kidney disease (CKD). The 14% of cases developed CKD while remaining in ACVIM class B2. No dogs in the control group developed CKD or MVD. Correlations between worsening renal function (WRF -sCr elevation ≥0.3 mg/dl or 25% from baseline), furosemide administration, UPC levels, radiographic parameters of heart enlargement and echocardiographic parameter were investigated. Only a statistically significant difference in IRIS class between the groups according to WRF and in the echocardiographic parameter left atrium to aortic root (LA/Ao) according to furosemide amount were observed. Both these results were expected. None of the cases included experienced renal damage (WRF or IRIS class change or UPC change) concomitant to episodes of CHF. The persistence of normal renal condition regardless of CHF events and therapy administration was unexpected. In conclusion, experiencing CHF seems not to directly affect renal function. To authors' opinion, the use of WRF, better than single sCr and UREA levels, may be useful in the long term management of aged patients affected by MVD. However, the small number of cases included in this study represents a great limit. We consider this work a pilot study.

ESVIM-P-2 PET OWNERS USE OF THE INTERNET FOR THEIR PETS' HEALTH. A. Chatard, M. Hugonnard. VetAgro Sup, Marcy l'etoile, France

Disclosures: Sara Hoummady received a grant from MP Labo for his PhD Work about dog aging and Marc Blondot work at the Paris guide dog school. Laryngeal dysfunction is most commonly associated with aspiration pneumonia, however its role in other lower airway diseases has not been investigated. Laryngoscopic and bronchoscopic findings in dogs examined by the author between 2001 and 2014 were evaluated for the presence or absence of laryngeal abnormalities. Dogs that presented for evaluation of inspiratory difficulty or panting were excluded from analysis. Clinical diagnoses of inflammatory airway disease, airway collapse, airway infection, eosinophilic bronchopneumopathy or a combination of these disorders were obtained through bronchoscopy and bronchoalveolar lavage fluid analysis. Detection rates for laryngeal abnormalities were compared among disease groups using Chi Square analysis and Fisher's exact test, with significance set at P < 0.05. A total of 138 dogs were evaluated and varied in age between 4 months to 15.5 years (median 8 years). Weight ranged from 1.5-63.4 kg (median 13 kg), with 31 dogs <5 kg, 28 dogs from 5.1-9.9 kg, 24 dogs from 10-20 kg, 45 dogs from 20.1-40 kg, and 9 dogs >40 kg. Laryngeal hyperemia or swelling was found in 73/138 dogs (53%), and detection rate did not differ among disease processes. Laryngeal function was considered suspect in 59/138 cases, prompting administration of doxapram, which normalized function in 30/59 dogs. Laryngeal paresis or paralysis was reported in a total of 26/ 138 dogs (19%). A substantial number of dogs with chronic cough displayed evidence of abnormal laryngeal structure or function, suggesting that a complete laryngoscopic examination should be performed in all dogs evaluated for cough.

ESCG-P-4 ASSESSMENT OF IMAGE QUALITY PRODUCED BY

ALI was successfully performed in 5/5 patients, with no adverse effects. Average study duration was 15.7 AE 4.1 hour and mean image acquisition count was 22.572 AE 17.315. Gastric and small intestinal transit times were 79.2 AE 39.8 minute and 119.4 AE 43.7 minute, respectively. Median (range) image quality scores were 9 (8-10), 8 (6-10) and 6 (5-9), for the stomach, SI and colon, respectively. Image quality scores were significantly higher in the stomach and SI than in the colon (P < 0.001). Visualized lesions were consistent with GI ulcers (2 dogs), inflammatory bowel disease (1 dog), and bilious vomiting syndrome (1 dog). One dog receiving chronic NSAIDs had a normal study.

ESVC-P-8 QUANTIFICATION OF SYSTOLIC AND DIASTOLIC RIGHT

In conclusion, STE provides a rapid and non-invasive evaluation of RV function that may be used for clinical investigations in canine cardiology. Doppler-derived +dP/dt and -dP/dt from mitralregurgitation are considered indexes for assessment of systolic and diastolicfunction respectively, that have less load dependence than the ejection phaseindexes. This study aimed to determine correlation between Doppler-deriveddP/dt and other systolic and diastolic echocardiographic indexes, and if theycan be used to identify dogs with and without remodeling, with or withoutcongestive heart failure (CHF) and for evaluation of chronic mitral valvedisease (CMVD) severity. Fifty-seven dogs with CMVD (stages B1, B2, C+D) wereincluded prospectively in an observational cross-sectional clinical study anddistributed in groups regarding the presence of remodeling and CHF, to evaluate+dP/dt and -dP/dt, and distributed according to TDI-diastolic pattern tocompare ÀdP/dt. Group C+D (2142 mmHg/s, P 25 -P 75 = 2023-2456) had +dP/dt significantly lower compared to B1 (2865 mmHg/s, P 25 -P 75 = 2383-3308) and B2 (2721 mmHg/s, P 25 -P 75 = 2241-3186) (P = 0.0023). GroupC+D also had lower ÀdP/dt, compared to B1 (968.5 mmHg/s AE 266.8 and 1198 mmHg/s AE 165.7; P = 0.0115). Dogs with CHF compared to those without CHF, presented lower +dP/dt (2142 mmHg/s, P 25 -P 75 = 2023-2456; 2858 mmHg/s, P 25 -P 75 = 2299-3241; P = 0.0007) and ÀdP/dt (968.5 mmHg/ s AE 266.8; 1155 mmHg/s AE 199.0; P = 0.0041). Regardingdiastolic function, ÀdP/dt was lower for the restrictive pattern group (769.7 mmHg/s AE 124.1) compared to those without diastolic dysfunction, (1132 mmHg AE 204.0), delayedrelaxation (1229 mmHg AE 186.9) and pseudonormal patterns (1107 mmHg AE 223.4) (P < 0.0001).When +dP/dt<1800 mmHg/s, the post-test chance for the dog with CMVD to haveCHF is twice the chance than not having it. For ÀdP/dt<800 mmHg/s theposttest chance of having CHF is eight times higher than not having it. In conclusion, Doppler-derived +dP/dt and ÀdP/dt may contribute respectively, for systolic and diastolic assessment ofdogs with CMVD.
Disclosures: No disclosures to report. In people anemia is frequent in patients with heart failure (HF) and it is associated with poor outcomes. The most likely pathogenic factors include iron deficiency, chronic kidney disease (CKD), and cytokine production, although other factors may contribute. Little is known about the prevalence of anemia in dog with cardiovascular disease.
Disclosures: No disclosures to report. The objective of this study was to evaluate left atrial (LA) function by left atrial total fractional area change (LA-FACtotal) and left atrial ejection fraction (LAEF) in dogs affected with chronic mitral valve disease (CMVD) naturally acquired with and without congestive heart failure (CHF). Our hypothesis was that LA-FACtotal and LAEF decrease with severity of CMVD. Eighty dogs were included in a prospective observational cross-section clinical study, grouped according to CMVD severity based on echocardiographic evaluation and clinical signs. The dogs were equally distributed in each group: A, B1, B2 and C, according to American College of Veterinary Internal Medicine staging system. Indicators of LA function were calculated with the following equations: LA-FACtotal = 100 9 (LAmaximum area ÀLAminimum area)/LAmaximum area, measured by apical four view; and LAEF = 100 9 (LAmaximum volume À LAminimum volume)/ LAmaximum volume, by biplane area-length method from the left apical four and two-chamber views. LA-FACtotal showed lower values (P < 0.0001) in group C (31.88%, P25%-75% = 26.47-41.12) compared with groups A (52.75%, P25%-75% = 48.08-56.07), B1 (48.38%, P25%-75% = 42.57-51.91) and B2 (46.15%, P25%-75% = 41.17-50). Group C had lower LAEF (40.69%, P25%-75% = 34.89-52.09) than groups A (68.12%, P25%-75% = 64.96-69.91), B1 (58.72%, P25%-75% = 52.25-64.60) and B2 (56.98%, P25%-75% = 52.08-61) (P < 0.0001). Left atrial function, assessed by LA-FACtotal and LAEF, was reduced in dogs with CMVD and CHF compared with healthy and asymptomatic CMVD groups.

ESVC

Twenty-one dogs affected by MVD (cases) were included and 20 healthy dogs (controls) were randomly selected among the eligible population. The 33% of cases experienced at least one episode of congestive heart failure (CHF), but none of these patients developed chronic kidney disease (CKD). The 14% of cases developed CKD while remaining in ACVIM class B2. No dogs in the control group developed CKD or MVD. Correlations between worsening renal function (WRF -sCr elevation ≥0.3 mg/dL or 25% from baseline), furosemide administration, UPC levels, radiographic parameters of heart enlargement and echocardiographic parameter were investigated. Only a statistically significant difference in IRIS class between the groups according to WRF and in the echocardiographic parameter left atrium to aortic root (LA/Ao) according to furosemide amount were observed. Both these results were expected. None of the cases included experienced renal damage (WRF or IRIS class change or UPC change) concomitant to episodes of CHF. The persistence of normal renal condition regardless of CHF events and therapy administration was unexpected. In conclusion, experiencing CHF seems not to directly affect renal function. To authors' opinion, the use of WRF, better than single sCr and UREA levels, may be useful in the long term management of aged patients affected by MVD. However, the small number of cases included in this study represents a great limit. We consider this work a pilot study.

ESVE-P-4 VALIDATION OF AN ENZYME FLUORESCENCE ASSAY (ELFA) TO MEASURE TOTAL THYROXINE IN DOGS AND CATS.

Disclosures: Sara Hoummady received a grant from MP Labo for his PhD Work about dog aging and Marc Blondot work at the Paris guide dog school. Laryngeal dysfunction is most commonly associated with aspiration pneumonia, however its role in other lower airway diseases has not been investigated. Laryngoscopic and bronchoscopic findings in dogs examined by the author between 2001 and 2014 were evaluated for the presence or absence of laryngeal abnormalities. Dogs that presented for evaluation of inspiratory difficulty or panting were excluded from analysis. Clinical diagnoses of inflammatory airway disease, airway collapse, airway infection, eosinophilic bronchopneumopathy or a combination of these disorders were obtained through bronchoscopy and bronchoalveolar lavage fluid analysis. Detection rates for laryngeal abnormalities were compared among disease groups using Chi Square analysis and Fisher's exact test, with significance set at P < 0.05. A total of 138 dogs were evaluated and varied in age between 4 months to 15.5 years (median 8 years). Weight ranged from 1.5 to 63.4 kg (median 13 kg), with 31 dogs <5 kg, 28 dogs from 5.1-9.9 kg, 24 dogs from 10-20 kg, 45 dogs from 20.1-40 kg, and 9 dogs >40 kg. Laryngeal hyperemia or swelling was found in 73/138 dogs (53%), and detection rate did not differ among disease processes. Laryngeal function was considered suspect in 59/138 cases, prompting administration of doxapram, which normalized function in 30/59 dogs. Laryngeal paresis or paralysis was reported in a total of 26/ 138 dogs (19%). A substantial number of dogs with chronic cough displayed evidence of abnormal laryngeal structure or function, suggesting that a complete laryngoscopic examination should be performed in all dogs evaluated for cough.
42 section matches
Saccharomyces boulardii (Sb)is a non-pathogenic yeast used in the prevention and treatment of gastrointestinal disorders in human beings and horses. The aim of this study was to evaluate the effect of Sb in healthy dogs and dogs with chronic enteropathies (CE). Sb was formulated in 10x10 9 CFU capsules. Its concentration and viability within the capsules was controlled by yeast culture in subsequent steps until expiration date. Four healthy dogs (HD) and 18 dogs with CE (10 inflammatory bowel disease -IBD, 8 protein losing enteropathy -PLE) were included. In HD Sb was administered for 10 days (1x10 9 CFU/kg BID); daily clinical evaluation was performed to assess possible adverse effects and quantitative stool cultures for yeasts were performed before, during and after the administration. In dogs with CE a randomized double blind placebo-control study was performed, administering Sb (1x10 9 CFU/kg BID) or placebo (Pl). Sb or Pl administration was added to standard therapeutic protocols (diet, antibiotics and immunosuppressive drugs), to evaluate its efficacy for the treatment of IBD and PLE. Complete blood work, abdominal ultrasonography, gastro-duodenal and colon endoscopy and histopathological evaluation of intestinal samples were performed at diagnosis and after 60 days of treatment. Validated score system for the clinical signs (CECCAI), ultrasonography, endoscopy and histopathology were applied. Significance was set for P < 0.05. Results in HD showed the absence of Sb in the faeces before treatment, its presence after one day, its steady state (10x10 7 CFU/g) after 5 days and its complete elimination 4 days after withdrawal of treatment. No adverse effects were reported. In CE dogs the clinical score improved significantly in dogs receiving Sb compared to dogs receiving Pl (P = 0.009). In PLE dogs the albumin concentration increased significantly (P = 0.034) in the group receiving Sb with respect to Pl. The daily frequency of defecation in the Sb group was significantly lower with respect to Pl after 45 (P = 0.032) and 60 (P = 0.004) days of treatment. No statistical differences were found between dogs receiving Sb and Pl after treatment, based on the endoscopic evaluation of duodenum and colon. No statistical differences were found between the two groups on the duodenal ultrasonographic and histological evaluation after treatment. In conclusion, Sb can be safely used in dogs with CE, in addition to standard treatment, to achieve a better control of clinical signs and a significant increase in albumin concentration compared to the standard therapy alone.

Conflicts of interest:

There are no other conflicts to disclose. Fecal S100A12 and fecal calprotectin concentrations have been described as biomarkers in dogs with chronic enteropathies [1]. However, to date there has been no direct comparison of these two markers in dogs with chronic diarrhea. The aim of this study was to evaluate the performance of these two markers in this situation.
Thirty one dogs presented for a history of chronic diarrhea were prospectively enrolled. The initial diagnostic workup for all patients included a serum biochemistry profile, fecal parasitology, abdominal ultrasound examination, and gastrointestinal endoscopy with collection of endoscopic biopsies. The severity of clinical signs was evaluated using the CCECAI scoring index and patients were grouped by having a CCECAI of < 12 or ≥ 12. Fecal calprotectin and S100A12 were quantified as previously described [1] . Correlations were evaluated with the Spearman rank correlation test. For both markers a receiver operating characteristics (ROC) curve was used to select cut-off value that allowed the best discrimination between dogs with a CCECAI<12 and dogs with a CCECAI ≥ 12. Sensitivity and specificity were calculated.
It is concluded oral cobalamin supplementation can effectively increase serum cobalamin concentrations in geriatric cats with idiopathic chronic enteropathy, but that following cessation of supplementation concentrations decrease rapidly and can become subnormal again within as few as 4 weeks.
ESVC-O-20 CARDIORENAL SYNDROME IN DOGS WITH CHRONIC VALVULAR HEART DISEASE: A RETROSPECTIVE STUDY. E. Martinelli 1 , P. Scarpa 2 , C. Quintavalla 1 , C. Locatelli 2 , P. Brambilla 2 . 1 University of Parma, Parma, Italy, 2 University of Milan, Milan, Italy
The aim of this study was to define the prevalence of chronic kidney disease (CKD) complicating chronic valvular heart disease (CVHD) in dogs and to investigate the relationship between class of cardiac insufficiency (ACVIM) and class of renal insufficiency (IRIS).
No conflicts of interest reported. Ligneous membranitis is a rare chronic inflammatory disease associated with congenital plasminogen deficiency. It has only been described in six unrelated dogs. The objective of this study is to report the presentation, clinicopathological and post mortem findings in three related Scottish terrier puppies with ligneous membranitis.
The cost of examinations reported in this study were covered by Hill's Pet Nutrition Belgium. Systemic Lupus Erythematosus, SLE, is a chronic autoimmune disorder with varying clinical manifestations and diagnosis is based on both clinical signs and laboratory findings. Other systemic rheumatic diseases, referred to as SLE-related diseases or immune-mediated rheumatic disease (IMRD), are also described. The most common clinical signs in dogs are stiffness and pain from varying joints. One hallmark of SLE and SLE-related diseases in both dogs and humans is high titres of circulating antinuclear antibodies (ANA), which can be demonstrated by the indirect immunofluorescence (IIF) ANA test. Earlier studies have shown that canine IIF ANA positive samples may be divided into two main subgroups: homogenous (ANA H ) and speckled (ANA S ) IIF ANA fluorescence pattern. In humans, further determination of the specificity of ANA positive sera is frequently employed to characterize the ANA reactivity. Some of these ANA specificities have been demonstrated in man to strongly associate with different systemic autoimmune diseases and also with different IIF ANA staining patterns. Presence and character of antinuclear antibodies in canine SLE-related diseases are not well described.
One of the authors, Erik Lattwein, is employed by EUROIMMUN where the analyses were performed. Chronic kidney disease (CKD) has a high prevalence in cats. Routine renal markers, serum creatinine (sCr) and urea are not sensitive or specific enough to detect early CKD. Serum Cystatin C (sCysC) has advantages over sCr for the detection of early kidney dysfunction, both in humans and dogs. A significant higher sCysC concentration in CKD cats has been demonstrated. The objective of this study was to determine the effect of age, gender and breed on feline sCysC and to establish a reference interval for feline sCysC.
The sera for virus neutralization were provided by Merial, France. defined herein as presence of sneezing, nasal-and/or ocular discharge, conjunctivitis and/or keratitis), but also oral cavity lesions, chronic stomatitis, limping syndrome and, rarely, virulent systemic disease. The aims of the present study were to compare cats suspected of FCV (FCV-SC) based on clinical symptoms and healthy controls (controls) and to investigate potential risk and protective factors, such as co-infection with feline herpesvirus-1 (FHV-1), Mycoplasma felis, Chlamydophila felis, Bordetella bronchiseptica and feline retroviruses, vaccination, gender, age, breed, housing and corticosteroid and antibiotic treatment. Oropharyngeal, nasal and conjunctival swabs from 200 FCV-SC and 100 controls were collected into transport medium, processed within 96 hours after collection and analyzed for FCV by virus isolation and for all tested pathogens using molecular assays. The samples were collected by randomly selected veterinary practices in 20 different areas of Switzerland (10 FCV-SC and 5 controls/ area). To record clinical data, retroviral status and vaccination history of the cats, a questionnaire was filled out by the private veterinarian. The seven tested pathogens were found in the investigated population. The prevalence (FCV-SC vs. controls) was: FCV 45% vs. 8%; FHV-1 20% vs. 9%, C. felis 8% vs. 1%, B. bronchiseptica 4% vs. 2%, M. felis 48% vs. 31%, Feline leukemia virus 2% vs. 1% and Feline immunodeficiency virus 2% vs. 1%. FCV-SC were positive for FCV significantly more often compared with controls (OR 9.2) and shed more FCV. Co-infections with up to four pathogens were detected; FCV-SC were significantly more frequently co-infected (40%) compared with controls (14%). Gingivostomatitis and oral ulceration but not URTD were highly associated with FCV infection. In contrast, C. felis was associated with URTD; FHV-1 was associated with nasal and ocular discharge and M. felis with conjunctivitis and ocular discharge. Risk factors for FCV infection were housing in groups (especially ≥4 cats), an intact gender, Maine Coon breed and corticosteroid therapy. FCV-positive cats with gingivostomatitis were older and more commonly vaccinated than FCV-positive cats without gingivostomatitis. Moreover they shed more FCV than cats with URTD. Vaccination and primary immunization defined as two vaccinations 2-6 weeks apart with the same vaccine brand were protective factors against FCV but not FHV-1 infection. Vaccination was associated with a decreased incidence of URTD in FCV-infected cats (OR 0.3). Further analyses will investigate cross-neutralization patterns of the prevailing FCV isolates.
Conflicts of interest: Financial support of Royal Canin. Canine chronic enteropathies (CCE) include diet-responsive, antibiotic-responsive, and immunosuppressive-responsive enteropathies (IRE). This prospective study was designed to evaluate a commercial hypoallergenic dry diet a containing oligopeptides as the only protein source for the management of dogs with IRE and as an alternative to immunosuppressive therapy over a 10 week period.

ESCG-P-3 A VERY LOW MOLECULAR WEIGHT POULTRY FEATHER HYDROLYZED-BASED EXTRUDED DIET

Nineteen dogs across France and Quebec entered the study. Dogs with food or antibiotic-responsive chronic enteropathy, hypoproteinemia, or treated with immunomodulating drugs were excluded from the study. Dogs were included in the study after complete clinical, ultrasonographic, endoscopic evaluation and histopathological evaluation of intestinal biopsies showing signs of intestinal inflammation. The owners were instructed to feed exclusively the study diet a .

Conflicts of interest:

The author works at Texas A&M University, whose GI Lab currently offer a commercial assay for faecal Alpha1-Proteinase Inhibitor. Canine chronic enteropathy (CE) is a common, but poorly understood syndrome, with variable response to therapy and prognosis. There is a need for novel biomarkers that are specific for intestinal disease and that provide objective measures of disease severity, progression, and prognosis. Serum citrulline is a useful biomarker in human intestinal disease as it is specific to the small intestine and indicates globally reduced enterocyte mass and absorptive function in various disease states. It is used to determine, quantitatively, intestinal integrity at the enterocyte level and is not influenced by nutritional or inflammatory status. The aim of this study was to determine whether serum citrulline can be used as a biomarker for CE in dogs.
One of the co-authors (Marco Caldin) has a diagnostic laboratory offering citrulline assays. Chronic enteropathy (CE) is a multi-factorial disease, which involves aberrant immune responses to commensal bacteria or dietary antigens. Macrophages have an important role in human disease but little information is available in canine intestine. Data to date have relied solely on macrophage identification using MAC387, an antibody directed against calprotectin, which recognizes both macrophages and neutrophils. In this study an alternative antibody for macrophages, AM-3K, directed against a scavenger receptor (CD163) was used and distribution of both markers was compared. This antigen is of interest as positive cells accumulate in intestine of humans with CE.
No conflicts of interest reported. Chronic diarrhea and vomiting are common clinical signs in dogs. Primary (e.g., inflammatory, infectious, neoplastic, mechanical, or other) and secondary gastrointestinal diseases (e.g., exocrine pancreatic, hepatic, renal, or endocrine disease) are possible underlying causes.
The aim of this study was to evaluate the final diagnoses in dogs with chronic diarrhea and/or vomiting and to determine the prevalence of various primary and secondary gastrointestinal diseases in dogs with these gastrointestinal signs.
Medical records of 209 dogs presented between July 2011 and August 2013 with chronic diarrhea (D), vomiting (V) or both (diarrhea and vomiting [VD]) were retrospectively reviewed. Dogs were included if a minimum work-up (hematology, plasma biochemistry profile, and fecal parasitology) had been performed and if a final diagnosis was recorded (155/209).
In this study, food responsive enteropathy (36%) was the most commonly diagnosed cause of chronic gastrointestinal signs. Chronic pancreatitis was the most frequent cause of secondary gastrointestinal disease (46%). Diarrhea was significantly associated with primary and vomiting with secondary gastrointestinal disease.
This study showed that pro-MMP-2 and -9 could be detected in the intestinal mucosa of healthy dogs using zymography, which seems to be a useful tool to evaluate the role of MMP-2 and -9 in the pathogenesis of canine chronic enteropathies, including inflammatory bowel diseases.
No conflicts of interest reported. There are only few laboratory markers being evaluated for diagnosing and/or monitoring canine chronic enteropathies, including inflammatory bowel disease (IBD). S100A12 belongs to the S100/calgranulin-protein family and has been proposed to play a central role in both innate and acquired immune responses. It has been reported to be increased in stool samples, serum and/or intestinal mucosa in human patients with IBD. Myeloperoxidase (MPO) is an enzyme found mostly in granulocytes. Intestinal mucosal levels of MPO have been shown to be increased in animal models and human IBD. To date, S100A12 and MPO levels in intestinal mucosal samples have been reported neither from healthy dogs nor from dogs suffering from IBD. To start investigating this aspect in dogs, the objective of this study was to evaluate mucosal S100A12 and MPO levels in the small and large intestines by using enzyme-linked immunoassay (ELISA) and spectrophotometric methods, respectively. For the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. The samples were taken and snap frozen in liquid nitrogen during necropsy from 12 healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota.

ESVIM

A prospective cross-sectional observational study was conducted and 20 dogs diagnosed with BP caused by opportunistic bacteria were included. 13 dogs with chronic (> 30 days) tracheobronchitis caused by Bordetella bronchiseptica were included as controls for virus analysis. Diagnosis was confirmed by thorough clinical examinations as well as with cytological and bacterial analysis of bronchoalveolar lavage (BAL) or transtracheal wash (TTW) samples. Canine parainfluenssavirus (CPIV), Canine adenovirus, Canine herpesvirus, Canine distempervirus, Canine respiratory coronavirus (CRCoV) and Canine pneumovirus were analysed in BAL or TTW samples using RT-PCR assay.
CPIV was detected in 7/20 (35%) and CRCoV in 1/20 (5%) respiratory samples in dogs with BP. Respiratory viruses were not detected in dogs with chronic tracheobronchitis. There were no significant differences in the duration of hospitalization (p = 0.427) or arterial paO 2 at presentation (p = 0.343) between BP dogs with and without a viral co-infection. These results indicate that co-infections with respiratory viruses are common also in household dogs with BP. Additionally, viral co-infections did not cause a more severe course of BP.

Conflicts of interest:

No conflicts of interest reported. Persistent renal proteinuria is considered an early marker of chronic kidney disease (CKD) and it is listed among the initiation factors and progression factors according to KDOQI guidelines. Nevertheless, few data are available about the prevalence of proteinuria in cats affected with CKD, in which it is assumed that nephropathy is mainly characterized by tubulointerstitial damage.
No conflicts of interest reported. Azotemia in dogs with chronic heart failure may reflect impaired renal function not only because of inadequate renal perfusion, but also due to organic renal injury. Impaired renal function is observed in 50% of dogs with heart failure. Altered renal hemodynamics due to decreased cardiac output results in renal hypoperfusion, and resultant elevation of blood urea nitrogen and creatinine, defined as azotemia. Azotemia is a prognostic factor in dogs with mitral regurgitation, therefore, preservation and/or restoration of renal function is thought to improve prognosis. Medical treatment for heart failure, however, includes angiotensin converting enzyme inhibitors and loop diuretics, which has been shown to increase the risk of developing azotemia. We hypothesized that mitral valve repair surgery ameliorates renal function by improvement of systemic hemodynamics. The change in renal function in dogs with mitral regurgitation was assessed by evaluating time-dependent changes in glomelular filtration rate by inulin clearance before and after cardiac surgery. Eighteen dogs with severe mitral regurgitation with azotemia (plasma urea nitrogen level > 28 mg/dL, plasma creatinine level >1.9 mg/dL) were included in this study. The glomerular filtration rate in all dogs were evaluated by determining inulin clearance before and 3 months after surgery. Serum atrial natriuretic peptide level, plasma NT-pro brain natriuretic peptide level, plasma urea nitrogen concentration, and plasma creatinine concentration were measured at each time point as well as during the initial staging of heart failure based on the International Small Animal Cardiac Health Council (ISACHC). Left atrial/aorta ratio by echocardiography and vertebral heart size by thoracic radiographs were also measured. Glomerular filtration rate significantly increased 3 months after surgery (40.0 ml/min/m 2 [25.6 -123.0], 2.7 ml/min/kg [1.0 -5.3]) compared to before surgery (38.4 ml/min/m 2 [12.6 -50.3], 2.2 ml/min/kg [0.7 -3.8]) (P < 0.05). The ISACHC stage of heart failure was improved at 3 months after surgery compared to before surgery. In addition, serum atrial natriuretic peptide level, plasma NT-pro brain natriuretic peptide level, plasma urea nitrogen concentration, LA/Ao and VHS significantly decreased after surgery (P < 0.05). The use of diuretics decreased after mitral valve repair surgery and consequently, a decrease in plasma urea nitrogen and creatinine levels were observed. Therefore, this suggests that the main cause of azotemia in dogs with mitral regurgitation may be due to inadequate renal blood flow and exacerbation by the use of diuretics.

ESCG-O-4 RISK FACTORS FOR FAECAL COLONIZATION WITH

Conflicts of interest: Dr Pomba currently receives research funding from the government and national programmes (Fundac ßão para a Ciência e a Tecnologia). In the past, she has occasionally received research support or honoraria for lectures from pharmaceutical companies including Zoetis and Atral Cipan. She is vice-chair of the Antimicrobial Working Party There are few reports in the literature reporting long-term relapse rate, owner compliance and clinical severity of dogs with chronic enteropathies. The goal of this study was to compare clinical activity index (CCECAI), number of relapses and compliance rates 1-3 years after diagnosis.
Conflicts of interest: Dr Allenspach has received research funding from BBSRC, American Kennel Club, Comparative Gastroenterology Society, Probiotics Ltd UK, Laboklin GmBH Germany, and Bioiberica Sp. she has also undertaken paid consultancy work for Bioiberica Spain and Hoffmann-Laroche, Switzerland. Despite the high prevalence of canine pancreatitis in postmortem studies and the introduction of new diagnostic tests, it is believed that the disease, particularly in its chronic form, remains under recognised due to the non-specific nature of presenting signs. Histology is considered to be the gold standard for diagnosis of canine pancreatitis, however, most clinicians are reluctant to take pancreatic biopsies due to significant risks to the patient.

Conflicts of interest:

The author receives a salary as Editor of the BSAVA journal Companion, and has undertaken unrelated paid consultancies for Bayer and Merial. The author also receives a salary from Avacta Animal Health, and duties involved working directly on this project. Canine chronic enteropathy (CCE) can cause significant long-term morbidity. In some cases this is due to intestinal inflammation, resulting from idiopathic inflammatory bowel disease (IBD). Currently, the diagnosis of idiopathic IBD and assessment of disease severity relies on results of subjective clinical indices, laboratory data, diagnostic imaging and intestinal histopathology, whilst ruling out known causes of inflammation. In humans with IBD, a number of faecal biomarkers including lactoferrin, aid with diagnosis and determining disease activity. It may therefore be valuable to develop similar non-invasive objective methods to aid diagnosis and clinical assessment of disease severity in dogs with intestinal inflammation due to idiopathic IBD.
No conflicts of interest reported. Sarcoplasmic reticulum (SR) Ca 2 + -ATPase and its regulatory proteins are pivotal determinants of myocardial active relaxation via calcium uptake against the SR-cytoplasmic gradient. The lowered density of the SR Ca 2 + -ATPase has been well demonstrated in many species during chronic hemodynamic overload. The genes linked to SR calcium uptake were reported not only being expressed in peripheral blood but serving as potential cardiac biomarkers in dogs with chronic mitral regurgitation, such as SR Ca 2+ adenosine triphosphatase isoform 2a (SER-CA2a), phospholamban (PLN), and HS-1 associated protein X-1 (HAX-1). The aim of this study is to determine whether the target genes expressed in the blood will be translatable to the myocardial setting as cardiac biomarkers.
Dr Bussadori receives royalties from ESAOTE (Florence, italy) related to an european patent (nr 071129712) he developed for Xstrain software. The study was not funded by a research grant. Cardiac cachexia which is characterized by progressive weight loss and depletion of lean body mass, is an independent predictor of survival in human patients with congestive heart failure. Chronic degenerative mitral valve disease (CDMD) is one of the most common cardiac diseases in dogs. The aims of this study were to evaluate the prevalence and the effects of cardiac cachexia in survival of dogs with CDMD.
The following conflicts of interest apply: The diet used in this study is manufactured by Royal Canin.whilst VB is employed by Royal Canin. VB and SS are employed by Royal Canin. AJG's Readership is funded by Royal Canin. Obesity and obesity-related metabolic dysfunctions are increasing in humans as well as in dogs. Obese dogs become affected by chronic diseases at young age, have a decreased quality of life and a shorter life-span. The aim of the study was to describe the metabolic and hormonal response to a feed-challenge test in lean and overweight dogs.
No conflicts of interest reported. Calprotectin (S100A8/A9 complex) belongs to the S100/calgranulin family, and is primarily released from activated neutrophils and macrophages. Serum calprotectin concentrations (CP) were shown to be increased in dogs with inflammatory diseases such as inflammatory bowel disease, pancreatitis, systemic inflammatory response syndrome, and sepsis. Canine CP thus appears to be a biomarker of inflammation. Considerable day-today variation of fecal canine CP was found in both healthy dogs and dogs with chronic gastrointestinal disease. However, the biological variation of canine CP in serum has not been reported. The aim of this study was to determine the biological variation of serum canine CP and its minimum critical difference (MCD).
The study was financially supported by the Swedish Juvenile Diabetes Foundation, the Fredrik and Ingrid Thuring Foundation, the Magnus Bergvall Foundation, the Lars Hierta Memorial Foundation, and the Foundation for Research, Agria Insurance Company. Feline acromegaly is an increasingly recognised endocrinopathy among diabetic cats, caused by chronic excessive growth hormone secretion by a functional somatotrophinoma in the pars distalis of the anterior pituitary gland. The majority of human somatotrophinomas are sporadic, however up to 20% of familial isolated pituitary adenomas are caused by germline mutations of the aryl-hydrocarbon-receptor interacting protein (AIP). Feline acromegaly has phenotypic and biochemical similarities to human familial acromegaly with AIP mutations, such as male predominance, somatotroph macroadenoma and resistance to octreotide therapy.
BALF samples were collected between 2008 and 2014 from 10 asymptomatic client-owned dogs and 55 dogs with various respiratory conditions, including 16 dogs with confirmed bordetellosis, 18 dogs with eosinophilic bronchopneumopathy (EBP), 8 dogs with chronic bronchitis and 13 dogs with bacterial bronchopneumonia, and were retrospectively assessed with a A. vasorum qPCR assay. Amongst those 65 dogs, only one BALF, from a dog with EBP, yielded a positive qPCR result. In this dog, faecal analysis was negative.
Conflicts of interest: ZHP and SG are supported by the EC FP7 Marie-Curie programme: NephroTools. The device development was supported by the FP7 activity: PLACE-it.NG is owner of a patent covering FITC-sinistrin and the technology for its measurement. Excretion of urinary biomarkers of renal damage should occur at an early stage of chronic kidney disease (CKD), thus facilitating earlier diagnosis of renal disease. Albumin and cystatin C in the renal ultrafiltrate are mostly reabsorbed by the proximal tubular cells, therefore increased urinary excretion of albumin and cystatin C (UAC and UCysC) would be expected to correlate with the presence of renal tubular damage and CKD. The aim of this study was to establish biological validity of two particle enhanced turbidimetric assays (PETIAs) for the measurement of albumin and cystatin C (previously validated for use in feline urine) by comparing the UAC and UCysC between non-azotaemic cats and cats with azotaemic CKD.
Results: Twenty-two dogs were included. No complications were reported during the laparoscopic procedure. The median number of portal triads per needle biopsy specimen was 6 (range, 4 to 8) compared to 20 (range 14;25) with wedge biopsy specimen. The median length of needle biopsy specimens was 10 mm; (range, 6 to 16 mm) and 6 mm for all wedge biopsies. On the basis of biopsy interpretation, the diagnosis was overall similar with the two methods: 8 dogs had vacuolar hepatopathy, 4 acute cholangitis, non-specific acute form in 3 dogs. Chronic hepatitis with cirrhosis was found in 2 cases, 3 dogs had diffuse neoplasia and 2 miscellaneous hepatic disorders. The fibrosis was considered to be severe in 3 dogs, moderate in 3 dogs and mild in 11 dogs. No quantitative and qualitative difference was observed between the two types of biopsies specimen.
No conflicts of interest reported. Alpha 1 -proteinase inhibitor (a 1 -PI) is a proteinase-resistent protein that can be quantified in fecal, urine, and serum samples from dogs. Recently, increased fecal and urinary canine a 1 -PI (ca 1 -PI) concentrations have been described in dogs with gastrointestinal diseases (e.g., inflammatory bowel disease [IBD] , but also in dogs with exocrine pancreatic insufficiency) and in dogs with chronic hepatitis or chronic kidney disease, respectively. Decreased serum ca 1 -PI concentrations have been reported in dogs with IBD, protein-losing enteropathy (PLE), and hypocobalaminemia. Treatment protocols for dogs with IBD and/or PLE commonly include corticosteroids, but the effect of corticosteroid therapy on serum ca 1 -PI concentrations have not yet been reported. The aim of this study was to evaluate the effect of hydro-cortisone on serum ca 1 -PI concentrations in healthy dogs.
The medical records of 13 cats diagnosed as having spontaneous digestive perforation between 2010 and 2013 were reviewed. The mean age of cats was 7.6 years (9 months to 17 years). Five cats had concurrent illnesses including viral upper respiratory tract disease, pancreatitis and chronic kidney disease. The most frequently reported signs included anorexia (85%), vomiting (61%), and lethargy (54%). Histological examination was performed in 11 cats and diagnosed alimentary lymphoma in 54% and inflammatory lesions in the other 46% of them. Six cats had received anti-inflammatory within the previous 3 months. Half of them were finally diagnosed with lymphoma. Five cats with lymphoma received chemotherapy.Three cats died early in the postoperative recovery period, 5 cats were euthanized 12 to 146 days after surgery, and 2 cats were still alive at the end of this study.
This study showed that using ELISA and spectrophotometry allow the detection of canine intestinal mucosal S100A12 and MPO, respectively. The levels on S100A12 and MPO seem to differ between certain parts of the intestinal mucosa of healthy dogs. Both assays appear to be useful to further evaluate the role of S100A12 and MPO in the pathogenesis of canine chronic enteropathies, including IBD.
The design of our feline diabetes mellitus DSS emerges from the syndrome of polyuria-polydipsia, with the possibility of spotting the accompanying pathologies. Fuzzy logic is used for dealing with knowledge representation and uncertainty. The fuzzy rules proposed to represent this knowledge emerge from anamnesis, clinician's input, clinical and paraclinical description, and confirmation diagnostic tests. Clinical signs such as polyuriapolydipsia, persistent hyperglycemia, polyphagia, weight fluctuations, administration of drugs with a diabetogenic potential, were considered decisive in the pattern of diagnosis establishment. Registered medical records of 29 cats, 16 males and 13 females, whit ages from 7 to 18 years old, were analyzed in order to validate the DSS. Using Matlab software, the DSS was implemented and tested. For any case with polyuria-polydipsia the system provides, via a friendly graphical user interface, the diagnosis with the highest probability. The set of diagnoses which can be generated by the DSS consists in: a) diabetes mellitus; b) diabetes mellitus induced by (b.1) hypersomathotropism, (b.2) hyperthyroidism, (b.3) hyperadrenocorticism and (b.4) diabetogenic medication; c) diabetes mellitus in association with (c.1) chronic kidney failure and (c.2) heart failure; d) ketoacidodic diabetes mellitus; e) pancreatitis. The DSS was applied with success on all 29 cases, revealing the following diagnoses / no of cases: (a) -8, (b.1) -2, (b.2) -1, (b.4) -8, (c.1) -3, (c.2) -3, (d) -4.
No conflicts of interest reported. Activins are cytokines belonging to the transforming growth factor (TGF)-b superfamily. It is thought that activins may be the key intermediary in TGF-b1 mediated fibrotic response. Activin A has been suggested to participate in the pathogenesis of human idiopathic pulmonary fibrosis (IPF), but studies regarding the role of activin B are still spares. Canine IPF (CIPF) is a chronic, incurable interstitial lung disease occurring particularly in West Highland White Terriers (WHWTs). During the disease course, acute exacerbations (AEs), with poor prognosis, can occur. Histopathologically AEs of CIPF are featured by diffuse alveolar damage, which is also a key feature in acute respiratory distress syndrome (ARDS). Our objective was to study the expression of activin A and B by immunohistochemistry in the lung tissue of CIPF WHWTs (n = 5), CIPF WHWTs with concurrent AE (n = 4), and dogs of various breeds with ARDS (n = 4), and to compare these findings to healthy WHWTs (n = 3). In addition, western blot analysis of activin B from bronchoalveolar lavage fluid (BALF) of CIPF WHWTs (n = 6) and healthy WHWTs (n = 6) was conducted. We demonstrated that activin B, but not activin A, is strongly expressed in the altered alveolar epithelium in lungs of diseased WHWTs as well as in ARDS lungs. Furthermore, activin B was detected in BALF of CIPF WHWTs, most notably in samples from dogs with AE, but not in BALF of healthy WHWTs. This novel finding suggests that activin B participates in the pathophysiology of CIPF and might act as a potential marker of alveolar epithelial damage.
No conflicts of interest reported. Dogs of the breed Nova Scotia Duck Tolling Retriever (NSDTR) are affected by several immune-mediated diseases, in particular steroid-responsive meningitis-arteritis (SRMA) and an immune-mediated rheumatic disease (IMRD). IMRD is a systemic lupus erythematosus-related disease characterized by chronic stiffness and pain in several joints. The aim of this study was to investigate the morbidity in NSDTRs and to test the hypothesis that NSDTRs are predisposed to SRMA and IMRD. Insurance data from a Swedish insurance company (Agria insurance company, Stockholm, Sweden) from 1995-2006 was used for the study. Approximately one third of Swedish dogs are insured by Agria and the insurance database is a validated tool for epidemiological studies. Assessment of morbidity was based on veterinary care events. Disease diagnoses were grouped in both general and specific disease categories. Individual diagnoses that were likely to represent IMRD were combined. Morbidity was defined as incidence rates and presented as number of cases per 10 000 dog years at risk (DYAR). Relative risk (RR) for NSDTRs compared to other breeds combined was calculated. The study included 445 336 dogs, 2890 were NSDTRs. The most common general causes of veterinary care for NSDTRs were injuries followed by gastrointestinal and musculoskeletal disorders with significant increased risk (RRs between 1.2 and 1.3) for NSDTRs compared to other breeds. The highest relative risk for NSDTRs was for systemic lupus erythematosus (RR 19.0). Compared to other breeds, NSDTRs had an increased risk for SRMA (RR 11.5) and IMRD (RR 11.8) with an incidence rate of 19.6 cases per 10 000 DYAR for SRMA and 8.8 cases per 10 000 DYAR for IMRD. The incidence rate for SRMA and IMRD in NSDTRs were also compared to dogs of other retriever breeds. The comparison revealed that NSDTRs also had a significant increased risk for both SRMA (RR 20.8) and IMRD (RR 10.1) when compared to other retrievers only. This study is the first to investigate the morbidity for IMRD in NSDTRs, which is important for further research and breeding practice. For several reasons the incidence rates might be underestimated and exact numbers should be interpreted with caution. However underestimation of incidence rates should not differ between dogs of different breeds, therefore not affecting the risk calculations. It can be concluded that NSDTRs are predisposed to the diseases SRMA and IMRD with an increased risk compared to other breeds and to other retrievers.
The aim of the present study was to investigate the frequency of Bb, Mycoplasma canis (M. canis) and M. cynos in canine EBP. Therefore, presence of Bb, M. canis and M. cynos were retrospectively assessed by quantitative polymerase chain reaction (qPCR) in bronchoalveolar lavage fluid (BALF) samples from 18 dogs with EBP (mean age = 4.5 y, mean body weight = 21.1 kg) as well as in 8 dogs with aspecific chronic bronchitis (7.3 y, 22.3 kg). Based on clinical signs, a clinical severity score (CSS, 0-5) was assigned each EBP dog.
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Abstract

Recommended treatment of chronic hepatitis B with interferon-α and/or nucleos(t)ide analogues does not lead to a satisfactory result. Induction of HBV-specific T cells by therapeutic vaccination or immunotherapies may be an innovative strategy to overcome virus persistence. Vaccination with commercially available HBV vaccines in patients did not result in effective control of HBV infection, suggesting that new formulations of therapeutic vaccines are needed. The woodchuck (Marmota monax) is a useful preclinical model for developing the new therapeutic approaches in chronic hepadnaviral infections. Several innovative approaches combining antiviral treatments with nucleos(t)ide analogues, DNA vaccines, and protein vaccines were tested in the woodchuck model. In this paper we summarize the available data concerning therapeutic immunization and gene therapy using recombinant viral vectors approaches in woodchucks, which show encouraging results. In addition, we present potential innovations in immunomodulatory strategies to be evaluated in this animal model.

Immunological Control of HBV Infection

Recent studies indicate that several mechanisms may be involved in the loss of the function of HBV-specific T cells during chronic hepatitis B. It was shown that high-level viremia negatively influences the virus-specific immune responses. High viral replication in the liver with viral load higher than 10 7 copies/mL is correlating with hyporesponsiveness of virus-specific CD8 + T cells in patients with chronic hepatitis B [23] . Moreover, the prolonged exposure to viral antigens occurring during the chronic viral infections can trigger the T cells to become tolerant and prone to apoptosis. The interaction between programmed death 1 (PD-1) receptor and its ligand PD-L1 (also known as B7-H1) plays an important role to prevent an overreaction of the immune system [24] . Recent studies revealed that inhibitory molecules such as PD-1 and CTLA-4 are markedly upregulated on virus-specific T cells, resulting in exhaustion (e.g., lack of IFNγ production and proliferation) [25] . Simultaneously, this mechanism can contribute to the development of the chronic infection by impairment of the effective antiviral response. This hypothesis was previously proven for hepatitis C virus (HCV) [26, 27] and human immunodeficiency virus (HIV) infection in humans [28] [29] [30] , as well as lymphocytic choriomeningitis virus (LCMV) infection in mice [31, 32] , and more recently for HBV [33, 34] . Furthermore, several studies imply that functional defects of antigen presenting cells (APCs), mainly dendritic cells (DCs), may contribute to the impaired T cell response in chronic hepatitis B patients [35] [36] [37] [38] [39] [40] [41] . In vitro studies showed that DCs isolated from HBV chronic carriers produce lower amount of antiviral cytokines, such as type I interferons and TNFα, in comparison to healthy controls [35, 36] . In addition, those DCs are less efficient in T cell activation and stimulation of T cell proliferation [35, [39] [40] [41] . The novel report demonstrated that myeloid DCs from chronic HBV patients express increased level of inhibitory PD-L1 molecule and therefore may down regulate functions of HBV-specific T cells [39] . Several investigations underline the significance of CD4 + CD25 + regulatory T cells in pathogenesis of persistent viral infections [42] . In HCVand HIV-infected patients, it was shown that regulatory T cells may downregulate HCV-and HIV-specific CD8 + and therefore influence the disease progression [43] [44] [45] . The role of regulatory T cells in HBV infection is still not clear. Nevertheless, the increased numbers of CD4 + CD25 + regulatory T cells were detected in the blood and the liver of patients with chronic severe hepatitis B [46] . In addition, the liver itself is an organ with tolerogenic properties that might contribute to the immunological tolerance during chronic HBV infection [47, 48] . Finally, viruses developed the strategies to efficiently evade the host immune response resulting in persistent infections. Viral immune escape due to the mutation of CD4 + , CD8 + , and B cell epitopes in a given HLA background have been observed in patients infected with HIV, HCV, and HBV [49] [50] [51] [52] [53] [54] .
Several studies demonstrate that the treatment with lamivudine alone, or in combination with interleukin-12 (IL-12), result in the restoration of the HBV-specific CD4 + and CD8 + immune response in chronic HBV-infected individuals. However, the therapeutic effect was not sustained in those patients [55] [56] [57] .

Clinical Trials of Therapeutic Immunization

Over 20 years, continuous efforts have been undertaken to develop a therapeutic vaccine for chronic hepatitis B to enhance the virus-specific immune responses and overcome persistent HBV infection [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] .
Yang et al. presented the novel DNA vaccine for treatment of chronic hepatitis and combined the immunizations with lamivudine treatment [71] . The multigene vaccine contains five different plasmids encoding most of HBV antigens and human IL-12 gene as a genetic adjuvant. The combination therapy led to sustained antiviral response in 6 out of 12 HBV chronically infected patients. The responders were able to clear HBeAg and had undetectable viral load at the end of a 52-week follow-up. Those effects were correlating with a detectable T cell response to at least one of the HBV antigens. [71] . Nevertheless, further studies are needed to evaluate this strategy on a larger cohort of HBV chronic carriers.

Transgenic Mouse Model for Studies on Therapeutic Immunization

In this paper we would like to introduce woodchucks as a useful preclinical model for designing of the new therapeutic vaccines in chronic hepadnaviral infections. We will summarize the available data concerning therapeutic immunization approaches in woodchucks and present potential innovations in immunomodulatory strategies that yet to be evaluated on this animal model.

Therapeutic Vaccination Approaches in the Woodchuck Model

Recently described advancements in the characterization and monitoring of the woodchuck immune system during the WHV infection, made this animal model particularly useful for development of the immunomodulatory approaches in chronic hepatitis B. The natural occurrence of chronic WHV infection in woodchucks, that is closely related to HBV infection in humans, allows to evaluate the potentially new therapeutic strategies directly in chronic WHV carriers. Up to date, several studies of diverse therapeutic vaccinations have been carried out in woodchucks ( Table 2) . The pioneer investigations based on therapeutic vaccines based on WHV core [96] or surface antigens in combination with a helper peptide FIS [120] , or with potent Th1 adjuvants like monophosphoryl lipid A [121] did not lead to satisfactory results. Those experiments proved that vaccinations could induce specific B-and/or T cell responses in chronic WHV carriers. However, this alone was not sufficient to achieve the control of virus replication. It is assumed that high level viremia, during the chronic hepatitis B, can inhibit the therapeutic effect of the vaccination. Treatment of chronic HBV patients with lamivudine could transiently restore HBV-specific T cell immune response [55, 56] . Therefore, reduction of viral load by the nucleos(t)ide analogues pretreatment might support the efficacy of immunization to enhance the virus-specific immune responses. This hypothesis was tested in three experimental trials of the combination therapies in chronic WHV carriers.
The first study performed by Hervás-Stubbs et al. was based on lamivudine therapy [108] . Five chronically WHVinfected woodchucks were treated orally with the drug for 23 weeks. At week 10, after decline of WHV DNA by 3-5 logs, three animals were vaccinated with 3 doses of serum-purified WHsAg combined with T-helper FIS peptide derived from sperm whale myoglobin. The vaccination induced T-helper responses against WHV antigens, shifting the cytokine profile from Th2 to Th0/Th1. However, no beneficial effect on WHV viral load and WHsAg levels was observed in comparison to nonimmunized animals. After withdrawal of the lamivudine treatment the values of viremia returned to the pre-treatment levels.
Recently, a novel therapeutic approach for treatment of chronic hepatitis B in a woodchuck model was described. The therapy combined the antiviral treatment with immunization with plasmid DNA and antigen-antibody immunogenic complex vaccines together [112] . DNA vaccines are considered to stimulate both humoral and cellular immune response, polarizing T cells in the direction of Th1 response [122] . Immunization of the naïve woodchucks with the plasmids encoding WHV core and preS2/S genes (pWHcIm and pWHsIm, resp.) induced the lymphoproliferative responses against the antigens and provided a protection against WHV challenge [123] . In addition, the DNA vaccine expressing HBsAg proved to elicit the vigorous T cell responses in chronic HBV carriers, however, this effect was only transient [70] . The HBsAg/anti-HBs IC vaccine is currently under the investigation in chronic HBV patients [77] [78] [79] .
To evaluate the efficacy of previously mentioned immunotherapy in woodchucks, firstly 10 chronic WHV carriers were treated with 15 mg of lamivudine, daily for 21 weeks. At week 10, four animals were pretreated with cardiotoxin and then received three immunizations with DNA vaccine containing three plasmids expressing WHsAg, WHcAg, and woodchuck IFNγ (pWHsIm, pWHcIm and pWIFN, resp.). Simultaneously, the other four woodchucks received three doses of the combination of DNA vaccine and WHsAg/anti-WHs immunogenic complex. Two chronic WHV carriers served as lamivudine monotherapy control. Lamivudine treatment resulted in only a slight decrease of WHV DNA levels in the woodchucks serum (0,7 and 0,32 log, resp.). Surprisingly, the DNA vaccination did not lead to any additional therapeutic effect beyond that observed for lamivudine treatment alone. In contrast, the triple combination of antiviral treatment, plasmid DNA encoding WHcAg, WHsAg, and wIFNγ and IC vaccines was able to decrease WHV viral load up to 2,9 log and the serum WHsAg up to 92%. Moreover, three of the four treated animals developed anti-WHs antibodies. Nevertheless, these effects were not sustained and all parameters reached the baseline levels shortly after withdrawal of lamivudine treatment. In addition, the vaccination did not induce WHVspecific T cell responses in the majority of woodchucks, even in animals that exhibited virological responses. Significant Hepatitis Research and Treatment 7 lymphoproliferative responses against WHV antigens were detected only in one animal after three immunizations with DNA vaccine [112] . The study demonstrated the benefit of using the combinatory therapy in chronically WHVinfected woodchucks. However, the transient therapeutic effects, suggest that this strategy needs further optimization.

Improvement of Adenoviral Vectors

Immunization of chronically WHV-infected woodchucks with plasmid DNA vaccine in combination with entecavir treatment showed a marked therapeutic effect. Addition of the recombinant adenoviruses to this regimen could be a new, more potent approach in treatment of chronic hepatitis B. We will apply DNA prime-AdV boost approach in WHV chronically infected woodchucks in combination with nucleos(t)ide analogs and evaluate its therapeutic potential.

Adenoviral Vectors for Gene Transfer Strategies in Treatment of Chronic Hepatitis B

So far, several trials of virus-mediated gene therapy for treatment of chronic hepatitis and HCC were performed in chronically WHV-infected woodchucks and in cell culture systems. Those strategies were mainly based on delivery of antiviral cytokines, such as IFNα, IFNγ, IL-12 by recombinant adenoviruses, to reduce viral replication or modulate the immune response ( Table 3) .
Transduction of primary woodchuck hepatocytes from chronic WHV carriers with helper-dependent AdV encoding woodchuck IFNα (wIFNα) resulted in the reduction of WHV proteins expression in vitro [169] . In vivo studies on chronically WHV-infected woodchucks, demonstrated that a single injection of 1 × 10 12 vp of this vector into the liver's portal vein could inhibit WHV replication by 1 log up to 11 weeks after the treatment [163] . The same approach with helper-dependent AdV expressing woodchuck IFNγ (wIFNγ) did not show any antiviral effect, even though the transduction led to the production of biologically active interferon [163] . Another study combined intravenous delivery wIFNγ by recombinant adenoviral vector with nucleos(t)ide analogues therapy. Chronic WHV carriers were treated with clevudine and emtricitabine (FTC), together, for 8 weeks and after the initial drop in viral load one group of animals received additionally two i.v. injections of 3 × 10 10 PFU of Ad-IFNγ. Delivery of wIFNγ induced inflammation, caused by T cell infiltration, and increased hepatocyte turnover. However, this effect did not induce additional antiviral outcome in comparison clevudine/emtricitabine biotherapy alone [164] . Similarly, poor therapeutic effect was observed for gene therapy based on both wIFNγ and wTNFα. Intravenous injection of those recombinant adenoviruses during clevudine treatment led to decrease of replicative intermediates of WHV DNA in the liver, beyond what could be achieved by clevudine alone. Nevertheless, 6 weeks after injection there was no significant difference between the groups of WHV carriers receiving AdV expressing the cytokines or beta-galactosidase as a control [165] . The benefits of using the immunomodulatory genes in this study are difficult to assess, since it was reported that adenovirus infection alone is sufficient to transiently suppress the WHV replication in chronically infected woodchucks [170] . The lack of therapeutic effect by direct delivery of IFNγ is consistent with in vitro data obtained from persistentlyinfected woodchuck primary hepatocytes. Treatment of the cells with wIFNγ, even in the presence of wTNFα, was not able to inhibit the WHV replication. Moreover, high concentration of those cytokines resulted in the loss of the cells during the culture [171] . This observation underlines the cytotoxic effect of Th1 cytokines on the woodchuck hepatocytes. Rapid downregulation of the IFNγ expression, after transduction of the liver cells with viral vector, could be one of the mechanisms to protect the organism from the potential toxicity of this cytokine in vivo [163] . In addition, several reports indicates that the level of wIFNγ and wTNFα is higher in the liver of chronic WHV carriers in comparison to naïve animals [172, 173] . Therefore, continuous presence of inflammatory cytokines in the liver during the chronic WHV infection could result in hyporesponsiveness of hepatocytes to such a therapy.
The novel strategy to treat chronic WHV hepatitis is based on adenovirus-mediated delivery of murine IL-12 (mIL-12) gene into hepatocytes [166] . Interleukin-12 is a proinflammatory cytokine produced naturally by antigen presenting cells. IL-12 stimulates production of IFNγ and TNFα by T and natural killer (NK) cells and enhances their cytotoxic activity [174] . In the study, mIL-12 gene expression could be regulated by inducible promoter that was responding to progesterone antagonist RU486. Eight chronic WHV carriers received single dose of 2 × 10 10 i.u. of AdV expressing mIL-12 (HC-Ad/RUmIL-12) by intrahepatic injection at laparotomy. Two weeks after, the expression of mIL-12 was induced by the administration of RU486. The IL-12 treatment resulted in intense and sustained suppression of WHV replication in the liver as well as decreased viral loads in the serum. This effect, however, was visible only in the animals with basal viremia lower than 10 10 WHV copies per milliliter of serum. Animals, which responded to the therapy, developed a vigorous T cell response to WHcAg, measured by woodchuck IL-2 production, and demonstrated WHeAg and WHsAg seroconversion. Moreover, the FoxP3 levels in the livers of those animals were decreased, while in nonresponder woodchucks FoxP3 values were significantly upregulated [166] . This finding suggests that the intrahepatic expression of IL-12 may inhibit the regulatory T cells in the liver during the chronic WHV infection. Indirect induction of inflammatory cytokines, such as IFNγ and TNFα by IL-12, seems to be a more efficient strategy in breaking the tolerance to virus antigens than direct delivery of those cytokines. It suggests that probably additional events occur in the liver after AdV-mediated IL-12 transfer that supports the antiviral effects of this therapy.

Gene Transfer Strategies for the Treatment of Hepatocellular Carcinoma

The results presented here indicate that viral-mediated gene therapy in treatment of chronic hepatitis B and HCC needs further optimization. However, treatment of the woodchucks with viral vectors allowed to achieve a long-lasting expression of the cytokines and their higher concentration preferably in the liver. Therefore, this strategy is proven to be more effective than an approach based on using of the soluble cytokines. In addition, adenovirusmediated gene transfer is proven to be a safe and a welltolerated strategy in the woodchucks.

Conclusion

The current progress indicates the feasibility of therapeutic approaches for treatment of chronic HBV infection. There is a general agreement that a combination of antiviral treatment and immunomodulation is essential to achieve a sustained control of HBV infection. However, many scientific questions are still not answered. The question how HBV infection leads to defective immune responses to HBV proteins remains to be investigated. This issue is the key to a more rational design of new therapeutic approaches. Recently, HBV proteins were found to suppress host innate responses [188] . It has to be clarified whether an early blockage of innate immune responses may further negatively influence the priming of adaptive immune responses. In addition, different groups reported consistently that TLR2 and TLR4 signalling may be impaired in chronic HBV infection patients [189, 190] . Thus, it is worthy to test whether an enhancement of innate immune responses in chronic carriers is necessary for restoration of specific immune responses. With the increasing number of available vaccine formulation, a more crucial question raised recently: what is the optimal combination of these vaccines. Obviously, it is necessary to test the mutual influences of different types of vaccines to maximize their effects and avoid the negative interference between the vaccines. Finally, the future design of therapeutic vaccines needs to be considered in nonnaïve hosts since patients have undergone other infections. It is yet not possible to foresee how the pre-existing infections and immunological backgrounds will influence the effect of therapeutic vaccines. Understanding these issues will be helpful for the translation of recent progresses for clinical use of therapeutic vaccines.

HBV Infection and Current Treatment Strategies

World Health Organization estimates that about 2 billion people worldwide have been infected with hepatitis B virus (HBV). Since the introduction of preventive vaccination programs against hepatitis B in over 170 countries, the number of new infections is continuously decreasing. Despite the success of prophylactic vaccines, chronic HBV infection is still a global health problem. Over 360 million people are persistently infected with HBV, of whom 1 million die each year from HBV-associated liver cirrhosis or hepatocellular carcinoma (HCC). The outcome of HBV infection varies greatly from person to person. In most of the cases the infection is cleared spontaneously, however, 5%-10% of adults develop chronic infection. By contrast, 40%-90% of children which are born to HBV-infected mothers will progress to develop a persistent liver disease [1] . In the recent, years a marked progress has been made in the treatment of chronic hepatitis B. Currently, the two types of antiviral therapies are approved: treatment with pegylated interferon alpha 2a (PEG-IFNα) or nucleos(t)ide analogues, such as adefovir, entecavir (ETV), lamivudine, telbivudine, and tenofovir [2] [3] [4] [5] . However, the efficacy of those therapies in preventing liver cirrhosis and HCC is still limited. Treatment with PEG-IFNα leads to a sustained antiviral response in only one third of patients, regardless of combining the therapy with polymerase inhibitors. On the other hand, the treatment with nucleos(t)ide analogues significantly suppresses HBV replication that leads to a decrease of necroinflammation in the liver. However, those antivirals cannot completely eradicate the virus. After withdrawal of the drug, the rebound of viremia is observed in the majority of patients. Furthermore, the long-term treatment is subsequently associated with the appearance of drugresistant HBV strains that is often the cause of the therapy failure [6, 7] . Therefore, the new approaches in treating chronic hepatitis B are urgently needed.

Immunological Control of HBV Infection

It is well documented that an appropriate adaptive immune response is required to efficiently control the HBV infection. T cell-mediated immune response directed against 2 Hepatitis Research and Treatment hepatitis B virus antigens is crucial for resolution of the infection [8] [9] [10] [11] [12] . HBV-specific CD8 + T cells are able to clear HBV-infected hepatocytes by secretion of Th1 antiviral cytokines, such as interferons (IFNs) and tumor necrosis factor alpha (TNFα), and direct cytotoxic mechanisms (perforin/granzyme, ligand-ligand induced cell death, e.g., Fas-Fas-L) [12] [13] [14] [15] [16] . An early, vigorous, polyclonal, and multispecific cellular immune response against the viral proteins is associated with the clearance of hepatitis B in acutely-infected patients. In contrast, chronic HBV carriers demonstrate weak, transient, or often undetectable CD8 + T cell response that correlates with HBV persistence [17] [18] [19] [20] [21] . Humoral immune response, especially neutralizing antienvelope antibodies, play a key role in preventing HBV spread to noninfected hepatocytes [20, 22] .

Clinical Trials of Therapeutic Immunization

The strategies designed to specifically stimulate HBVspecific T cell responses were also not successful [69] [70] [71] . The lipopeptide-based vaccine containing a single cytotoxic T lymphocyte (CTL) epitope derived from HBV nucleocapsid was able to induce a vigorous primary HBV-specific T cell response in naïve subjects [76] . However, in HBV chronic carriers, the vaccine initiated only poor CTL activity and had no effect on viremia or HBeAg/anti-HBe seroconversion [69] . The DNA vaccine expressing small and middle envelope proteins proved to elicit the HBV-specific cellular immune response in chronic HBV carriers, however, this effect was only transient [70] .

Hepatitis Research and Treatment

3 [77] . Immunogenic complexes (ICs) stimulate robust T cell responses by increasing uptake of HBsAg through Fc receptors on APCs and, therefore, modulate HBsAg processing and presentation. It was demonstrated that this vaccine administered to HBeAg-positive patients led to decrease of HBV DNA in serum, HBeAg seroconversion, and development of anti-HBs in part of the subjects [78] . Currently, the IC-based vaccine is the only one that entered phase III of clinical trials in chronic hepatitis B patients [79] . Even though the IC-based vaccine led to antiviral effect, clearance of HBV was not observed in treated patients. It seems that the vaccine alone is not sufficient to achieve the full control over HBV. Therefore, some steps have been undertaken to combine the IC-based vaccine with nucleos(t)ide analogues treatment, (Wen et al., personal communication) . The ongoing clinical trial will show whether IC are effective as a therapeutic vaccine in chronic hepatitis B.

The Woodchuck as a Preclinical Model for Pathogenesis and Therapy of Chronic Hepatitis B

The Eastern woodchuck (Marmota monax) is naturally infected by woodchuck hepatitis virus (WHV). WHV was discovered in 1978 as a virus closely related to HBV [89] and classified as a member of Hepadnaviridae family. WHV and HBV show a marked similarity in the virion structure, genomic organization, and the mechanism of replication, but differ in several aspects, for example, regulation of transcription (Table 1 ) [90] . WHV causes acute self-limiting and chronic infection similar to HBV infection in the pathogenesis and profiles of the virus-specific immune response [91] . This feature of the woodchuck model makes it so significant for investigation of the new therapeutic approaches in chronic hepatitis B. Experimental infection of neonates or adult woodchucks with WHV reflects the outcome of HBV infection in humans. In adult woodchucks infection with WHV usually leads to the resolution of infection and only 5%-10% of animals will develop the chronic hepatitis. The exposure of woodchuck, neonates to WHV results in development of chronic WHV infection in 60%-75% of the cases [92] . The continuous replication of WHV in the liver during the chronic infection is nearly always associated with development of HCC in the woodchucks [93, 94] . After diagnosis of HCC the survival prognosis of the animals is estimated on about 6 months, like in humans. The common features of HBV-and WHVinduced carcinogenesis give the opportunity to examine the new anti-HCC therapies in the woodchucks [95] .

Proteins

Replication of HBV DNA occurs by reverse transcription of an RNA intermediate within cytoplasmic nucleocapsids [22] The same mechanism [97] Genetic diversity 8 major genotypes [105] 1 major genotype (minor sequence differences) [91] Integration into host chromosome Yes [22] Yes, often close to N-myc oncogene region [106] Clinical course of infection were characterized ( Figure 1) . In contrast to self-limiting infection, WHV chronic carriers demonstrate weak or no virus-specific T cell responses against the identified epitopes [72, 73, 99] . The establishment of the assays for monitoring of cellular immune response in woodchucks is of great importance for a reliable evaluation of therapeutic and immunomodulatory strategies for treatment of chronic hepatitis B in the woodchuck model [96, 102, 103 ].

Therapeutic Vaccination Approaches in the Woodchuck Model

The second trial evaluated the therapy with a very potent antiviral drug: clevudine (previously called L-FMAU) combined with a WHsAg-based immunization [74, 109, 110] . A large cohort of thirty 1-2-year-old chronically WHVinfected woodchucks was enrolled in the study. Half of the animals were orally treated with clevudine (10 mg/kg/day) for 32 weeks; the other 15 woodchucks received placebo. After withdrawal of clevudine treatment, 8 animals from each group were vaccinated with the four doses of formalin inactivated alum-adsorbed WHsAg and 7 were injected with the saline as a control. Combination of the drug and vaccine therapy resulted in marked reductions WHV DNA (6-8 logs) and WHsAg in serum during the 60-week monitoring period, in contrast to the vaccine only and placebo groups, where both markers remained at high levels. Combination therapy did not enhanced anti-WHs responses beyond those measured for vaccine alone. However, treatment with clevudine and vaccine together led to more sustained and robust lymphoproliferative responses to WHsAg and additionally to WHcAg, WHeAg, and WHxAg. Moreover, combination therapy delayed the onset of the liver disease and prevented HCC development in up to 38% of treated chronic WHV carriers in the long-term follow-up study [111] .
The results from the previous studies clearly confirm the poor efficacy of the lamivudine therapy in woodchucks [108, 112, 124] . A new strategy evaluated the potency of an entecavir treatment and increased number of immunizations [Lu et al., unpublished results] . Chronically WHV-infected woodchucks were pretreated with the entecavir for 21 weeks; 10 weeks in a daily and 11 weeks in a weekly manner. During the weekly administration of the drug, one group of animals received 6 immunizations with two-plasmid DNA vaccine (pWHsIm and pWHcIm),the second group received combination of DNA vaccine together with purified WHV core and surface antigens, and the third group remained untreated. The entecavir therapy resulted in rapid and significant decrease of the viral load and WHsAg levels in serum of the animals. The effect was especially pronounced in animals that additionally received vaccines. In woodchucks treated only with entecavir, the increase of viremia was observed already during the weekly administration or immediately after withdrawal of the drug. By contrast, in both groups of animals, that were immunized with DNA or DNA/proteins vaccines, the delay before the rebound of WHV replication was significantly prolonged. In addition, entecavir treatment was effective to suppress WHV replication and enhanced the induction of WHV-specific T cell responses. An increased CTL activity was detected in individual woodchucks after DNA or DNA/proteins vaccinations. Moreover, two animals completely eliminated the virus from the blood and were WHV DNA negative in the liver [Lu et al., unpublished results] .
Altogether, the results obtained in the woodchuck model concerning combination of nucleot(s)ide therapy and immunization proved the synergistic effect of both therapeutical approaches. The therapeutic effects observed during such therapies were significantly increased and prolonged in comparison to the monotherapy alone. In addition, those therapeutic approaches could stimulate the WHV-specific T cell responses, usually impaired in WHV chronic carriers [72, 73] . A combination of antiviral treatment and vaccination is required for the improvement of virus specific T cell responses. Designing of the future therapeutic approaches should include pretreatment with the potent antiviral drugs, such as entecavir or clevudine, that proved their efficacy in the woodchuck model.

Therapeutic Immunization Using Recombinant Viral Vectors and Prime-Boost Strategy

Vaccines based on recombinant viruses have gained a great interest because of their ability to stimulate robust humoral and cellular immune responses. Viral vectors were investigated as prophylactic and therapeutic vaccines against many human pathogens such as measles virus, herpes simplex virus (HSV), human papillomavirus (HPV), HIV, and rabies [126] [127] [128] [129] [130] . However, the utility of those recombinant vaccines in the treatment of chronic hepatitis B was not yet evaluated. Preliminary results obtained from the study in chronically HBV-infected chimpanzees immunized with retroviral vector, based on Moloney murine leukemia virus, encoding HBcAg suggest that further investigation of viral-vector based vaccines should be taken into consideration [131] . In the experiment, one of the three therapeutically immunized chronic carrier chimpanzees cleared the virus and showed HBeAg seroconversion. Significant ALT elevations observed in this animal implicate restoration of HBV-specific cytotoxic and humoral responses without causing fulminant hepatitis. Moreover, the other two chimpanzees demonstrated high anti-HBe titers after the therapy and one of them HBcAgspecific CTLs [131] . This study demonstrates not only the benefit of using the recombinant viral-vectors for treatment of chronic HBV infection in primate model, but also the possible advantage of using core antigen-based therapeutic vaccines. Even though the retroviral vector vaccination was well tolerated in the chimpanzees, several clinical trials suggest that gene therapy with traditional retroviral vectors can lead to oncogenesis [132, 133] . Therefore, the usage of another recombinant virus as a carrier of the proteins could be beneficial.

Gene Transfer Strategies for the Treatment of Hepatocellular Carcinoma

A recent study presents gene therapy with semliki forest viral vector expressing high levels of murine IL-12 (SFV-enhIL-12) on remission of HCC in chronically WHV-infected woodchucks. In the research, the vector was delivered by surgery into multiple sites of HCC tumors in the liver [75] . A total of nine woodchucks were enrolled in the experiment. Six of the woodchucks, two animals each, received different doses of SFV-enhIL-12: 3 × 10 9 vp, 6 × 10 9 vp, and 1, 2 × 10 10 vp, and three animals served as a control and received saline injections. The tumor size was monitored by ultrasound examination for 23 to 24 weeks. In all woodchucks, reduction in tumor volume was observed, however, this effect was transient and dose dependent. Animals treated with the highest dose of SFV-enhIL-12 showed the most spectacular reduction of the tumor size 71% and 80%. Nevertheless, the tumors started to grow between 6 and 14 weeks after the treatment. The antitumoral effect was associated with the induction of the immune response towards the tumor antigens, demonstrated by T cell proliferation assay, upregulation of leukocyte markers expression, and cytokine production, such as IFNγ, TNFα, IL-6, and IL-12. In addition, the therapy resulted in transient induction of lymphoproliferative responses against WHcAg and WHsAg and led to short-term reduction in WHV viral load [75] .
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Abstract

The progressive loss of immunological memory during aging correlates with a reduced proliferative capacity and shortened telomeres of T cells. Growing evidence suggests that this phenotype is recapitulated during chronic viral infection. The antigenic volume imposed by persistent and latent viruses exposes the immune system to unique challenges that lead to host T-cell exhaustion, characterized by impaired T-cell functions. These dysfunctional memory T cells lack telomerase, the protein capable of extending and stabilizing chromosome ends, imposing constraints on telomere dynamics. A deleterious consequence of this excessive telomere shortening is the premature induction of replicative senescence of viral-specific CD8+ memory T cells. While senescent cells are unable to expand, they can survive for extended periods of time and are more resistant to apoptotic signals. This review takes a closer look at T-cell exhaustion in chronic viruses known to cause human disease: Epstein-Barr virus (EBV), Hepatitis B/C/D virus (HBV/HCV/HDV), human herpesvirus 8 (HHV-8), human immunodeficiency virus (HIV), human T-cell leukemia virus type I (HTLV-I), human papillomavirus (HPV), herpes simplex virus-1/2 (HSV-1/2), and Varicella-Zoster virus (VZV). Current literature linking T-cell exhaustion with critical telomere lengths and immune senescence are discussed. The concept that enduring antigen stimulation leads to T-cell exhaustion that favors telomere attrition and a cell fate marked by enhanced T-cell senescence appears to be a common endpoint to chronic viral infections.

T-cell Exhaustion in Chronic Viral Infection

T-cell exhaustion is a state whereby normal T-cell functions are impaired as a consequence of chronic antigen stimulation. The details of T-cell exhaustion are still being defined, but include: 1-impaired memory T cells; 2-impaired CD8+ T-cell effector responses; 3-impaired CD4+ T-cell responses; 4-resistance to apoptosis; and 5-accelerated immune senescence ( Figure 3 ).
The theory that CD8+ T-cell functions are exhausted during chronic viral infections was originally described during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice [44] . Unlike acute viral infections, some LCMV viral isolates persist within their host and do not cause lethality, thereby mimicking chronic infection [45] . The host's CTL response disappears following infection with these chronic strains of LCMV. The original proposal advocated that the virus was never effectively cleared because T cells were overwhelmed fighting the virus and the CTL response was eliminated entirely. It is now clear that exhausted T cells show progressive losses in antiviral functions. Proliferative capacity is diminished, followed by sequential loss of interleukin-2 (IL-2) production, cytolytic killing, decreased tumor necrosis factor (TNF) and interferon-gamma (IFNγ) production, and diminished degranulation, all of which can result in clonal deletion [46] . Dampened cytolytic responses during chronic viral infections have been shown in viral-specific CD8+ T cells of HIV, EBV, and cytomegalovirus (CMV) infection [47] . This includes loss of perforin, along with granzyme proteases that are vital for CTL's ability to eliminate antigen-presenting cells and to control the level of activated CD8+ T cells through apoptosis during chronic infection [48] . There is also an extensive profile of gene dysregulation between exhausted T cells and naïve or effector T cells, including defects in host cell signaling molecules, calcium binding, cytokine signaling, apoptosis/cell death, and migration [6] . Impaired CTL function is attributed, in part, to the presence of co-inhibitory markers that are part of the normal, yet transient, response to acute infection. These inhibitory markers serve to dampen and inhibit further immune responses, but continue to be expressed on the surface of exhausted T cells. A more advanced stage of T-cell exhaustion, marked by damped T-cell effector functions, occurs when more inhibitory markers are found on the surface of the T cell, along with a greater variety of inhibitory markers expressed [49, 50] . The most well-known and highly expressed inhibitory marker on exhausted T cells is Programmed Death receptor (PD-1), a member of the CD28 co-stimulatory family. PD-1 modulates the T-cell response to infection, reduces CTL effectors, and decreases proliferation of specific effector memory CD8+ T cells [51, 52] . Additional markers found on exhausted T cells include natural-killer cell receptor (2B4), T-cell immunoglobulin and mucin protein-3 (TIM-3), natural killer receptor BY55 (CD160), Ly49 family members, lymphocyte-activation gene 3 (LAG-3), cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), and Prostaglandin E Receptor 4 (Ptger4) [6] . Most of these possess immuno-tyrosine inhibitory motifs or switches that decrease active cell signaling in exhausted T cells. For example, TIM-3 prevents effector TH1 responses and expression of TNF and IFN-γ. This, in turn, has adverse effects on non-T-cell populations, such as dendritic cells, natural killer cells (NKs), and macrophages [53] . It is important to note that studies on inhibitory markers were carried out using the LCMV model in mice. Thirteen inhibitory receptors were found to be differentially regulated on exhausted T cells, which may not represent a complete description in human cells. For instance, the mouse glycoprotein 49B (GP49B) was up-regulated on exhausted T cells, but a human homolog has yet to be identified.

Telomere Dynamics and T-cell Exhaustion during Chronic/Productive Viral Infections

Exhausted T cells have been found in individuals infected with a multitude of human chronic viruses and in mice with LCMV (as described above). Telomere shortening in viral-specific T cells has a hierarchical ranking system, with some antigens producing telomere shortening at a faster rate than other antigens. CMV infection seems to take the highest spot in this hierarchy, having a more differentiated state and shorter telomeres than any other virus, such as HIV, EBV, and HCV [38] . It has yet to be confirmed, but one could infer that viral infections that lead to a more extensive exhaustive state would be prone to faster telomere shortening and immune senescence.
Like HIV, hepatitis B and C (HBV and HCV) viruses are productive, chronic viruses and display T-cell exhaustion from telomere shortening. HBV and HCV are the major causes of acute liver infection; however, only HBV and HCV are responsible for a large proportion of global, chronic liver disease. Anywhere from 60-90% of HBV and HCV infections reroute to chronic infection. A delayed immune response to the ever-changing viral mutational landscape allows the virus to adapt and persist, especially in HCV infections. In fact, chronic viral liver disease is nearly completely due to the adaptive immune response where immunopathology, consisting of a low-level CTL response, destroys hepatocytes and leads to liver cirrhosis and hepatocellular carcinoma (HCC) [69] .
Early exposure in life to HBV infection can possibly lead to shorter telomere lengths in CD8+ T cells and early development of immunosenescence [83] . While only an indirect measurement of HBV infection was performed, these individuals lived in HBV endemic areas where HCC development was high and had T-cell immune markers of HBV infection (TCRVβ12). These individuals also demonstrated low CD3+ and CD4+ T-cell counts, suggesting immune impairment from long-term exposure to HBV. Chronic HCV patient CD8+ T cells were shown to have shorter telomeres with hallmarks of DNA damage, including γ-H2AX (marker of DNA double-stranded breaks) and p53 serine phosphorylation [84] . Immune impairment was seen in the CD8+y-H2AX+ T cells, with failed signaling through the JAK/STAT signaling pathway. The majority of T cells that were y-H2AX-positive were highly differentiated, with either CD27−CD57− or CD27−CD57+ phenotype, but did not express PD-1 or TIM-3, possibly owing to the fact that HCV-specific CD8+ T cells were not analyzed. It could be that these T cells, since not HCV-specific, could owe their phenotypes to bystander effects from secreted pro-differentiation factors released during the course of the chronic viral infection. This has been described for CMV infection, in which release of interferon-alpha (IFN-α) causes CMV-specific CD4+ T cells to differentiate and lose telomere lengths [85] . IFN-α can be released in response to CMV infection and inhibits telomerase activity, leading to telomere shortening [86] . So, while a direct examination of telomere lengths and senescence has not been examined for HBV-(or HCV-) specific CD8+ T cells, it can be suggested that telomere dynamics and slowed replication are altered in exhausted HBV-specific T cells.

Telomere Dynamics and T-cell Exhaustion during Chronic/Latent Viral Infections

Similar to HTLV-I, Epstein-Barr virus (EBV) is a chronic, latent virus that is oncogenic and linked to several cancers. It is estimated that nearly 90% of the world population is infected with EBV. EBV is responsible for a wide plethora of human diseases, including those associated with defective B cells, such as acute infectious mononucleosis (AIM), and a wide variety of non-B-cell diseases. Given the prevalence of EBV infection in the population, it is likely that most individuals harbor EBV-specific CD8+ T cells. Studies have shown PD-1 expression on EBV CD8+ T cells in healthy individuals, which were increased in EBV CD8+ T cells from EBV mono-infection or EBV/HIV co-infection. High expression of TIM-3 was also found on EBV CD8+ T cells in EBV/HIV co-infected individuals [113] . The level of PD-1 expression on EBV CD8+ T cells was nearly equal to that of HIV CD8+ T cells. In EBV+ diffuse large B-cell lymphoma (DLBCL), administration of PD-L1 caused enhanced proliferation and cytokine expression, and restored antitumor immunity [114] . EBV exhausted T cells have also been linked to the emergence of several conditions and a wide variety of EBV-associated diseases. This includes chronic fatigue syndrome (CFS), multiple sclerosis (MS), and systemic lupus erythematosus (SLE). Signs of EBV-specific T-cell exhaustion included elevated PD-1 and decreased cytotoxic T-cell functions in EBV CD8+ T cells in SLE, diminished T-and B-cell memory response in CFS, and reduced EBV CD8+ T-cell functionality and exhaustion in MS [115] [116] [117] . PD-L1 has also been found to be elevated in EBV-positive HL and PTLD [118] . In the latter, it was shown that an EBV latent gene product, LMP1, could elevate PD-L1 gene promoter expression.
A clearer understanding of the effect of telomere dynamics during chronic EBV infection came from studying EBV-specific CD8+ T cells during and after AIM. During and immediately following Viruses 2017, 9, 289 12 of 23 AIM, EBV-specific CD8+ T cells were able to prevent telomere erosion by up-regulating telomerase [11] . In the majority of individuals, telomere lengths remained unchanged immediately following AIM; however, in longitudinal studies in infected patients (15 months to 14 years), a marked telomere attrition was noted in EBV-specific CD8+ T cells [11] . These cells continued to be exposed to EBV antigen but were unable to up-regulate telomerase. This, and the fact that long-term clones of EBV-specific T cells decrease over time, suggest that chronic EBV exposure leads to replicative senescence of EBV CD8+ T cells [119] .
In other situations in which EBV infection is not controlled, researchers examined telomere dynamics. Chronic EBV infection and replication often occurs in HIV-infected immunocompromised individuals. In fact, AIDS-related non-Hodgkin lymphoma (AIDS-NHL) is thought to evolve due to accumulation of EBV-specific T cells that have lost their cytotoxic immune function (i.e., exhausted) and are no longer able to control EBV infection [120] . In these individuals, EBV-specific CD8+ T cells showed marked decreases in telomere length, which seemed to preclude a senescent-like phenotype towards EBV infection, as suggested due to a large decrease in IFN-γ-producing EBV CD8+ T cells, which was similarly seen in the elderly [121] . A similar situation occurs in X-linked lymphoproliferative syndrome (XLP), in which there is an inflated immune response to EBV infection. An examination of EBV-specific CD8+ T cells in survivors demonstrated loss of telomerase, shorter telomere lengths, and a more differentiated state (CCR7−/CD27−) [122] .
Information is also needed on replicative senescence and T-cell exhaustion in chronic HPV infection. However, the low antigenic loads of HPV infection likely contribute to the fact that T-cell exhaustion may not occur to the same extent, or is difficult to detect, in HPV-infected individuals compared to other chronic viral diseases. This is because long-term existing T cells require the presence of viral antigen to survive and the direct amount of antigen dictates T-cell exhaustion [127] . HPV favors the use of non-preferred codons to reduce the frequency of HPV virus and antigen that is exposed to the immune system [128] . Indeed, HPV-specific T cells are absent or in negligible numbers in the peripheral blood of patients with cervical cancer [129] . This may result from the absence of T cells in the infected area or cross-reactivity of HPV epitopes with other pathogens [130] . An additional consideration is that for some chronic latent viruses, viral-specific CD8+ T cells may be located outside the blood stream. In the case of HPV, HPV-specific CD8+ T cells may be absent from peripheral blood and instead be localized in mucosal tissues [131, 132] . Therefore, telomere length measurements and T-cell exhaustion markers may need to be analyzed on memory CD8+ T cells from the mucosa. There are some reported studies suggesting T-cell dysfunction; however, most studies have not directly assessed the phenotype of HPV-specific CD8+ T cells, i.e., inhibitory markers or cytokine profile. T cells and dendritic cells in cervical tissue were positive for PD-1 in high-grade HPV infection [133] . This was seen in conjunction with a reduction in IFN-γ and IL-12 from helper T cells. It has also been shown in HPV-associated recurrent respiratory papillomatosis (RRP) that HPV-specific T cells are dysfunctional, with decreased STAT5 activity and reduced IL-2; however, this has been linked to T-cell anergy and not exhaustion, as these cells did not express PD-1 [134] . PD-1 therapy was able to boost vaccines directed against HPV16's E6 and E7 genes by boosting tumor-infiltrating CD8+ T cells [135] . HPV-associated head and neck squamous cell carcinoma (HNSCC) is generally more responsive to chemotherapy than HPV-negative HNSCCs. It was found that HPV+ HNSCCs had increased PD-1 expression on infiltrating T cells and that in mice given vaccine therapy, PD-1 expression was elevated and had a more favorable outcome [136] . However, these cells were likely not exhausted, since over half of them did not express TIM-3.

Concluding Remarks

In conclusion, sustained antigenic stimulation due to chronic viral infection imposes considerable constraints on the immune system. Chronic proliferation of viral-specific memory T cells can lead to telomere shortening, activation of the DDR, and replicative senescence. T-cell exhaustion occurs, leading to loss of CTL effector and memory cell function. These constraints make it difficult to effectively clear the virus and can lead to re-activation of the virus later in life, cancer (as is the case for oncogenic viruses), disease reemergence, and host death, making alleviation of T-cell exhaustion a priority in antiviral therapy.

Replicative Senescence in Chronic Viral Infection

During acute viral infection, a rapid immune response occurs between the infected host and the viral pathogen [1] . Resolution involves either viral clearance and host memory, host death due to overwhelming inflammation and/or extensive viremia, or a transition to a chronic infectious state. Unlike acute viruses, chronic viruses persist in a semi-stable relationship within their host, generating antigenic stimulation for several months to decades. These chronic viral infections can be categorized into: 1-Latent (lack of substantial viral production between initial and late stages); 2-Productive (persistent viral production between beginning and late stages); and 3-Slow infection (increasing viral production from incubation period to late stages) [2] (Figure 1 ). These stages are established by restricting viral propagation and reprogramming viral gene expression. In conjunction with viral adaptation, the host controls the immune response to prevent overwhelming chronic inflammation that could otherwise become harmful to various tissues. Enduring hyper-antigenemia (even at low to undetectable levels), which occurs during persistent viral infection, imposes a permanent stress on the immune system [3] . The magnitude of the CD8+ T-cell response following initial infection can be substantial and it is essential that most of the expanded cells die after antigen clearance to maintain lymphoid homeostasis [4] . However, for an efficient memory pool to persist, selected CD8+ T cells that have escaped apoptosis must retain sufficient replicative potential to allow successive rounds of proliferation in response to antigen recall throughout the host's life. Unlike normal memory T cells, which persist due to the levels of interleukin-7 (IL-7) and IL-15, exhausted T cells only require the presence of viral antigen to continue proliferating [5] . This is partly due to losses in interleukin-2 receptor-β (CD122) and interleukin-7 receptor (CD127) that limit generation of virus-specific T cells [6, 7] . Because viral antigen is intermittently or constantly supplied to these cells, viral-specific T cells never cease proliferating. Depending on the length of infection, this could result in progressively shorter telomeres and an agerelated decline in T-cell responses.
The average telomere length for naive CD4+ and CD8+ T cells is about 2.5 kb longer than effector or memory T cells [8] . It would be easy to infer then that during clonal expansion, memory T cells are at a distinct replicative disadvantage compared to early effector T cells due to a theoretical loss of telomere sequence following the initial encounter with antigen. However, this does not appear to be the case. Antigen-specific T and B cells can up-regulate telomerase activity during the initial The average telomere length for naive CD4+ and CD8+ T cells is about 2.5 kb longer than effector or memory T cells [16] . It would be easy to infer then that during clonal expansion, memory T cells are at a distinct replicative disadvantage compared to early effector T cells due to a theoretical loss of telomere sequence following the initial encounter with antigen. However, this does not appear to be the case. Antigen-specific T and B cells can up-regulate telomerase activity during the initial response to acute infection, thereby preserving the clonal potential of initial memory T cells for subsequent encounter [17, 18] . However, despite the preservation of telomere length, telomerase activity is not retained after resolution of acute viral infection [19] . Enduring antigen-specific T cells are then susceptible to telomere defects, including senescence and death, during subsequent clonal expansions or proliferation ( Figure 3 ). On average, peripheral blood mononuclear cells (PBMCs) lose about 50 bp of telomeric DNA per year, but this loss has the potential to be increased during chronic viral infection, such as in the case of patients receiving antiretroviral treatment (ART) for HIV-1, where loss of up to 250 bp per year of telomeric DNA has been documented [20] . Telomere attrition and the DNA damage response during chronic viral infection. During an initial viral encounter, a robust T-cell response occurs followed by telomerase activation and retention of telomere lengths. Subsequent antigen encounters lead to inactivation of the telomerase promoter and loss of telomerase expression. Telomere attrition, caused by chronic exposure to viral antigen, is exacerbated over time (dotted arrows). Enduring hyper-antigenemia results in telomere crisis and the activation of the DNA damage signal. This results in T-cell exhaustion, inhibition of T-cell proliferation, and cell cycle arrest. The eventual outcome is programmed cell death (apoptosis) or replicative senescence. A deleterious consequence of excessive telomere shortening is the premature induction of replicative senescence of CD8+ T cells [21] . While senescent cells are unable to expand, they can Telomere attrition and the DNA damage response during chronic viral infection. During an initial viral encounter, a robust T-cell response occurs followed by telomerase activation and retention of telomere lengths. Subsequent antigen encounters lead to inactivation of the telomerase promoter and loss of telomerase expression. Telomere attrition, caused by chronic exposure to viral antigen, is exacerbated over time (dotted arrows). Enduring hyper-antigenemia results in telomere crisis and the activation of the DNA damage signal. This results in T-cell exhaustion, inhibition of T-cell proliferation, and cell cycle arrest. The eventual outcome is programmed cell death (apoptosis) or replicative senescence. Somatic cells, like those of naïve, effector, and memory T cells, have a finite proliferative capacity, due largely to the inability of DNA polymerase to replicate the distal ends of chromosomes. Pluripotent stem cells and the majority of cancer cells can circumvent this problem by up-regulating telomerase, a reverse transcriptase that can retrogradely add a six base pair (TTAGGG) nucleotide sequence onto the ends of chromosomes [13] . Cellular replication in the absence of telomerase expression results in progressive telomere shortening. Elegant studies demonstrated that a single dysfunctional telomere is sufficient to cause telomere end replication problems and increase Viruses 2017, 9, 289 4 of 23 chromosomal instability, degradation, and fusion [14, 15] . Dysfunctional telomere-induced foci (TIF) engage a DNA damage response (DDR) and trigger activation of the ATM Serine/Threonine Kinase (ATM)/p53-dependent senescence program ( Figure 2 ) [16] . Unlike most somatic cells, T cells, which include CD8+, CD4+, naïve, and memory, can reactivate telomerase through mitogenic stimulation [17] [18] [19] . These cells initially harbor nearly undetectable levels of telomerase. Upon antigenic stimulation, as is the case with acute viral infection, telomerase is reactivated. This process is reiterated during second antigenic stimulation, but by the third and all subsequent stimulations, T cells are less responsive to mitogenic stimulation and the telomerase gene promoter is inactivated [20] .
Immune senescent T cells bear several trademarks: shortened telomeres, decreased T-cell-specific surface glycoprotein-CD28 (CD28) and T-cell activation antigen CD27 (CD27), co-receptors for T-cell activation, increased β-1, 3-Glucuronyltransferase-1 (CD57), and loss of proliferation [25] [26] [27] . While CD57+ cells may not be terminally differentiated, it does serve as a marker for highly differentiated CD8+ T cells [28] . CD57+ cells have usually undergone more cell divisions, have an increased risk for activation-induced cell death, and are increased in individuals with chronic immune activation [28] . It is believed that either phenotype, CD57+ or CD28−, can lead to senescence and a loss of replication in some of the cells bearing these markers [27] . The fact that these hallmarks of senescence increase with age and are seen on lymphocytes of healthy, elderly individuals suggests a diminished replication potential of these cells. CD28 loss often precludes CD27 expression, which marks a more terminally differentiated cell. An accumulation of CD8+CD28−CD27− cells occurs during normal aging and chronic-antigen stimulation. These cells have shortened telomeres and lower telomerase activity than less differentiated T cells [29] [30] [31] . Loss of CD28 can be delayed in HIV-specific T cells transduced with telomerase, suggesting that telomere dynamics provide some intracellular signal for extinction of CD28 expression [32] . A consequence of accumulating CD8+/CD28− T cells is that they can lose their function, have reduced expression of effector molecules (granzyme B and perforin) and reduced cytotoxic T-lymphocyte (CTL) activity [33] . These cells have been found in various chronic viral infections, such as HIV, Epstein-Barr virus (EBV), and Hepatitis C virus (HCV), with each viral-specific CD8+/CD28− T cell responding with varying degrees of the differentiated phenotype [34] . An additional marker of senescent cells is increased expression of Killer Cell Lectin-Like Receptor G1 (KLRG1). Similar to CD57, KLRG1 is expressed on the surface of NK cells and "antigen-experienced" T cells, and leads to defects in signaling and proliferation in highly differentiated T cells [35] .
Critical telomere shortening induces the DNA damage response (DDR), and if the DNA damage is not repaired either programmed cell death (apoptosis) or replicative senescence occur. In the absence of apoptosis, cells harboring dysfunctional telomeres are at risk of developing genomic defects, with numerous chromosomal instabilities, such as DNA deletions, amplifications, and aneuploidy [36, 37] . In some T cells progressing to senescence, apoptotic markers have been found, such as low expression of the anti-apoptotic gene, B-Cell CLL/Lymphoma 2 (Bcl-2), and up-regulation of cell death markers, such as Tumor Necrosis Factor Receptor Superfamily Member 6 (CD95), which lead to p53-mediated cell death [38] . As an example, HIV-specific CD8+ T cells that had undergone replicative senescence, defined by loss of proliferative capacity and shorter telomeres, were found to be more sensitive to apoptosis [39] . These cells were not CD28− or C-C chemokine receptor type 7 (CCR7)-deficient, but did harbor CD57 expression, and therefore may not be terminally differentiated. CD57 expression is believed to make cells more susceptible to activation-induced cell death by apoptosis [39] . It appears that, in some cases, replicative senescent T cells do undergo apoptosis. This contradiction can be expounded upon as (1) apoptotic resistance is acquired through altered differentiated states during chronic stimulation, (2) many subpopulations of cells exist within the same individual, and (3) senescent cells can, in some settings, be prone to apoptosis progression [27] .

T-cell Exhaustion in Chronic Viral Infection

Viruses 2017, 9, 289 3 of 23 sequence onto the ends of chromosomes [8] . Cellular replication in the absence of telomerase expression results in progressive telomere shortening. Elegant studies demonstrated that a single dysfunctional telomere is sufficient to cause telomere end replication problems and increase chromosomal instability, degradation, and fusion [9, 10] . Dysfunctional telomere-induced foci (TIF) engage a DNA damage response (DDR) and trigger activation of the ATM Serine/Threonine Kinase (ATM)/p53-dependent senescence program ( Figure 2 ) [11] . Unlike most somatic cells, T cells, which include CD8+, CD4+, naïve, and memory, can reactivate telomerase through mitogenic stimulation [12] [13] [14] . These cells initially harbor nearly undetectable levels of telomerase. Upon antigenic stimulation, as is the case with acute viral infection, telomerase is reactivated. This process is reiterated during second antigenic stimulation, but by the third and all subsequent stimulations, T cells are less responsive to mitogenic stimulation and the telomerase gene promoter is inactivated [15] . Less is known about T-cell exhaustion in CD4+ T cells. It is clear that exhausted CD4+ T cells have an altered phenotype compared to effector and memory CD4+ T cells. Exhausted CD4+ T cells express similar inhibitory markers and transcriptional factors as their counterpart CD8+ cells, including PD-1, but express a higher ratio of some inhibitory markers such as CTLA-4, CD200, and B-and T-lymphocyte associated protein (BTLA) [54] . Exhausted CD4+ T cells have diminished TH1 responses and altered effector functions, the latter related to their distinct transcriptional program. Regulatory T cells (Tregs) also have altered functions during chronic viral infections. Chronic viral infection leads to up-regulation of PD-1 on the surface of Tregs and enhanced Treg proliferation [55] . This allows for greater inhibitory function against CD4+ and CD8+ T cells. In addition, expression of CTLA-4 on the surface of Tregs aids in enhancing their immune suppressive actions [56, 57] .

Telomere Dynamics and T-cell Exhaustion during Chronic/Productive Viral Infections

HIV infects CD4+ T cells and is responsible for acquired immune deficiency syndrome (AIDS). Due to advances in antiretroviral therapy (ART), HIV-infected individuals are living longer. During chronic HIV infection, the immune system bears a striking resemblance to those of otherwise healthy, elderly people, with higher incidences of infection and loss of effector functions [58] . This is despite the fact that there is a precipitous drop in infectious HIV between acute and chronic phases, with over 99% of HIV antigen coming from HIV particles in later stages. This suggests that a consequence of ART is that HIV-specific T cells are exposed to viral antigen for a longer period of time, leading to increases in HIV-specific T-cell exhaustion and possible immune senescence. Indeed, markers of both T-cell exhaustion and immune senescence have been documented in HIV-infected individuals. These include enhanced expression of inhibitory markers (such as PD-1, CTLA-4, and LAG-3), reduction in effector cytokines, reduced proliferative and self-renewal potential, and a lack of CD4+ T-cell help [59] [60] [61] . In fact, the expression of PD-1 correlates with impaired T-cell function, reduced CD4+ T cells, increased viral load and a sensitizing of the cells to apoptosis [62, 63] . This is significant, as HIV-induced apoptosis of uninfected "bystander" cells contributes to HIV-associated T-cell death and disease progression, a process that is not completely understood [64, 65] . The significance of the interplay between T-cell exhaustion and HIV disease is demonstrated by the fact that aggressive forms of the disease harbor more cells with advanced T-cell exhaustion and PD-1 therapy reduces the exhausted phenotype, leading to increased T-cell effector functions and a reduction in virus load [66] .
While ART treatment can effectively control CD4+ T-cell counts and viremia, an unintended consequence is a rise in CD8+ T-cell levels [67] . These CD8+ T cells have been linked to an increase in complications and non-AIDS-related malignancies. Researchers have discovered that while there is an increase in the immune senescence phenotype (CD8+ T cells that are CD28−/CD27), most of these are CMV-specific. This is due to the fact that, during chronic HIV infection, there is an increase in viral-specific, non-HIV CD8+ T cells. In fact, most HIV-specific CD8+ T cells have an intermediate phenotype in which they still express CD27. Another report determined that an increase in CD57 on HIV-specific CD8+ T cells serves as a better marker of immune senescence since it correlates with replicative capacity [68] . HIV-specific CD8+/CD57+ cells were more susceptible to apoptosis and point to a general trend that in all cells (regardless of infectious state) CD57 expression is indicative of more cellular divisions that result in shorter telomeres.
Telomere length measurements in HBV and HCV infection have been performed in the host cell hepatocytes. Telomere lengths have been found to be shorter in hepatocytes from chronic HCV infection, especially in HCC, which leads to chromosomal instability [73] [74] [75] . Studies on replicative senescence in HCV-specific CD8+ T cells are difficult due to the relative scarcity of HCV-specific cells in the periphery [76] . Despite this difficulty, an abundance of data supports the notion that telomere attrition occurs in total T cells from chronic HBV and HCV infections. Telomerase RNA levels were found to be lower in the peripheral blood lymphocytes (PBLs) of HBV-and HCV-infected individuals that may contribute to immunosuppression [77] . Telomere length assessments in HCC patients infected with HBV or HCV demonstrated shorter telomere lengths not only in liver biopsied tissue, but also in PBLs [78] . Telomere attrition was not seen in HBV/HCV-noninfectious HCC. This suggests that long-term HBV/HCV infection causes adverse effects on telomere lengths in PBLs. In support of this, chronic HCV patients have shorter telomeres in CD4+ and CD8+ T cells with the memory phenotype [79] . Telomere lengths were comparable to those of normal aging of 10 years, which was enhanced to 15 years with more severe fibrosis disease [79] . Telomere shortening has not been found in cell-free serum DNA from chronic HBV infections [80] .
When telomere lengths were followed over 15 years in individuals infected with HCV or dual HCV/HIV, researchers found differences in telomere lengths compared to lengths of non-HCV-infected Viruses 2017, 9, 289 9 of 23 individuals [81] . Telomere lengths in CD4+ T cells were shorter in HCV-infected individuals and shorter in both CD4+ and CD8+ T cells in dual infection. Telomere lengths did not correlate with a more differentiated cell, again owing to the possibility that length measurements were taken on total PBMCs from the patients and were not sorted for viral-specific T cells. Among the genes altered during chronic HBV infection was telomeric repeat binding factor 2, interacting protein (TERF2IP) [82] . TERF2IP is known to regulate telomere dynamics and TERF2IP expression was decreased in HBV-specific CD8+ T cells. In addition, expression of genes associated with cellular senescence were found down-regulated in exhausted HBV-specific CD8+ T cells, including ATM, several DNA repair proteins (RAD proteins), and POLH (DNA Polymerase Eta), a polymerase involved in translesion repair [79] .
Exhausted viral-specific T cells have been described for both HBV and HCV. Exhausted HCV-specific T cells have been found in the liver, spleen, and blood, and the exhausted inhibitory receptor phenotype varies according to the inhibitory marker and level of expression. CD127, or the IL-7 receptor, is up-regulated on effector and memory T cells, and is a marker for the activated state during HBV acute infection [87] . Low CD127 expression is seen on exhausted CD8+ T cells in other chronic viral infections, such as HIV [88] . In HCV-specific T cells that expressed low CD127 and high inhibitory markers, impaired proliferation was seen and could only be reinvigorated once PD-1/PD-L1 was blocked. In addition, blockade of TIM-3 enhanced T-cell proliferation and CTL responses [89] . Not only was TIM-3 present on the surface of chronically infected HCV T cells, but CD57 was also present in conjunction with TIM-3 [89] . Given the co-localization of these two markers on the same cell, it suggests that during chronic HCV infection, exhausted T cells undergo senescence.

Telomere Dynamics and T-cell Exhaustion during Chronic/Latent Viral Infections

Less is axiomatic between viral-specific T-cell exhaustion in latent viral infections, including whether or not latent viral infection can lead to shortened telomeres, replicative senescence, and/or apoptosis. The majority of studies on T-cell exhaustion were performed in the LCMV model, which, though correctly depicting chronic productive viruses, does not accurately reflect chronic latent viruses. Often, these viruses are hidden from immune surveillance and/or produce a low abundance of viral antigens. Studies using an adapted influenza A virus to mimic chronic antigen stimulation have shown that CD8+ T-cell exhaustion still occurs [90] . This model allowed for the absence of large viral loads and lymphocyte infection, and demonstrated T-cell exhaustion, albeit with altered mechanisms. While evidence of T-cell exhaustion and/or senescence is found in latent chronic viral infections (as outlined below), there is still a large gap in research describing telomere dynamics during these infections. A majority of the chronic latent viruses discussed are known to be oncogenic, and have demonstrated altered telomerase activity and shorter telomeres in the infected cell [91] . However, only EBV studies have demonstrated telomere attrition in EBV-specific T cells, highlighting the fact that more studies need to be done to examine the relationship between long-term, chronic viral infections and their effect on viral-specific immune responses in regard to telomere attrition and senescence.
Unlike HTLV and EBV infections, there is little to no information regarding telomere shortening and how it relates to T-cell exhaustion during some chronic viral infections. This is the case for chronic Kaposi's sarcoma-associated herpesvirus (KSHV) infection (also known as human herpesvirus-8 (HHV-8)) and human papilloma virus (HPV) infection. To date, no study has been done to examine T-cell exhaustion or senescent markers, nor telomere length on HHV-8-specific memory cells. Given the fact that T-cell exhaustion has been found in all chronic viral infections, it is conceivable that this is also the case in HHV-8 patients. Patients with Kaposi's sarcoma (KS) had lower amounts of HHV-8-specific CD8+ T cells than HHV-8 carriers [123] . This was seen in both HIV-related KS and classic KS, suggesting global HHV-8-specific T-cell dysfunction in diseased patients. In addition, PD-1 was expressed on natural killer cells (NK) of KS patients and PD-L1 staining was also found on some cells of HHV-8-associated diseases [124, 125] . Senescent markers, such as decreased CD57+/CD28− on CD4+ and CD8+ T cells, have also been found in HHV-8-infected individuals with HIV-associated KS [126] .

Concluding Remarks

Evidence is mounting that high levels of antigen stimulation result in excessive proliferation, driving cells into a state of replicative senescence due to telomere attrition. The benefits for addressing viral T-cell exhaustion and immune senescence in patients with chronic viral infections and chronic inflammatory or auto-immune diseases are great so as to finally eradicate the chronic virus. Therefore, it is relevant to the ongoing efforts to develop therapeutic vaccines aimed at stimulating CD8+ T-cell responses and current immunotherapy based on adoptive transfer of expanded virus-specific CD8+ T cells.
There are still many questions when it comes to the therapeutic potential of blocking T-cell exhaustion. One concern is whether fully exhausted T cells can be reactivated. If exhausted T cells have reached a state of terminal differentiation, they may have undergone permanent cell cycle arrest and irreversible cellular senescence. In this case, it is important that anti-exhaustion therapy (such as drugs to block immune inhibitory markers, as discussed below) be given at the proper time, before the cells become permanently differentiated. In the latter case, it would then be imperative to target these cells for removal through enhanced cell death, since reactivation is not possible. Furthermore, in a subset of CD8+ T cells exposed to chronic viral infection, expression of the T-cell factor-1 (TCF-1) gene has been shown to be elevated [144] . Because these cells retain properties of both exhausted and memory T cells, TCF-1+ cells may represent a pole of cells that can be reactivated and proliferate during chronic infection, when inhibitory markers (such as PD-1) are blocked.
Another concern is whether anti-exhaustion therapy can fully reactivate viral-specific pools of memory T cells and whether exhausted T cells retain all their effector functions. Another caveat is determining how many and which immune inhibitory receptors should be targeted. As shown above, the extent of up-regulated inhibitory markers on T cells varies between viruses, so it would be easy to infer that different therapies would be more or less effective for different viruses. For example, a drug targeting CTL-4, ipilimumab, was approved by the Food and Drug Administration (FDA) and has shown success in patients with metastatic melanoma. However, in some viral infections, targeting CTLA-4 has led to increased viral production and a reduction in anti-viral therapy [145] . In chronic HCV infection, PD-1 therapy led to only small increases in proliferation, suggesting that a cocktail of therapies against various inhibitory markers would be more successful. Use of an anti-PD-1 antibody had great effects in the simian version of HIV (simian immunodeficiency virus-SIV). SIV CD8+ T cells increased in the blood and led to enhanced proliferation and effector functions and a drop in SIV viral levels [146] . PD-1 was also shown to be effective in an in vivo model of HBV, whereby blocking PD-1/PD-L1 interactions led to increased IFN-γ and enhanced effector functions that cleared the HBV virus [147] . The FDA has already approved two anti-PD-1 therapies, pembrolizumab and nivolumab, for use in metastatic melanoma [148] . Their use has not yet been approved for chronic viral infections but there are ongoing clinical trials, such as the use of nivolumab for treating HTLV-I-associated ATL. In addition, a phase 2 trial for nivolumab on HPV-associated squamous cell carcinoma of the anal canal (SCCA) patients showed complete or partial responses in some patients [149] . A combination of anti-PD-1 and anti-CTLA-4 therapies produced an enhanced ability to control EBV infection, leading to reductions in lymphomas, increased EBV effector responses, and decreases in EBV-infected B cells [150] . Additional evidence for blocking more than one inhibitory receptor is seen in cancers, such as non-small cell lung cancer patients that develop resistance to anti-PD-1 therapy [151] . This has been linked to an enhancement of TIM-3 on the cell surface of PD-1-resistant cells.
Furthermore, there are a few concerns that will need to be addressed in upcoming studies regarding T-cell exhaustion and immune senescence: 1-An important limitation of studies previously performed in some chronic viral infections is the use of total PBMCs instead of specific sub-populations and the use of Southern blotting of telomere restriction fragments (TRF) to measure telomere lengths. These assays give a general overview but do not allow distinction between different cellular subtypes and individual chromosomes. This is important because a single chromosome harboring a critically short telomere can initiate senescence [14] . Studies will need to be repeated to look at the individual chromosome level. 2-Most of the molecular determinants for T-cell exhaustion have been discovered with the use of LCMV infection in mice or have been interpreted from viral studies carried out in mice. However, mice have extremely different telomere dynamics than humans. For one, unlike humans, mice have extremely long telomeres [153] . A second concern is the lifespan differences between the two species. Humans live considerably longer (over 80 years) than mice (2-3 years), and therefore the effects of progressive T-cell exhaustion, telomere shortening, and senescence should be far more pronounced and dramatic in humans [154] . 3-Given the shorter lifespan and often sterile experimental laboratory conditions, mice are not exposed to as many distinct pathogens as humans are. Therefore, mice do not have as many distinct, viral-specific, long-term memory T cells as humans. 4-HIV and HTLV are retroviruses that require reverse transcriptase for viral production [155] . Most retroviral therapies, consequently, would also target telomerase. In these cases, the telomere shortening effect could be even greater than in other viral infections that do not rely upon a reverse transcriptase for proliferation.
An additional consideration to keep in mind is that while telomerase loss and telomere shortening have been described in immune senescence, the molecular mechanisms responsible for shutting down telomerase expression have not been elucidated. It is intriguing that repeated cellular stimulation in immune cells results in transcriptional silencing of the telomerase promoter. Therefore, while the outcome (telomere shortening and immune senescence) may be similar in chronic infections, the cell sub-populations and the underlying signaling mechanisms involved are worth investigating. Understanding the regulatory mechanisms controlling telomerase expression in chronic infection will allow the design of specific targeted therapies and may apply to some other chronic stimulation disorders.
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iNTRODUCTiON

Bronchiectasis is a complex chronic respiratory condition traditionally characterized by recurrent infection, airway inflammation, and progressive decline in lung function. The defining symptom common to children with bronchiectasis is chronic wet cough. Region-specific studies suggest that geographic locality and socioeconomic environment play a large role in determining the likely etiology of bronchiectasis in children. Cystic fibrosis, primary immune deficiency, defects in mucociliary clearance mechanisms (primary ciliary dyskinesia, congenital malformations), and aspiration of a foreign body increase susceptibility to respiratory infection and are known to be associated with bronchiectasis in children. However, on a global scale, lower respiratory infection in the absence of known underlying conditions accounts for the greatest number of bronchiectasis cases (1) . The focus of this review will be bronchiectasis with no known underlying disorder in children.
Early diagnosis and intensive treatment protocols can stabilize or even improve the clinical prognosis of children with bronchiectasis (6, 7) . Over the decades, considerable advances have been made in identifying the pathogens associated with recurrent or chronic infection. However, understanding the host immunologic mechanisms that contribute to recurrent infection and prolonged inflammation has been identified as an important area of research that would contribute substantially to effective prevention strategies for children at risk of bronchiectasis (8, 9) . This review will begin by exploring the role of the child's immune response in establishing an environment conducive to the recurrent infection and chronic inflammation characteristic of bronchiectasis. This will be followed by a discussion of the important pathogens associated with bronchiectasis in children and the obstacles in treating and preventing these infections. The review will conclude with "the road forward" areas of research identified by the authors as important for the advancement of understanding and addressing the pathogenesis of bronchiectasis in children.

PATHOPHYSiOLOGY OF BRONCHieCTASiS

Over recent years, considerable efforts have been made to understand airway inflammation associated with bronchiectasis. Some parallels exist between bronchiectasis and other chronic respiratory disorders, including cystic fibrosis and chronic obstructive pulmonary disease (COPD), in relation to airway inflammation. As a result, inflammatory processes involved in cystic fibrosis and COPD are sometimes used in an attempt to understand the pathophysiology of bronchiectasis. However, cystic fibrosis and COPD have distinctly different etiologies from bronchiectasis in children and extrapolating data, particularly to the pediatric setting, should be done with caution. Nevertheless, similarities between the chronic respiratory conditions suggest that bronchiectasis arises from exaggerated and/or dysregulated inflammation in response to challenge from respiratory pathogens. Thus, the "vicious circle" hypothesis of self-perpetuating infection, inflammation, and tissue damage first described by Cole in 1986 (10) remains the most likely explanation for the pathogenesis of bronchiectasis. What remains in question is, what causes the highly controlled immune response to become dysregulated?

HUMORAL iMMUNe ReSPONSeS

The role of the humoral response in the pathogenesis of bronchiectasis is unclear. There are considerable published data showing chronic colonization and active infection of the lower airways with NTHi, despite the presence of an active humoral immune response (42, 51, (53) (54) (55) . Adults with chronic respiratory conditions, including bronchiectasis, chronic bronchitis, and COPD, also exhibit a comprehensive humoral immune response against NTHi, with ample amounts of specific total IgG, IgG subclasses, IgA and IgM present in circulation (43, 45, 56) . These antibodies are functional against NTHi in vitro and, when combined with serum complement, form a potent NTHi-clearance mechanism. Although the amount of circulating immunoglobulin is age dependent, children also appear to be proficient at producing NTHi-specific IgG (42, 51, 57, 58) . It is thought that this strong, universal humoral response is one of the main reasons why NTHi rarely causes systemic infection. However, high systemic antibody levels do not appear to correlate with protection from respiratory infections (42, 45) .

NON-TYPeABLe Haemophilus influenzae

There are a number of virulence factors employed by NTHi that provide an environment conducive to infection and chronic colonization (41) . Some of these, including production of biofilm and secretion of proteases, have been mentioned previously in this review. Biofilm has recently been described in children with bronchiectasis (67) and recent evidence from in vitro studies suggests that NTHi may manipulate activated neutrophils into facilitating the production of biofilm (81) . NTHi also produces human IgA proteases that, in vitro, contribute to invasion of and survival within human respiratory epithelial cells (59) .

iNiTiATiON OF LOweR ReSPiRATORY iNFeCTiON

Nasopharyngeal carriage of S. aureus is high in infants <3 months old but declines rapidly as carriage of the three main respiratory bacteria increases, reaching a low point at 1-2 years (132, 137) . Carriage increases again to reach its highest prevalence in children >5 years old (138, 139) , as NP carriage of the FiGURe 1 | The extended vicious circle hypothesis to explain high rates of chronic endobronchial disorders such as bronchiectasis. The high and early burden of multiple pathogen species and strains in the nasopharynx of Indigenous children and children in low income countries (high-dose versus low-dose exposure) likely contributes to their high burden of acute and chronic lower respiratory infections. Carriage without inflammation is usually cleared but may result in pneumonia or invasive disease in the presence of virulent strains and/or underlying disease (unshaded boxes). Dense and diverse colonization causes neutrophilic inflammation, mucosal tissue damage, increased mucus secretion, and decreased mucociliary clearance, leading to chronic mucosal disease, recurrent lower airway infection, and bronchiectasis (shaded boxes). Persistent nasal discharge and chronic cough perpetuate this vicious circle, particularly where there is poor hygiene and overcrowding, by increasing opportunities for transmission. High rates of transmission events result in an accumulation of strains at a rate greater than can be cleared by the immune response (particularly in infants and young children) or by damaged mucosa, and the vicious circle (bold arrows) is repeated. other three bacteria declines. If aspiration of S. aureus from the URT contributes to bronchiectasis, then S. aureus lower airway infection may be more common in older children.

MULTiPLe STRAiN CARRiAGe AND PeRSiSTeNCe OF iNFeCTiON

It is probable that the high and early burden of pathogens in the nasopharynx of Indigenous children and children in low income countries contributes to the high burden of acute and chronic lower respiratory infections. Figure 1 , adapted from Cole's original model (10) and modified to explain chronic lung disease (145) , illustrates an "extended vicious circle" hypothesis to explain high rates of chronic endobronchial disorders such as bronchiectasis.

MiCROBiOMe

Respiratory viruses are an important cause of exacerbations in other chronic respiratory illnesses including asthma and COPD (156, 157) . It has been postulated that viruses may alter immune responses and promote respiratory exacerbations from bacterial infection (158) . Furthermore, bacteria/virus coinfections reportedly result in more severe symptoms (158, 159) . Australian Indigenous children carry a high burden of respiratory bacteria from a very young age (135, 160) . Increased nasopharyngeal NTHi density has been shown in the presence of any one of several respiratory viruses in Indigenous children with acute OM; the most commonly detected viruses being rhinovirus, polyomavirus, and adenovirus (161) . Adenovirus has also been associated with suppurative lung conditions in children, particularly with respect to bacterial coinfection (162) .

CONCLUDiNG STATeMeNT

Suboptimal adaptive immune responses, in addition to dysregulated local inflammatory responses, likely contribute to an environment conducive to chronic or recurrent infection. An effective management strategy for bronchiectasis in children requires an understanding of the adverse immunologic events leading to recurrent infection and persistent inflammation. Promoting an environment that supports efficient pathogen clearance and rapid resolution of inflammatory responses should be forefront in our future endeavors to combat a condition that should be largely preventable.

iNTRODUCTiON

In underprivileged populations, including populations within affluent societies (such as Indigenous populations of Australia, New Zealand, and Alaska), severe lower respiratory infection early in life is the most likely cause of bronchiectasis (2) (3) (4) . Globally, it is estimated that severe bacterial or viral pneumonia accounts for approximately 60% of the cases of postinfection pediatric bronchiectasis. Measles and tuberculosis combined account for 25% (1) . Although pneumonia is a significant risk factor for bronchiectasis, only a proportion of children develop bronchiectasis following an episode of pneumonia (5) . The mechanisms involved in progressing from acute lower respiratory infection to chronic inflammation and persistent infection are poorly understood.

AiRwAY iNFLAMMATiON

Pediatric studies are important to our understanding of the pathogenesis of bronchiectasis. Inflammation in the absence of bacterial infection in children with relatively newly recognized disease could mean that neutrophilic inflammation is an indication of abnormal immune regulation, rather than a symptom of chronic infection.

THe ROLe OF NeUTROPHiLS iN BRONCHieCTASiS

There are currently no data regarding neutrophil function in children. However, impaired neutrophil function has been described in adults with bronchiectasis, and this is associated with more severe disease (29) (30) (31) . King and colleagues (30) demonstrated a high prevalence of impaired bacterial-specific oxidative burst function by circulating neutrophils in a large group of adults with bronchiectasis. Furthermore, patients with a low capacity for neutrophil-generated oxidative burst also demonstrated a reduced capacity for intracellular bactericidal activity by the neutrophils. These data, however, were not reflected in two small studies of adult bronchiectasis where no impairment in oxidative burst by circulating neutrophils was found (29, 32) , although impairment of airway neutrophils was observed (29) . The discrepancies between these data may be attributed to the methods used to induce oxidative burst (bacterial phagocytosis versus synthetic peptide) or the clinical characteristics of the study cohorts (age and severity of disease). In the study by King and colleagues, phagocytosis and intracellular killing were investigated using a bacterial challenge, which may represent an alternate and more accurate pathway of activation compared with using peptide as the challenge. Despite the conflicting data, both studies found evidence of impaired microbicidal activity, albeit in different populations of neutrophils, suggesting that functional phagocytosis accompanied by impaired intracellular killing may be one strategy employed by pathogens to establish an intracellular niche and avoid host clearance mechanisms. The possibility that impaired phagocytosis and intracellular killing mechanisms correlate with disease severity highlights the need to investigate the role of neutrophil function in the progression of bronchiectasis in children who are in the early stage of chronic disease.

eosinophils

Eosinophils comprise a small but potent proportion of the leukocyte population in circulation and in the lungs. Recruitment and activation of eosinophils are associated with several respiratory disorders including asthma and eosinophilic bronchitis, as well as parasite infection. Although rarely reported in association with bronchiectasis, our studies have identified a high prevalence of airway eosinophilia in Australian Indigenous children with bronchiectasis, which correlates with circulating eosinophils (13, 16) . Limited investigations implicated a possible role for viruses in elevated airway eosinophils (16) ; however, it is likely that airway eosinophilia in children with bronchiectasis has multiple etiologies, including parasite infection, coexistent asthma, and hypersensitivity to fungi. In adults with chronic airway diseases, including bronchiectasis, eosinophilia is thought to be associated with more severe disease (33) . Therefore, it is important to understand the etiology of airway eosinophilia and its contribution to the perpetuation of chronic inflammation and the pathogenesis of bronchiectasis.

CeLL-MeDiATeD iMMUNe ReSPONSeS

In vitro challenge assays of blood mononuclear cells indicate that the cell-mediated immune response to NTHi may be compromised in children with bronchiectasis. This was demonstrated by a reduced capacity to produce IFN-γ in response to NTHi (42) . Importantly, this compromised cell-mediated immune response was strongly associated with airway inflammation, specifically, elevated levels of IL-1β and IL-6 (16), indicating a possible link between localized inflammation and systemic adaptive immunity. The mechanisms driving the association between airway inflammation and NTHi-driven IFN-γ in children with bronchiectasis have not been determined. However, IL-1β and IL-6 are integral to the initiation and phenotype of the adaptive immune response. IL-1β drives the inflammatory cascade by localizing neutrophils and promoting the production of inflammatory modulators such as IL-6 and IFN-γ-inducible protein 10 (IP-10; CXCL10). IL-6 plays a complex role in the inflammatory response, from promoting inflammation to wound healing. Dysregulation of IL-6 pathways is associated with chronic inflammation (46) . In addition to its inflammatory modulating properties, IL-6 is integral to initiating the adaptive response and in directing its primary phenotype. In the lung, IL-6 polarizes the adaptive immune response in favor of the humoral response by inhibiting IL-12 production (47, 48) and by promoting the differentiation of B-cells into antibody-producing plasma cells (49) . These data suggest that IL-6 and IL-1β may be more than simply markers of inflammation, but rather indicative of suboptimal T-helper pathways that result in impaired clearance mechanisms and persistent infection. Collectively these data support an association between impaired cell-mediated immune responses to NTHi, dysregulated airway inflammation, and the pathogenesis of bronchiectasis in children. These data in children complement extensive studies of NTHispecific immune responses in adults (43, 44) to show that a Th1 polarized cell-mediated immune response contributes to protective immunity against NTHi. In these studies, King and colleagues showed that circulating CD4+ T-helper cells from healthy adults responded to an in vitro NTHi challenge with increased expression of IL-2 and IFN-γ, in a predominantly classic Th1 manner. In contrast, the cytokine response from adults with bronchiectasis and chronic NTHi infection was polarized in favor of IL-4 and IL-10 and complemented by low expression of IL-2 and IFN-γ.
Several factors may contribute to the differences in cytokine profiles generated in response to NTHi, including a reduction in the size of the pool of CD4+ memory T-cells. However, King and colleagues (44) showed that polarization of cytokine profiles was in direct relation to a skewing of the cytokines produced, rather than an overall reduction in the absolute number of CD4+ T-cells. In addition to CD4+ T-cells, CD8+ cytotoxic T-cells have been implicated in chronic infection with NTHi (44) . CD8+ T-cells have the capacity to switch between IFN-γ (Th1) and IL-4 (Th2) polarized responses (50) . CD8+ phenotype switching has not been investigated with respect to chronic NTHi infection; however, CD8+ T-cells from adults with bronchiectasis demonstrated a non-specific capacity to produce IFN-γ that was not realized in response to a specific challenge with NTHi (44) . Phenotype switching is one mechanism that may explain this.

HUMORAL iMMUNe ReSPONSeS

Secretory IgA is the main immunoglobulin associated with mucosal immunology. While data on NTHi-specific secretory IgA are lacking, a small study of 25 adult patients indicated that deficient secretory IgA was not associated with bronchiectasis or chronic bronchitis (56) . In contrast, in vitro studies have shown that NTHi produces human IgA proteases that may facilitate its internalization and persistent survival within lung epithelial cells (59) . It is plausible that impaired antibody function rather than deficient levels of antibody may contribute to recurrent infection with NTHi in bronchiectasis. Alternatively, the presence of bacterial biofilm or host bronchial secretions may impede the activity of antibodies; however, there are no data to confirm or refute this in children or adults with bronchiectasis. Collectively, these data indicate that chronic infection with NTHi is unlikely attributed to an overall deficiency in antibody production. However, NTHi is a heterogeneous species and can induce the production of strain-specific antibodies (54, 60) . Thus, it has been suggested that antibody specificity may explain the inability of a previous infection to protect against a future infection with NTHi. Contrary to this hypothesis, a considerable degree of functional antibody cross-reactivity has been demonstrated between different NTHi strains (45) . Furthermore, in children and adults, there is a high turnover of strain carriage, and multiple strains are often carried concurrently (61, 62) . Hence, it would be expected that a high level of antibody diversity is circulating at any one time, which should significantly restrict the number of infections regardless of strain specificity. These data do not preclude the role of humoral immunity in protection against respiratory infections with NTHi as humoral immunity likely plays a significant role in protection from systemic and airway disease. However, high prevalence of recurrent infection in the presence of high levels of antibody supports the argument that an increased susceptibility to infection with NTHi is more closely linked to the cell-mediated immune response than the humoral immune response.

PeRSiSTeNT iNFeCTiON AND BRONCHieCTASiS

Respiratory pathogens employ a variety of strategies to avoid clearance by host defense mechanisms. When successful, these strategies inhibit the host from effectively clearing infection and contribute to an environment supportive of chronic infection and associated inflammation. Some of the primary strategies employed by common respiratory pathogens include formation of protective structures such as biofilm (H. influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae) (63, 64) , secretion of immune-blocking agents such as IgA proteases (H. influenzae) (65), and the secretion of toxins which damage mucus-clearing structures (including cilia) of the epithelium (66) . Secreted proteases can damage the structure of the bronchial wall, including cilia, hampering sputum clearance from the lungs and promoting inflammatory processes by the host. Biofilm has been reported in the lower airways of children with bronchiectasis (67) and can impede the action of antibiotics (68) . Some pathogens associated with chronic respiratory infections, including H. influenzae and Mycobacterium tuberculosis, can avoid the host humoral response by manipulating the host's own phagocytic cells; hijacking antimicrobial mechanisms and establishing an intracellular niche (69) .

NON-TYPeABLe Haemophilus influenzae

Haemophilus influenzae is a Gram-negative pleomorphic coccobacillus and non-capsulated (non-typeable) strains (NTHi) are common colonizers of the upper respiratory tract (URT) (40) . As the bacterium most commonly isolated from the airways of children and adults with bronchiectasis, NTHi likely contributes substantially to recurrent respiratory infections. In fact, the significance of NTHi in pediatric bronchiectasis was noted 60 years ago (79) with the conclusion that "non-capsulated H. influenzae is responsible for keeping the chronic inflammatory process smoldering in bronchiectatic individuals. " NTHi is also an opportunistic pathogen associated with other respiratory infections such as otitis media (OM), sinusitis, and pneumonia in children (80) .

MULTiPLe STRAiN CARRiAGe AND PeRSiSTeNCe OF iNFeCTiON

The high level of concordance between bacterial strains (NTHi ribotypes and pneumococcal serotypes) in the nasopharynx and lungs of Australian Indigenous children with bronchiectasis and concurrent carriage and lower airway infection (72) suggests recent aspiration of NP secretions, given the high turnover of NP strains. However, multiple strains of NTHi and S. pneumoniae have consistently been found more frequently in BAL compared to NP specimens (72, 89) . This suggests accumulation of strains in the lower airways resulting from recurrent aspiration and failure to eliminate prior strains. Multiple NTHi strains have also been reported in sputum microbiology from adults with chronic respiratory conditions (54, 76) .

MiCROBiOMe

A recent study using 16S rRNA gene sequencing found that the microbiota in BAL specimens from 78 young children with chronic lung disease (36 had bronchiectasis) includes taxa present in both NP and OP specimens (153) . Diversity using Simpson's index was significantly lower in NP swabs compared to OP and BAL specimens, reflecting the more common dominance of individual operational taxonomic units (OTUs) in the NP microbiota (153) . Dominant OTUs (>50% relative abundance) in NP and BAL specimens included M. catarrhalis, H. influenzae, S. aureus, and mitis group streptococci (which includes S. pneumoniae); only Porphyromonas sp. was dominant in OP specimens (153) . These data support the hypothesis that the dynamics of bacterial populations in the nasopharynx are primarily responsible for dysbiosis in the lungs of children with bronchiectasis although both OP and NP analysis should be included in studies of the lower airways.
Respiratory viruses are likely an under-recognized factor contributing to acute exacerbations and persistent airway inflammation in children with bronchiectasis. Large, population-based pediatric studies investigating the effect of viruses on airway immunopathology are important to fully appreciate the contribution of viruses to chronic inflammation and the pathogenesis of bronchiectasis in children.

iMPACT OF vACCiNeS

Currently, there is no commercially available vaccine specifically targeting NTHi, the main bacterial pathogen in pediatric bronchiectasis. Oral vaccines against infection with NTHi have been successful in reducing the number and severity of exacerbations, as well as carriage, in adults with COPD and chronic bronchitis (173) (174) (175) (176) . However, a systematic review found the benefit was too small to advocate widespread NTHi oral vaccination of people with COPD (177) . A more effective oral vaccine may improve mucosal protection (178) and reduce the incidence and/or severity of respiratory infections caused by NTHi.
There are few published clinical trials assessing the impact of PCVs in children with bronchiectasis, although vaccination is currently recommended (78) . A clinical trial of PHiDCV in children with protracted bacterial bronchitis, chronic suppurative lung disease, and bronchiectasis (collectively referred to as chronic endobronchial disorders), with respiratory exacerbation as the primary outcome, is currently underway (190) . We have recently found that carriage serotypes of S. pneumoniae (mostly non-vaccine types in PCV-vaccinated populations) have a similar propensity to cause lower airway infection in Australian children with chronic endobronchial disorders (191) . From these data, it may be argued that PCVs will have little impact on lower respiratory infection in this population. However, PHiDCV may be effective against mucosal infection with NTHi (192) , and we await the full findings of the clinical trial (190) .

THe ROAD FORwARD

The past decade has seen tremendous advances in our understanding of the immunologic parameters associated with bronchiectasis. Bronchiectasis is a complex condition involving suboptimal adaptive immune responses and dysregulated inflammatory responses that culminate in recurrent and persistent infection. However, current management strategies for children continue to rely primarily on antibiotic therapy for the treatment or prevention of acute bacterial infection and physiotherapy for airway clearance. While these strategies are important, a novel, multidirectional approach is required to address the impaired adaptive immune responses and dysregulated inflammatory mechanisms responsible for chronic inflammation and persistent infection. Novel, plausible therapeutic directives may include targeting airway inflammation through the use of inhaled anti-inflammatories or improving adaptive immune responses to pathogens important to the pathogenesis of bronchiectasis. However, both approaches require a commitment to understanding the complex immunology associated with suboptimal immune responses and protective immune pathways, prior to developing and incorporating immunomodulating therapeutics to the management of bronchiectasis in children. Understanding the mechanisms driving the immunopathology of bronchiectasis has the potential to revolutionize therapeutics and management strategies for children. Here, we consider future research endeavors to advance management and prevention of bronchiectasis in children.

Understanding the Mechanisms Responsible for immune Dysfunction

Dysregulated airway inflammation appears to be linked to the functional phenotype of the adaptive immune response (16) ; however, the direction of this association, be it cause or effect, is unknown and likely complex and questions remain regarding the transition between acute and chronic responses. Inappropriate T-cell responses may increase susceptibility to infection with NTHi and prolong inflammation due to ineffective clearance mechanisms. Dysregulated IL-6 and IL-1β pathways in the lungs may promote an environment supportive of Th2 and Th17 polarized responses, increasing susceptibility to infection with NTHi and viruses. It is also plausible that chronic inflammation may contribute to impaired macrophage activation and T-cell responses, resulting in an environment of immune tolerance or exhaustion. Understanding immunologic mechanisms behind the chronic symptoms of bronchiectasis will greatly inform targeted management practices.
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Abstract

Daily oscillations of pulmonary function depend on the rhythmic activity of the circadian timing system. Environmental tobacco/cigarette smoke (CS) disrupts circadian clock leading to enhanced inflammatory responses. Infection with influenza A virus (IAV) increases hospitalization rates and death in susceptible individuals, including patients with Chronic Obstructive Pulmonary Disease (COPD). We hypothesized that molecular clock disruption is enhanced by IAV infection, altering cellular and lung function, leading to severity in airway disease phenotypes. C57BL/6J mice exposed to chronic CS, BMAL1 knockout (KO) mice and wild-type littermates were infected with IAV. Following infection, we measured diurnal rhythms of clock gene expression in the lung, locomotor activity, pulmonary function, inflammatory, pro-fibrotic and emphysematous responses. Chronic CS exposure combined with IAV infection altered the timing of clock gene expression and reduced locomotor activity in parallel with increased lung inflammation, disrupted rhythms of pulmonary function, and emphysema. BMAL1 KO mice infected with IAV showed pronounced detriments in behavior and survival, and increased lung inflammatory and pro-fibrotic responses. This suggests that remodeling of lung clock function following IAV infection alters clock-dependent gene expression and normal rhythms of lung function, enhanced emphysematous and injurious responses. This may have implications for the pathobiology of respiratory virus-induced airway disease severity and exacerbations.
I nfluenza virus, rhinovirus, coronavirus, respiratory syncytial virus, parainfluenza, adenovirus and metapeumovirus are among the respiratory viruses known to cause exacerbations in Chronic Obstructive Pulmonary Disease (COPD) and asthma 1-4 . In fact, respiratory viral infections account for about 50-70% of acute exacerbations in patients with COPD 5 . COPD exacerbations are often followed by subsequent clinical pulmonary deterioration, including significant declines in forced expiratory volume at 1 second (FEV 1 ) and increased hospitalization or mortality 6 . This is associated with worsening of COPD phenotypes, such as cough, fever and mucus production; the timing of which depends on the activity of the biological clock.
Circadian rhythms are biological oscillations that occur with a near-24-h period and are synchronized or entrained to environmental cues, such as the day-night cycle 7 . These rhythms are the manifestation of an autoregulatory molecular oscillator of interlocked positive and negative transcription factors collectively referred to as clock genes 8 . In mammals, the central pacemaker, localized to the suprachiasmatic nucleus (SCN) of the anterior hypothalamus, drives rhythms of physiology and behavior, and synchronizes internal timing with the external environment 7 . Apart from the central clock, peripheral tissues such as the liver, heart and the lung also contain autonomous circadian oscillators that coordinate tissue specific cellular functions and responses to environmental stimuli 7-12 . As an example, circadian rhythms in pulmonary function have been demonstrated in animal models and healthy individuals (highest at noon time and low during the early morning hours) [13] [14] [15] . The coordinated and synchronized activity of central and peripheral oscillators is referred to as the circadian timing system 9,12 . It has been shown that recruitment of leukocytes to tissues following infection is also regulated by the circadian timing system 16, 17 . For example, macrophages and mast cells exhibit robust rhythms of circadian clock and proinflammatory cytokine gene expression 18, 19 . The magnitude of immune and inflammatory parameters varies with time of day, and disruption of circadian rhythms (chronodisruption) has been implicated in cellular dysfunction, and in the pathogenesis of chronic metabolic disease, infection, and inflammatory diseases 20-22 .

We hypothesize that IAV infection exacerbates the effects of chronic CS-induced COPD/emphysema. Moreover, we hypothesize the negative consequences of IAV infection in chronic smokers and patients with COPD may be due to the combined influence of CS and influenza infection on circadian clock function in the lungs. To address these hypotheses, we measured body weight, mortality, locomotor activity, rhythms of clock and clock-controlled gene (CCG) expression in the lungs, lung function rhythms, inflammation and emphysematous responses of mice exposed to chronic CS with or without subsequent IAV infection. Further, to more directly assess the role of the timing system in response to infection, we measured activity, body weight, mortality, inflammation and emphysematous responses in the lungs of BMAL1 knockout mice and WT littermates following IAV infection.

Results

The negative effects of Influenza A virus infection on body weight, mortality and locomotor activity are exacerbated following chronic CS exposure. Chronic (6 months) air-and CS-exposed mice were infected intranasally with 120 HAU IAV (H3N2). Before infection (Day 0), we did not observe a significant difference in body weight among the experimental groups. The body weight of mice in both IAV-infected groups dropped significantly on days 1-3 post-infection, with a slightly more dramatic decline in the CS1Virus group (Fig. 1a ). This suggests that chronic CS-exposed mice exhibited greater mortality following IAV infection. Indeed, IAV infection in chronic CS-exposed mice produced a 14.1% reduction in survival within 9 days of infection (Fig. 1b) . As reported, there was no significant difference in the body weight and mortality between the chronic air-and CS-exposed mice 30 . We found that chronic air and CS-exposed mice infected with IAV showed significant reduction in body weight compared to corresponding controls at the end of chronic exposure or day 9 post-infection ( Supplementary Fig. 2b) . Thus, while IAV infection alone (Air1Virus) has modest effects, infection of mice following chronic CS exposure (COPD/emphysema model) significantly increases mortality.
Influenza A virus affects the phase and amplitude of circadian clock gene expression in the lungs. We have previously reported that core clock genes (bmal1, clock, per1-2, cry1-2, rev-erba) were rhythmically expressed in the lung of air-and CS-exposed mice both during acute and chronic CS exposure 30 . Of the known core clock genes, only rora gene expression was not rhythmic in lungs of chronic air-and CS-exposed mice 30 . The peaks of gene expression rhythms for most clock genes in air-exposed controls were similar to those previously reported 30, 31 . In both the chronic air-and CSexposed mice, the expression of bmal1, clock, and cry1 displayed nocturnal acrophases, peaking during the mid to late portion of the dark phase (ZT18-24; ZT0 5 lights on: ZT12 5 lights off) 30 . As anticipated, both chronic air-and CS-exposed mice showed peaks of per1, per2, cry2, rev-erba, and rev-erbb antiphase to bmal1 (between ZT6-ZT12) 30 . Chronic CS-exposed mice showed a modest reduction in the amplitude of bmal1 and rev-erba expression and substantially reduced amplitude of per1 expression 30 . Analysis of clock gene expression 9 days after IAV infection in air-exposed mice (Air1Virus) revealed significant rhythms of bmal1 (P , 0.001), per1, per2, cry1 and rev-erbb (P , 0.05) but not clock, cry2, rev-erba and rora in lung tissue ( Fig. 3a- Supplementary Fig. 5 and Table 1 ). Mice exposed to chronic CS and IAV infection (CS1Virus) only displayed rhythms of bmal1 (P , 0.01) and per1 (P , 0.05) expression in the lungs ( Fig. 3a- Supplementary Fig. 5 and Table 1 ). Previously reported data from air-exposed mice are shown here for comparison ( Fig. 3a- Supplementary Fig. 5 and Table 1 ). In Air1Virus infected mice, the peak expression of bmal1, per1, per2, cry1, cry2, and rev-erbb shifted to the middle of the light phase (ZT6; ZT05 lights on; ZT125lights off) and the amplitude of gene expression was altered at ZT6 when compared to air-exposed controls and chronic CS1Virus group ( Fig. 3a- Supplementary Fig. 5a -b). As in Air1Virus treated mice, expression of bmal1, clock and rev-erbb was shifted to peak at mid-day (ZT6) in CS1Virus exposed mice ( Fig. 3b and Supplementary Fig. 5b ). However, unlike Air1Virus treated mice, the phase of peak per1 and cry2 expression returned to ZT12 and the remaining clock genes [per2, cry1, rev-erba and rora] peaked at middle of dark phase (ZT18). Together, these data support the notion that IAV induces circadian disruption in the lungs, though the effects appear to be somewhat attenuated in mice exposed to chronic CS ( Fig. 3a-b and Supplementary Fig. 5a-b) .
We next determined the impact of chronic CS exposure followed by IAV infection on rhythms of proinflammatory cytokine release in BAL fluid. MCP-1, IL-6 and MIP-2 levels were measured in BAL fluid at day 9 post-infection across the 24h day. The CS1Virus group showed significant increases in the expression of MCP-1 at ZT6 and ZT24 when compared to Air1Virus and air-exposed controls (Fig. 4e ). We observed a significant decline in the levels of proinflammatory cytokines MIP-2 at ZT0 and ZT12 and IL-6 at ZT12 in the Air1Virus and CS1Virus groups 9 days postinfection compared to uninfected CS-exposed mice ( Fig. 4f-g) . Chronic CS-exposed mice showed significant increases in the levels of IL-6 and MIP-2 that peaked at ZT12 compared to air-exposed controls ( Fig. 4f-g) . Overall, IAV infection dampened the total number of neutrophils, MIP-2 and IL-6 levels, while it increased the total number of macrophages, lymphocytes and MCP-1 levels by day 9 post-infection. CircWave analysis confirmed significant diurnal rhythms of both IL-6 and MIP-2 levels in BAL fluid from chronic CS-exposed mice (P , 0.01). In contrast, chronic air-exposed, Air1Virus and CS1Virus groups did not show rhythmic expression of MCP-1, IL-6 and MIP-2 in BAL fluid as confirmed by CircWave analysis. The increase in levels of cytokines that we observed on day 9 post-infection was associated with influx of inflammatory cells in the lung in CS1Virus and chronic CS-exposed mice ( We have also analyzed proinflammatory cytokine release in BAL fluid by pooling time points according to photoperiod (ZT61ZT12 5 daytime and ZT18124 5 nighttime) for MCP-1, MIP-2, IL-6 and TGF-b1. Chronic CS-exposed mice showed a significant increase in the levels of MIP-2, IL-6 at ZT61ZT12 (daytime) and TGF-b1 at ZT181ZT24 (nighttime) compared to controls (Supplementary Fig. 6a-d) . Chronic CS-exposed mice infected with IAV showed a significant increase in the level of MCP-1 both at ZT61ZT12 (daytime) and ZT181ZT24 (nighttime). The levels of other cytokines including MIP-2, IL-6 and TGF-b1 were significantly reduced in CS1Virus group compared to CS-exposed mice at ZT61ZT12 (Supplementary Fig. 6a-d) . Thus, our data show that disruption of circadian clock function in the lung was associated with augmented MCP-1 levels during IAV-induced exacerbation in chronic CSexposed mice.
Influenza A virus exaggerates chronic cigarette smoke-mediated airspace enlargement/emphysema and altered rhythms of lung function. To investigate the role of IAV-induced circadian clock disruption of the lungs and the possible impact on lung airway remodeling and pulmonary function, we examined airspace enlargement/emphysema by lung histopathologic and functional measurements in chronic CS1Virus infected mice. There was a significant difference in lung histopathological changes between chronic CS1Virus infected mice and Air1Virus infected mice 9 days post-infection, which was confirmed with measurements of the mean linear intercept (MLI; Air1Virus 43.27 6 3.31 vs. CS1Virus 62.27 6 3.20; P , 0.001; Supplementary Fig. 9b ). These data suggest that IAV infection and chronic CS exposure can synergistically enhance airway remodeling in mice.
www.nature.com/scientificreports SCIENTIFIC REPORTS | 5: 9927 | DOI: 10.1038/srep09927 Figure 5 | Chronic CS-exposed mice infected with IAV show persistent inflammation, mucus hypersecretion, and pulmonary fibrosis. Data from chronic (6 months) air-or CS-exposed mice given intranasal inoculation of either saline (control group) or influenza A virus (IAV; treatment group) at ZT4-6 are shown. Lungs were harvested on day 9 post-infection. (a) Representative images of lung tissues stained with hematoxylin and eosin (H&E) to demonstrate parenchymal and bronchial airway inflammation. Bronchial inflammation scores were calculated for each treatment group. (b) Representative images of lung tissues stained with Periodic-acid Schiff (PAS) to visualize mucus overproduction induced by IAV in the bronchial epithelium of chronic air-and CS-exposed mice. Average mucus scores from 3-4 different areas per slide/treatment group (n54-5mice/group) was used to calculate the percentage of PAS positive cells. (c) Representative images of lung tissues stained with Gomori's Trichrome to visualize matrix accumulation/collagen deposition and quantified by Ashcroft fibrosis score. Original magnification x200. Data are representative of mean 6 SEM (n54-5 mice/group). * P , 0.05; ** P , 0.01; significant compared to air-or CS-exposed mice. # # P , 0.01; # # # P , 0.001; significant compared to air-exposed mice. response to IAV infection, we measured lung compliance, resistance and tissue elastance in WT mice infected with IAV on day 9 postinfection, uninfected BMAL1 heterozygous KO mice and uninfected homozygous BMAL1 KO mice. Lung compliance was significantly decreased in both WT-Virus and BMAL1 KO-Saline treated mice compared to WT-Saline and BMAL1 Het-Saline treated mice (Fig. 6c) . However, lung resistance and tissue elastance were significantly increased in both WT-Virus and BMAL1 KO-Saline treated mice (Fig. 6c) . BMAL1 KO-Saline treated mice displayed a significant increase in lung compliance and decrease in resistance and elastance when compared to WT-Virus infected mice suggesting an inherent defect in the lung mechanical properties of BMAL1 KO mice in the absence of infection (Fig. 6c) . In a separate experiment, we determined lung function at different ZT (ZT0-ZT18; n51 mice/ ZT time point) in 2-3 months old BMAL1 KO mice and WT littermates. We also performed differential cell counts in BAL fluid collected from WT and BMAL1 KO mice at all the four ZT time points. We averaged the cell counts data from different time points and found that Bmal1 KO mice had a significant increase in neutrophil counts compared to WT littermates ( Supplementary Fig. 10a ). Total cell counts and macrophage counts were not significantly different between BMAL1 KO and WT littermates (Supplementary Fig. 10a) .

Discussion

The proinflammatory cytokine MCP-1 response was also greater in the IAV infected mice exposed to CS. Similarly, chronic CS exposure also increased MIP-2 and IL-6 at ZT6/12 when compared to controls. However, IL-6 and MIP-2 levels were dampened in CS plus Virus group which could be due to immunosuppressive effects of CS which is in line with recent report in which CS exposure suppressed the production of cytokines and chemokines after pandemic H1N1 or avian H9N2 virus infection in mice 57 . It has been shown that the IAV-mediated inflammatory response begins early on day 3 and remains high until day 5-7 post-infection 58 . Subsequently, viral clearance occurs by day 10 in the lungs, thereby resolving the inflammatory phenotype, such as inflammatory cellular influx and proinflammatory mediators release observed during IAV infection in vivo 58 . Hence, we did not observe significant increases in pro-inflammatory mediators at day 9 post IAV infection in chronic air-and CSexposed mice. CS-exposed mice infected with IAV showed sign of severe pulmonary inflammation, lung permeability damage and mucus hypersecretion which are characteristic features of acute exacerbation of COPD 59, 60 . We have previously reported that mainstream CS exposure at a concentration of 300 mg/m 3 (TPM) for 8 weeks causes significant increase in PAS positive cells in mouse lungs 61 . In this study, we used a low dose side-stream smoke exposure (,90-100 mg/m 3 ) for 6 months, that unlike mainstream smoke, does not cause mucus production in the chronic CS-exposed mouse lungs when compared to Air1Virus and CS1Virus groups. A recent study identified a regulatory mechanism, whereby the lung epithelial clock and glucocorticoid hormones control both time-of-day variation and magnitude of pulmonary inflammatory responses to bacterial infection 62 . Similarly, rhythms of pulmonary function define time-of-day dependent sensitivity to steroids and b2-agonists in patients with nocturnal asthma and asthmatics who smoke [63] [64] [65] . Hence, it is possible that the mechanism that couples the circadian clock and bronchiolar glucocorticoid receptor to pulmonary innate immunity plays an essential role during COPD exacerbations by IAV infection. These data suggest that temporal increases in chemoattractants, leukocyte trafficking, proinflammatory cytokines/chemokines, and phagocytic ability before the activity phase is indicative of clock-controlled sensitivity and immunosurveillance. Collectively, these data suggest that the molecular clock and associated transcription factors, epigenetic regulators, and key regulatory signaling pathways play an essential role in cytokine gene expression through temporal gating of immune responses.

www.nature.com/scientificreports

In conclusion, we show for the first time that IAV infection can cause temporally gated circadian disruption associated with exaggerated lung inflammation and injurious response in the lungs, culminating in exacerbations of COPD/emphysema. Our in vivo model of COPD exacerbation clearly demonstrates how changes in molecular clock function and immune responses can affect morbidity and mortality, rhythms of locomotor activity, lung inflammation and small airway remodeling. The role of the clock gene BMAL1 in COPD exacerbation was tested using the IAV infection model. Overall, our findings clearly show that the circadian clock plays a crucial role in modulating immune-inflammatory response during viral respiratory infection in mice. The COPD exacerbation mouse model develops augmented inflammatory responses and lung damage due to involvement of clock-dependent mechanisms that in turn affect immune response and rhythms of lung function. Understanding molecular clock function and its physiological significance in different animal models of chronic lung disease, including our COPD exacerbation model, could hasten the development of novel chronotherapeutic approaches for the treatment and management of COPD and associated exacerbations.

Methods

Tobacco/cigarette smoke exposure and influenza A virus infection. Eight week-old mice were used for tobacco/CS exposure as previously described 30, 74, 75 . We used chronic (6 mo. exposure which causes pulmonary emphysema) CS exposure mouse models to determine the effect and mechanism of chronic CS exposure followed by influenza virus infection on circadian clock function and lung inflammation. Mice were exposed to CS using an environmental side-stream delivering Teague TE-10 smoking machine (Teague Enterprises, Davis, CA) for 6 months CS exposure in the Inhalation Facility at the University of Rochester Medical Center. The smoke was generated from 3R4F research cigarettes containing 11.0 mg of total particulate matter (TPM), 9.4 mg of tar and 0.73 mg of nicotine per cigarette (University of Kentucky, Lexington, KY). The total particulate matter (TPM) in per cubic meter of air in exposure chamber was monitored in real-time with a MicroDust Pro-aerosol monitor (Casella CEL, Bedford, UK), and verified daily by gravimetric sampling 30, 74, 75 . Control mice were exposed to filtered air in an identical chamber according to the same protocol described for CS exposure. For chronic 6 months CS exposures, 3R4F cigarettes were used to generate a mixture of sidestream smoke (89%) and mainstream smoke (11%) at a concentration of ,100 mg/m 3 TPM, so as to avoid the possible toxicity to mice at a high concentration of long-term CS exposure 30, 74 according to the Federal Trade Commission protocol (1 puff/min of 2 second duration and 35 ml volume). Each smoldering cigarette was puffed for 2 seconds, once every minute for a total of 5 puffs, at a flow rate of 1.05 L/min, to provide a standard puff of 35 cm 3 . Mice received 5-hour exposures per day, 5 days/week for the duration of exposure and were sacrificed at 6-hour intervals 24h after the last CS exposure.
After 6 months chronic Air/CS exposure, mice were intranasally infected under anesthesia (Avertin; 2,2,2-tribromoethanol; Sigma-Aldrich) with 120 hemagglutination units (HAU) of influenza A virus (IAV), strain HKx31 (x31; H3N2) in 25 ml sterile PBS as previously described 32, 76 . Mock-infected control group mice received 25 ml of sterile PBS alone. After infection, survival and body weight of all the experimental groups were monitored and recorded daily until post-infection day 9. Mice were euthanized post-infection day 9 at 6 hour intervals for 24 hours (5 time points: ZT0, ZT6, ZT12, ZT18 and ZT24). The 6-hr sampling interval was based on prior studies on circadian gene expression in mice 30, 37 . Schematic for chronic CS exposure combined with IAV infection and IAV infection in BMAL1 KO and wildtype littermates including parameters measured from these experiments are included in the Supporting information (see Supplementary Fig. 1a-b) .

An early morning surge in lung function and pronounced troughs in forced vital capacity (FVC), FEV 1 and peak expiratory flow (PEF) during the night are common in patients with COPD exacerbations, including chronic smokers 23, 24 . This could be due to cigarette smoke (CS)-mediated alterations in circadian clock proteins, levels of steroid hormones, surfactants in the lungs, mucus retention/secretion accompanied by increased inflammatory responses and a decline in normal rhythms of lung function 15, [25] [26] [27] . Disruption of pulmonary function rhythms during COPD exacerbations commonly results in emergency room visits at night or in early morning hours when lung function is low 15, 25, 26 . In addition to a decline in lung function, patients with COPD have other sleep-related abnormalities, such as insomnia, excessive daytime sleepiness and altered rhythms of airway caliber and resistance 28 . Lung function is also altered following viral infections, including influenza A virus (IAV 29 ) , and IAV is known to increase the intensity and duration of exacerbations in those patients with COPD. However, the effect of virus-induced COPD exacerbations on clock function in the lung and the role of the lung clock in the pathogenesis of COPD and associated exacerbations are unknown.

Results

We have previously reported that mice exposed to 10 days of CS (acute exposure model) during the active phase show reduced locomotor activity associated with increased inflammation 30 . Similarly, mice exposed to chronic CS showed significantly reduced activity beginning 5 days after the first exposure that persisted for much of the exposure period 30 . In the present study, chronic CS-exposed mice infected with IAV (CS1Virus) showed a significant reduction in locomotor activity after infection that persisted for the duration of measurement (9 days post-infection; Fig. 1c-d) . Chronic air-exposed mice infected with influenza (Air1Virus) showed an initial reduction in locomotor activity 1-5 days after infection with a trough 5-6 days after infection followed by a modest recovery by day 9 after infection ( Fig. 1c-d) . Actograms clearly reveal that the CS1Virus group showed significantly reduced locomotor activity postinfection (lower right panel) when compared to Air1Virus, air-and CS-exposed mice ( Fig. 1d and Supplementary Fig. 2a) . Limiting our analysis to the dark phase reveals that the primary influence of IAV infection was a reduction in nighttime activity, which was further suppressed in those mice previously exposed to chronic CS (Fig. 1c) . The period of locomotor activity in L:D during days 1-8 post IAV infection was very similar between air and CS groups, whereas the Air1Virus and CS1Virus groups showed increased variation in period between animals although the mean was not statistically different from controls (Fig. 1e) . Analysis of behavior as a function of daily distribution (day vs. night) emphasizes the mortality and behavior in chronic air or CS-exposed mice. Data from uninfected air-and CS-exposed mice from a previous experiment were included for comparison [panels c-e; 30 ]. (a) Chronic CS-exposed mice infected with IAV showed a modest reduction in weight, marked by a decline on days 1-3 post-infection relative to air-exposed mice infected with IAV. (b) Mortality was monitored for 9 days post IAV infection. Data are representative of mean 6 SEM (n530-40 mice/group). ** P , 0.01 significant compared to Air1Virus group. Analysis of differences in survival over time was determined by a Mantel-Cox test (P , 0.68). (c) Nocturnal activity for 9 days post-infection was plotted as ambulatory counts. IAV infection reduced locomotor activity in CS-exposed mice by 70-80% during days 1-7 post-infection when compared to CS-exposed mice treated with saline. Similarly, IAV infection reduced locomotor activity in CS-exposed mice by 70-80% on days 1-2 and day 4 postinfection but only 30-45% during days 5-9 post-infection when compared to Air1Virus treated mice. Data are mean 6 SEM (n56 mice/group) for each time point. ** P , 0.01; *** P , 0.001 significant compared to control groups (air or Air1Virus); $$ P , 0.01 significant compared to air-exposed mice; # P , 0.05; # # P , 0.01; # # # P , 0.001 significant compared to CS-exposed mice. (d) Representative double plotted actograms of total cage activity from chronic air, chronic CS, Air1Virus and CS1Virus treated mice. In panel d, gray shading indicates the dark phase (ZT12-24) and activity was not recorded during body weight measurements (ZT5-6). Red arrow head indicates day 0 IAV infection and asterisks denotes the time during which the mice were infected with IAV (ZT4-6). (e) Periodogram analysis of activity in L:D during days 1-8 post IAV infection. Period was very similar between air and CS groups, whereas the Air1Virus and CS1Virus groups showed increased variation in period though the mean was not significantly different from controls. Data are mean 6 SEM (n56 mice/group). Fig. 3a-d) . Control (air) and Air1Virus infected mice were primarily active (70-80% activity) during the dark phase and less active (20-30%) during the light phase from day 2 to day 9 post-saline infusion (Supplementary Fig. 3a and c) . In three of the four groups, we detected a transient change in the distribution of activity on day 1 post-infusion regardless of treatment, suggesting that the infusion procedure acutely and temporarily altered activity ( Supplementary Fig. 3a-d) . In agreement with our previous report, chronic CS-exposed mice displayed a distribution of locomotor activity similar to air-exposed mice with the majority (70-80%) of activity limited to the dark phase ( Supplementary Fig. 3b) 30 . Following IAV infection in chronic CSexposed mice nighttime activity declined and daytime activity increased from days 3-7 post-infection such that we detected no difference between day and night activity levels by day 7 (Supplementary Fig. 3d ). By day 8-9 post-infection the CS1Virus mice showed recovery of their normal nighttime (70-80%) and daytime (20-30%) distribution of activity ( Supplementary Fig. 3d ).
Infection with IAV increases macrophages, lymphocytes and CSmediated inflammatory responses in the lungs. As previously reported, similar levels of virus-specific antibodies were present in the blood of both Air1Virus and CS1Virus infected mice 32 . To investigate whether IAV infection after chronic CS exposure alters the inflammatory status in mouse lung, we measured the number of leukocytes in lung bronchoalveolar lavage (BAL) fluid 9 days postinfection. We observed more total cells, macrophages and lymphocytes near the end of the dark phase (ZT24) in the CS1Virus group when compared to Air1Virus, air-and CSexposed mice (Fig. 4a-c) . There was also a significant increase in neutrophils at ZT24 in chronic CS-exposed mice compared to airexposed control. That is, in the absence of infection, there was an increase in neutrophils; however, this was not observed on day 9 of infection ( Fig. 4d) .
To further examine the role of circadian regulatory factors on host responses to infection, we determined the total number of leukocytes in the airways. Consistent with prior reports, there was a significant increase in total cells, macrophages, lymphocytes and neutrophils in BAL fluid from WT-Virus infected mice compared to WT-Saline treated mice (Supplementary Fig. 7a-d) . However, we were unable to evaluate immune cells in lungs of infected BMAL1 KO mice due to 100% mortality in this group by day-9 post-infection (see Fig. 2b ). Because we observed a significant increase in inflammatory cellular influx into the lung of IAV infected WT mice, we next determined the level of proinflammatory cytokines/chemokines in BAL fluid. WT-Virus infected mice showed a significant increase in release of www.nature.com/scientificreports SCIENTIFIC REPORTS | 5: 9927 | DOI: 10.1038/srep09927 30 ]. Lung tissues were harvested every 6 h for 24 h beginning at ZT0 day 9 post-infection. (a) Expression of core clock genes (bmal1, clock, per1, cry1, and rev-erba) in mouse lung tissue. CircWave analysis confirmed statistically significant rhythms of clock gene expression in Air1Virus (P , 0.05 for per1 and cry1; P , 0.001 for bmal1) and CS1Virus (P , 0.05 for per1; P , 0.01 for bmal1) treated mice. (b) IAV infection adjusted the phase of clock gene expression in a gene-and treatment-(Air vs. CS) dependent manner. Center of gravity (COG) or peak phase for each clock gene was plotted on a horizontal phase map. In panels a and b, gray shading indicates the relative dark phase (ZT12-24). Data from air-exposed (open circle), Air1Virus (gray square) and CS1Virus (solid diamond) mice are representative of mean 6 SEM (n53-4 mice/group) for each time point. * P , 0.05; ** P , 0.01; *** P , 0.001 significant compared to Air group. # # P , 0.01; significant compared to Air1Virus group. Influenza A virus augments chronic cigarette smoke-mediated inflammation and fibrosis. Chronic CS exposure enhanced IAV induced inflammatory responses, as observed by increases in peribronchial and perivascular inflammatory cell infiltration in the airways (Fig. 5a) . Mucus hypersecretion in the airways is an important characteristic pathological feature during acute exacerbation of COPD and is a key facet of inflammation-induced airway obstruction 33 . We determined the degree of mucus production in the airways of Air1Virus and CS1Virus infected mice at day 9 post-infection. Mucus-producing PAS positive cells were significantly increased in airway epithelium of chronic CS1Virus infected mice compared to Air1Virus infected mice that displayed a paucity of mucus producing cells in the airway epithelium (Fig. 5b) . Trichrome staining demonstrated significant increases in airway fibrosis in mice exposed to CS1Virus with increase in collagen deposition observed around the airways (Fig. 5c) . Further, we detected a significant increase in a-SMA levels, a marker for activated myofibroblasts observed in fibrotic lung disease, in the fibrotic regions of the lung from both air and CS-exposed mice infected with IAV ( Supplementary Fig. 9a ). These characteristic features suggest that IAV infection of mice previously exposed to chronic CS results in exaggerated lung inflammation and fibrotic airway remodeling in vivo. It has been documented that after IAV infection, epithelial damage induced by the virus could be repaired in order to completely restore lung structure and function in vivo. Clara cell specific marker, CCSP was used to identify changes that occur during virus infection in the airway bronchial epithelial cell population. At day 9 post-infection, we observed a significant decrease in the expression of CCSP positive cells in the airway bronchial epithelium of chronic CS1Virus infected mice compared to Air1Virus, air-and CS-exposed mice ( Supplementary Fig. 9a ).
We then determined the impact of IAV infection on diurnal rhythms of lung function, including lung compliance, resistance and tissue elastance. We have previously reported that when examined as a function of time of day (day vs. night) chronic CS-exposed mice show significantly increased lung compliance and reduced elastance, but resistance is not significantly reduced 30 . When analyzed at specific time points across the day, elastance and resistance were only significantly reduced at ZT18 in chronic CS-exposed mice 30 . Lung compliance was significantly decreased at ZT12 and ZT24 in the CS1Virus group when compared to the Air1Virus group ( Fig. 6a-b ; Tables 2-3 and Supplementary Tables 1-3 ). There was a change in the phase and rhythms of lung compliance in Air1Virus group when compared to air-exposed and chronic CS1Virus infected mice ( Fig. 6a-b ; Tables 2-3 and Supplementary Tables 1-3) . However, lung resistance decreased significantly only at ZT6 in CS1Virus group as compared to Air1Virus group ( Fig. 6a-b ; Tables 2-3 and Supplementary Tables 1-3) . Tissue elastance was significantly decreased at ZT6 and significantly increased at ZT24 in the CS1Virus group when compared to the Air1Virus group ( Fig. 6a-b ; Tables 2-3 and Supplementary Tables 1-3) . Similarly, there were significant alterations in the phase and amplitude of lung resistance and tissue elastance rhythms in CS1Virus group and Air1Virus group when compared to air-exposed controls ( Fig. 6a-b ; Table 2-3 and Supplementary Tables 1-3) . Overall, when we combined the data from day (ZT6 and ZT24) and night (ZT12 and ZT18), lung compliance was significantly reduced whereas lung resistance and tissue elastance significantly increased during the dark phase (ZT12-18) in CS1Virus group compared to Air1Virus group ( Fig. 6a-b ; Tables 2-3 and Supplementary Tables 1-3) . Thus, IAV infection appeared to invert the phase of peak tissue elastance and resistance but not lung compliance such that each marker of pulmonary function peaked during the light phase (when the animal is generally sleeping; Fig. 6b ). Exposure to CS prior to IAV appeared to oppose this response, preventing the dramatic shifts produced by IAV infection.
BMAL1 KO mice show altered lung function and pro-fibrotic responses. To investigate the role of the molecular clock in the Figure 4 | Chronic CS-exposed mice infected with influenza A virus show increased inflammatory cell influx and proinflammatory cytokine release in BAL fluid. Data from chronic (6 months) air-or CS-exposed mice given intranasal inoculation of either saline (control group) or influenza A virus (IAV; treatment group) at ZT4-6 are shown. Data from uninfected air-and CS-exposed mice from a previous experiment were included for comparison [panels a-d; 30 ]. The total number of inflammatory cells was determined in BAL fluid from air, CS, Air1Virus and CS1Virus infected mice on day 9 post-infection. At least 500 cells in the BAL fluid were counted to determine (a) total cells, (b) total macrophages, (c) total lymphocytes and (d) total neutrophils. Data are representative of mean 6 SEM (n53-4 mice/group) for each time point. * P , 0.05, *** P , 0.001 significant compared to air-exposed mice; $ P , 0.05 significant compared to air-exposed mice; # # # P , 0.001 significant compared to CS-exposed mice. Levels of proinflammatory mediators (e) MCP-1, (f) MIP-2 and (g) IL-6 levels were measured in BAL fluid obtained from air, CS, Air1Virus and CS1Virus infected mice. IAV infection of chronic CSexposed mice alters diurnal rhythms of proinflammatory cytokine release in mouse lungs. Data are representative of mean 6 SEM (n53-4 mice/ group) for each time point. * P , 0.05; ** P , 0.01; *** P , 0.001 significant compared to air or Air1Virus groups; # P , 0.05; # # P , 0.01; # # # P , 0.001 significant compared to CS-exposed mice.

Discussion

Patients with COPD display daily rhythms of symptom exacerbation affiliated with sleep disruption [34] [35] [36] . Given the daily nature of these exacerbations and their impact on sleep quality, we hypothesized that disruption of the biological timing system occurs during the etiology of COPD. We have previously reported that rhythms of behavior (locomotor activity), hormone secretion (serotonin and corticosterone) and clock gene expression in the lungs and brain are altered in a mouse model of COPD due to environmental tobacco/cigarette smoke exposure 30, 37 . Influenza infection is also known to alter behavior 38, 39 and has a negative influence on lung function 29 . The immune-inflammatory system is regulated by the circadian clock at multiple levels with immune and inflammatory responses showing Data from chronic (6 months) air-or CS-exposed WT mice given intranasal inoculation of either saline (control group) or influenza A virus (IAV; treatment group) at ZT4-6 are shown. Data from uninfected air-exposed mice from a previous experiment were included for comparison [panels a-b; 30 ]. (a) Daily rhythms of compliance, resistance and elastance were measured in air, Air1Virus and CS1Virus mice. As in previous experiments, measurements were taken on day 9 post-infection. (b) COG or peak phase values for each measure of lung function were plotted on a horizontal phase map. Gray shading in panels a and b indicates the relative dark phase (ZT12-24). Data from air-exposed (open circle), Air1Virus (gray square) and CS1Virus (solid diamond) are representative of mean 6 SEM (n53-4 mice/group) for each time point. ** P , 0.01; *** P , 0.001, significant compared to Air1Virus; $ P , 0.05; $$$ P , 0.001 significant compared to air-exposed mice. (c) Influenza A virus infection altered lung function in WT mice. After day 9 post-infection, lung compliance, resistance and tissue elastance were determined in WT-Saline, WT-Virus, BMAL1 KO-Saline and BMAL1 Het-Saline (heterozygous) treated mice. Data are representative of mean 6 SEM (n510 WT-Saline; n 58 WT-Virus, n54 BMAL1 KO-Saline and n56 BMAL1 Het-Saline) for each time point. *** P , 0.001, significant compared to WT-Saline; # P , 0.05; # # P , 0.01 significant compared to WT-Virus. 36 . These individuals are also commonly diagnosed with sleep disorders including obstructive sleep apnea 41 . Psychological distress in those with COPD is associated with a decline in lung function, increased exacerbation frequency and worsening of cardiovascular disease, further disrupting sleep in these patients 34 . Though our experiments did not directly measure sleep per se (e.g. using polysomnographic recording), the considerable reduction in activity during the day and night in both WT and BMAL1 KO mice following IAV infection implies adaptive changes in the homeostatic or circadian sleep drive that lead to increased inactivity during IAV infection. This increase in sleep/ reduced activity level could be as a result of immune/inflammatory cell infiltration (e.g. macrophages) that can generate influenzainduced sleep enhancement 38 . Alternative compensatory immune mechanisms that generate an effective host defense response may also contribute to sleep propensity during viral infection 38 . It has been suggested that altered expression of Temt (thioether-S-methyltransferase) in the hypothalamus and basal forebrain during IAV infection may influence sleep patterns through its effects on prostaglandin metabolism 42 .
We show for the first time that IAV infection alters circadian clock gene expression in the lungs and reduces the amplitude of locomotor activity in a COPD/emphysema mouse model. The effect of IAV infection persisted longer (post-infection day 7-9) in chronic CSexposed mice, coincident with a decrease in body weight and increased mortality. BMAL1 KO mice infected with IAV also displayed a significant decline in body weight and survival (100% mortality), suggesting that proper function of the timing system is necessary for maintaining the innate immune response to infection. Chronic air-and CS-exposed mice infected with IAV after 6 months of exposure and WT and BMAL1 KO mice infected with IAV show widely varying changes in morbidity (weight) and mortality. This could be due to strain background-and age-dependent effects of IAV infection. The mice were 2-3 months old when we started the chronic air and CS exposure. At the end of chronic air and CS exposure (6 months) and before IAV infection chronic air-and CS-exposed mice were about 9-10 months old. Similarly, WT littermates and BMAL1 KO mice were 2-4 months old when they were infected with IAV. Hence, we speculate the two confounding factors such as strain background and age of these mice when they were infected with IAV play an essential role on the severity of IAV on the loss of body weight and mortality in vivo. This notion is supported by studies wherein circadian desynchronization due to experimental jet-lag increased inflammatory responses and mortality following LPS challenge 43 . It is evident that changes in the phase and amplitude of clock gene expression in the lungs are affiliated with impaired lung function (compliance, resistance and tissue elastance). Analysis of lung function rhythms clearly revealed a near inversion of peak phase rhythms of lung function in Air1Virus group compared to airexposed mice. Though generally similar, prior exposure to CS (COPD) did not fully attenuate the effects of IAV infection, as there were still slight differences in peak lung functions in these mice. These changes in lung function rhythms may underlie the reduced activity and increased mortality observed among IAV infected mice.
To assess the impact of IAV with or without CS exposure more directly, we examined the influence of cigarette smoke extract (CSE) Data are shown as mean 6 SEM (n53-4 per group). *P , 0.05, **P , 0.01, ***P , 0.001, significance of rhythmicity as determined by CircWave analysis in air or Air1Virus or CS1Virus exposed mice. NS, Not significant { Data from animals exposed to air for 6 months from a previous study were included here for comparison 30 . and influenza A virus (300 HAU/ml) on PER2::LUC expression in lung tissue explants. As reported earlier, CSE treatment at a very low dose (0.1%) tended to lengthen the period of PER2::LUC expression in lung tissue explants, though the effect was modest. Infection with IAV resulted in a significant increase in period when compared to uninfected controls. Surprisingly, CSE treatment combined with IAV infection attenuated the effect of IAV infection alone on the period but not the amplitude of PER2::LUC expression in lung explants. Circadian disruption due to chronic jet lag has been shown to alter lung mechanics and clock gene expression in the lungs in a sexually dimorphic manner 44 . As a whole these data strongly suggest that CS exposure and virus infection either alone or in combination can affect clock function in the lungs. CS combined with IAV infection affects both lung CCGs and pulmonary function akin to other models of circadian disruption, suggesting that even a subtle change in clock function may have significant impact on clock-dependent physiological processes in both the lungs and immune system 16, 20, 21, 45, 46 . Though implied, direct support for a functional link between rhythms of lung function and circadian clock gene expression in the lungs remains elusive. While not conclusive evidence for such a link, the combined and somewhat parallel impacts of IAV infection on pulmonary function and clock gene expression in the lungs support the notion that the lung clock contributes in a meaningful way to the timing of pulmonary physiology. It is well known that immune-inflammatory parameters change with time of day and disruption of circadian rhythms has been associated with infectious and inflammatory diseases 20, 21, 45 . Studies from animal models highlight the extent to which the core clock proteins (BMAL1, CLOCK and REV-ERBa) regulate fundamental aspects of the immune-inflammatory response 21 , such as toll-like receptor 9 (TLR9) 46 and repressing chemokine (C-C motif) ligand 2 (CCL2) expression 47 . Further, REV-ERBa has been shown to attenuate the activation of IL-6 expression 19, 47 . It has been shown that the core circadian clock protein was bound to nuclear factor kappa B (NF-kB) RelA/p65 activating NF-kB-dependent transcription 48 . Both transcription factors activator protein 1 (AP-1) and NF-kB share unique sequences that overlaps consensus sequence from rev-erba promoters demonstrating role of REV-ERBa in regulating oxidative stress and/or inflammation 49 . These reports suggest involvement of these proinflammatory gene regulatory transcription factors and role of molecular clock on exaggerated inflammatory responses observed in our mouse model of COPD. BMAL1 KO mice are behaviorally arrhythmic 50 and show signs of advanced aging and underlying pathologies, correlated with increased levels of ROS and cellular senescence 51, 52 . We have recently shown that CS exposure reduced mRNA and protein levels of BMAL1 and BMAL1-CC10 cre (epithelium specific Bmal1 KO) with augmented inflammatory responses and dysregulation of CSinduced oxidative stress. These data suggest the involvement of the molecular clock in regulation of CS-induced lung inflammation 30 , which supports previous work indicating that BMAL1 has an antiinflammatory function 19, 53 . Recently, Clock D19 circadian mutant mouse lung showed altered temporal Nrf2 activity complemented with reduced GSH levels, increased protein oxidation and a spontaneous fibrotic-like phenotype 54 . We found that BMAL1 KO mice also develop a pro-fibrotic phenotype in the lung which could be due to increased ROS levels and altered oxidative stress-mediated cellular senescence 51, 52 . BMAL1 deletion in myeloid cells demonstrated diurnal variation in the absolute number of specific monocytes (Ly6C hi ) in blood and in the spleen under normal conditions which was enhanced during inflamed peritoneum at ZT8 versus ZT0 53 . This study also revealed that BMAL1 binds to E-boxes in the promoters of Ccl2, Ccl8 and S100a8 (encoding S100 calcium binding protein A8) and recruits with it members of the polycomb repressor complex (PRC2) thereby allowing repressive histone marks to block transcription and attenuate Ly6C hi monocyte numbers and inflammation at the site of damage 53 . It has been shown that the number of leukocytes in the mouse circulation strongly correlates with circadian variability, such that leukocyte numbers peak at ZT5 and recruitment into the tissues peaks at ZT13 17 . Keller et al. demonstrated that the role of entire toll-like receptor 4 (TLR4) pathway in peritoneal macrophages is tightly regulated under the control of circadian clock and thus equip the immune cell to face exaggerated response at times 18 . In the present study, analysis of clock gene expression revealed a significant impact of IAV infection on the expression of core clock genes. There was also a time of daydependent increase in the number of total cells, macrophage counts and lymphocyte counts following IAV infection of CS-exposed mice. In the present study, the total numbers of inflammatory cells were significantly increased at ZT24 when compared to ZT0 though they represent the same circadian phase. Previous reports suggest that inflammatory cells in the lungs were significantly increased in BAL fluid of mice euthanized 24 h after the last exposure compared to 2 h post-last CS exposure 55, 56 . These findings suggests that CS exposure has a suppressive effect on the number of inflammatory cells recovered in the lavage, possibly due to capillary trapping or increased adhesion, which causes reduction in the numbers recovered from the air spaces at ZT0 compared to ZT24. This finding is supported by increased proinflammatory mediators released into the lung at 24 h compared to 2 h after the last CS exposure, which would attract more inflammatory cells into the lung interstitium during CS exposure.

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Several studies have examined the effects of acute CS exposure combined with IAV infection 58, [66] [67] [68] [69] . Only two prior reports determined the effects of IAV infection combined with chronic CS exposure model 70, 71 . Robbins et al. were the first to show chronic CS affects primary antiviral immune-inflammatory responses, yet secondary immune protection remained intact suggesting exaggerated inflammatory responses during viral infection might possibly influence decline in clinical status associated with COPD exacerbations 70 . Wortham et al. demonstrated NKG2D stimulation during chronic CS exposure plays an essential role in the development of NK cell hyper-responsiveness and influenza-mediated exacerbations of COPD 71 . Based on the published studies, it is evident that both acute and chronic CS exposure combined with influenza infection causes increased pulmonary and systemic inflammation in vivo which was accompanied by increased viral proliferation or reduced clearance 58, 67 . In this study, the dynamics of viral proliferation were not affected; instead we observed an exaggerated inflammatory response and apparent normalization of lung function rhythms in chronic CSexposed mice infected with IAV. The discrepancies observed in previous studies include CS exposure protocols, dose and duration of CS exposure (acute vs. chronic), viral dose (low and high) used, time of virus infection (ZT0-24) and duration post-infection of analyses. Regardless of these discrepancies, these studies support the fact that CS-induced inflammation plays a defining role in the initial inflammatory responses to IAV infection in the lungs. It is possible that IAV-mediated resetting of clock function in the lungs may influence the survival of mice previously exposed to chronic CS. The re-alignment of many of the clock genes following IAV infection may represent transient 'sensitization' of the timing system to an acute inflammatory mediator 12, 72 . It is worth noting that, outside of rora, only those clock genes associated with the repressive function of the clock (per1,2 and cry1,2) were in effect 'phase-reset' by IAV. This suggests a disparate influence of IAV infection on the oscillator, a phenomenon reported previously in response to dual entrainment by multiple cues in the liver 73 .

Methods

Animals. Male C57BL/6J (C57) and BMAL1 knockout (KO: B6.129-Arntl tm1Bra /J) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). C57BL/6J and BMAL1 KO mice were housed under a 12:12 light-dark (LD) cycle with lights on at 6 a.m. and fed with a regular diet and water ad libitum unless otherwise indicated. For chronic (6 months) CS exposure, mice were kept in a standard 12:12 L:D cycle with lights on from 6 am-6 pm throughout the experiment. Data from animals exposed to air/CS for 6 months from a previous study were included here for comparison 30 .
RNA isolation and quantitative PCR. Total RNA was isolated from non-lavaged lung tissue specimens (stored in RNAlater, Ambion, Austin, TX) using RNeasy kit (Qiagen, Valencia, CA). RNA yields were determined by UV absorbance using a Nanodrop instrument (ND-1000 Spectrophotometer, NanoDrop Technologies). cDNA was synthesized from 0.5 mg of total RNA using the RT 2 First Strand Kit (SABioscience, Frederick, MD). To validate the expression of diverse genes in lung tissue by quantitative real-time PCR (qPCR) (Bio-Rad CXF-96 real-time system) using the SYBR Green qPCR Master mix from SABioscience. In chronic Air/CS exposed mice infected with or without influenza A virus, this includes qPCR data from circadian genes at ZT0-ZT24 time point (n53-4 mice/group) in all datasets. All the specific primers were purchased from SABioscience. Expression of genes was normalized to RPL13 (60S ribosomal protein L13 gene) levels. The samples from chronic air-and CS-exposed mice represented in this study were obtained from our previous study that was conducted in parallel with the chronic air and CS combined with influenza A virus infection 30 . In chronic air exposure group, qPCR data gathered from circadian gene expression at the ZT24 time point (n52/air group) in all qPCR datasets. Relative RNA abundance was quantified by the comparative 2 -DDCt methods.
21 section matches

Background

This paper summarizes the Part 3 of the proceedings of the 8 th International Conference on Management and Rehabilitation of Chronic Respiratory Failure, held in Pescara, Italy on 7 and 8 May, 2015. It summarizes the contributions from numerous experts in the field of chronic respiratory disease and chronic respiratory failure. The outline follows the temporal sequence of presentations.
Moving across the spectrum of care for long-term ventilation Rationale As technology advances, therapeutic options for individuals with chronic respiratory failure requiring short-and longterm ventilator support increase. This section will review old and new indications for ventilator therapy, implementation and feasibility of these types of complex interventions, potential methods to improve their applicability and safety, and economic issues resulting from their use.

Palliative and end-of-life care

Most end-stage patients with chronic respiratory failure complain of dyspnoea in the last 3 months of life [35] . Breathlessness is often more severe in these patients than in those with advanced lung cancer [36] . As a consequence, NIV is being increasingly used to relieve dyspnea in these patients [37, 38] . Recent guidelines state the following: "As relief of dyspnoea with NIV may not relate to changes in arterial blood gases, it is appropriate to reassess the br0eathlessness experienced by patients receiving such ventilatory support at frequent intervals" [39] . Observational studies as well as clinical trials have recently confirmed the role of NIV in patients with chronic disease and poor life expectancy (with or without COPD), showing that this ventilatory technique may favourably reduce dyspnoea shortly after initiation, even without an associated episode of hypercapnic ARF [40] . About half of the patients survived the episode of respiratory distress and were discharged from the hospital.

Key points

Chronic respiratory failure is very frequently the final stage of the natural history of chronic obstructive pulmonary disease. The role of long-term non invasive positive pressure ventilation in improving survival in COPD patients with CRF is still discussed. Long-term night non invasive ventilation in these patients has some physiological and clinical benefits. Long-term non invasive ventilation should be reserved to individual patients.
Chronic respiratory failure (CRF) is very frequent in the end stage of the natural history of chronic obstructive pulmonary disease (COPD). Among other factors, inspiratory muscle dysfunction due to pulmonary hyperinflation may lead to ineffective alveolar ventilation resulting in chronic hypercapnia. Whether chronic hypercapnia is adversely associated with overall prognosis is still discussed, at least in patients on long term oxygen therapy (LTOT) [43] .

Clinical results

Although home NPPV is widely accepted for the treatment of chronic hypercapnia due to respiratory or neuromuscular disease, whether stable hypercapnic COPD patients should routinely be offered this therapy is still discussed [48] . Recently, the role of ventilator management on physiological parameters and outcome in stable hypercapnic COPD patients has become more evident. It is suggested that its benefits depend on the ability of NPPV to substantially reduce PaCO 2 through using "high" inflation pressures [49] . This was confirmed by prospective trials, showing an advantage of high over lower inspiratory pressure levels, with regard to improvements of lung function, blood gases, exerciseinduced dyspnoea and health status [50, 51] . A multicenter study showed a highly significant survival advantage of NPPV (compared with standard care) when it was targeted to maximize hypercapnia reduction [52] . The findings of that study may influence the attitude of clinicians on the use of NPPV in patients with stable hypercapnic COPD. However, the effect of elective home NPPV on exacerbation frequency in stable hypercapnic COPD remains to be determined.
The use of NPPV is a first line treatment of acute on chronic hypercapnic respiratory failure in COPD patients [8] . However, once acute hypercapnic respiratory failure is successfully managed and these patients are discharged, there is an 80 % re-hospitalization rate due to another acute exacerbation over the following year [53] . Furthermore, long-term survival in this patient cohort remains poor [54] . Three relatively small studies investigated the effect of home NPPV after acute hypercapnic respiratory failure successfully treated in COPD patients. One study showed that, compared to sham (continuous positive airway pressure) ventilation, NPPV significantly reduced the probability of recurrent acute hypercapnic respiratory failure [55] . Another study compared home NPPV versus standard therapy in chronic hypercapnic respiratory failure patients after acute exacerbation in order to prevent clinical worsening [56] . The authors demonstrated that the probability of clinical worsening was significantly lower in the group receiving home NPPV, with additional improvements observed in exercise capacity. The third, retrospective, study demonstrated better survival in COPD patients discharged after acute respiratory failure with home NPPV compared to those discharged without this form of therapy [57] .

Conclusion

There is conflicting evidence regarding the effect of NPPV on reducing health care utilization and mortality in acute on chronic respiratory failure due to COPD. We need to better assess when to initiate this therapy in patients with hypercapnia in this setting. Once stable hypercapnia is proven, NPPV may improve survival and health status. Therefore, despite recent studies adding some new data, the authors cannot recommend the widespread use of this therapeutic intervention after an episode of acute-on-chronic respiratory failure in COPD. There is simply not enough evidence to support it. Instead, this modality should be reserved for individual cases, treated in specialized centers experienced with NPPV for the treatment of stable hypercapnic COPD.

Introduction and rationale

Patients with chronic respiratory insufficiency requiring home mechanical ventilation (HMV) have a high, although underestimated prevalence in Europe [4] . Home mechanical ventilation requires patient and family cooperation, nevertheless clinical conditions, technology needs, lack of professional supervision, and acute exacerbations make its management a difficult task [4, 83] . Provision and maintenance is often carried out by external companies, without any accepted standardisation, and a regular feedback to the clinical centres is usually lacking [84] . The need to reduce healthcare costs has prompted the development of telemedicine for home assistance [85] . However, only few controlled studies evaluating its effectiveness are available so far. Identification and selection of HMV patients who may benefit from such tele-monitoring approach represent key factors [86] . There are real challenges when providing HMV, including patient and caregiver training, adequacy of respiratory care, and reimbursement.

Indications for TM in ventilator-dependent patients

Chronic respiratory failure [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] : In general, TM for chronic respiratory failure is feasible, tends to reduce hospitalisations, relapses, and urgent GP calls, helps facilitate titration of oxygen, and helps with changes in mechanical ventilation settings.

Equipment/technology available

The components of the technological dimension can be grouped into three sets, of variables: synchronicity, network design, and connectivity [85] : a) Synchronicity is used here to incorporate both timing and technology. b) Network design/configuration includes three modalities: Virtual Private Networks, the open internet, and social networks, in which information is posted and shared. c) Connectivity, wired and wireless, provides different levels of bandwidth and the attendant speed and resolution or quality of service.

Future considerations

Tele-monitoring could become be a key element (part of the 'total package') in the integrated management of the patient requiring home mechanical ventilation for chronic respiratory failure. Future outcome assessment could include: Ethics and palliative care in critically-ill respiratory patients (Michele Vitacca)

Key points

Due to the different evolution of end stage chronic respiratory diseases, and to the lack of physician's accuracy in judgment of prognosis in terminally ill patients, it may often be difficult to decide when to start palliative treatment [36, 127] . In these patients, the transition from the usual to the palliative and end of life care cannot be a step down, but should start simultaneously to the care, as soon as needed, and last until and after the death to ensure appropriate support to the family.

Mandatory ventilation

Patients receiving mandatory ventilation in the Intensive Care Unit (ICU) find themselves in an environment in which, understandably, the attending clinical team is focused on those with acute clinical issues. Their mobility is confined to the length of their ventilator tubing. Subsequent management outside the ICU will depend on the availability of resources such as a chronic assisted ventilator care unit, a long term acute care unit or a skilled nursing facility. The resource utilization is inversely related to the level of patient independence ( Fig. 1 ) [1] .

Prevalence of HMV

The prevalence of home ventilation is influenced by the increasing incidence of the underlying disorders, the increased knowledge of the healthcare providers (HCP) regarding the option of being safely ventilated outside of the ICU and the guidelines and recommendations of professional societies regarding LTMV [3] . It is also influenced by the attitudes and preferences of the patient and family as well as the availability of formal and informal (caregiver) support services. In Europe (Fig. 3) [4] the prevalence of HMV varies widely (France 17/100,000 to Poland 0.1 per 100,000), as does the distribution of diseases requiring ventilatory support (thoracic cage disorders, neuromuscular disorders and airway disorders). If the patient is unable to return home immediately, a chronic assisted ventilatory care (CAVC) unit will provide a safe, non-acute care environment with a rehabilitative focus, to optimize health related quality of life and promote autonomy. The CAVC unit requires a multidimensional continuum of services, by an interdisciplinary team trained both in ventilator management and rehabilitation. The preferred patient is medically stable, mentally alert, understands that ventilatory assistance is long term, is prepared to participate in comprehensive training and will relocate with appropriate supports. In order for a patient to return home, it must be safe and have the required utilities as well as trained care givers. The availability of home health care, technical support and organized followup is critical.

New indications for non-invasive ventilation (Nicolino Ambrosino) Key points

The use of non invasive ventilation (NIV) is an option in acute hypercapnic respiratory failure, cardiogenic pulmonary oedema, acute respiratory distress syndrome (ARDS), community-acquired pneumonia, and weaning failure Evidence supports NIV during complicated bronchoscopy, some cases of transoesophageal echocardiography, and in some interventional cardiology NIV can reduce the need for deep sedation or general anaesthesia NIV should be considered with caution in severe communicable airborne infections likely to progress to ARDS The role of assisted ventilation during exercise training is still controversial NIV should be applied under close monitoring, and endotracheal intubation should be promptly available in the case of failure. A trained team, careful patient selection and optimal choice of devices, can optimize outcome of NIV Non invasive ventilation (NIV) may be considered as one of the most important advances in respiratory medicine over the past 20 years, [8, 9] and is increasingly being utilized world-wide [10] . A PubMed search from January 1966 to March 2015 with the term "non invasive ventilation" defines NIV as "any form of ventilatory support applied without endotracheal intubation (ETI)". There is strong evidence (Level A) for the use of NIV to prevent ETI in acute on chronic respiratory failure, acute cardiogenic pulmonary oedema, and to facilitate extubation in patients with acute exacerbations of chronic obstructive pulmonary disease (COPD). Less evidence supports the use of NIV for patients with severe acute asthma exacerbations, post-operative or post-extubation acute respiratory failure (ARF), pneumonia, or acute respiratory distress syndrome (ARDS) [8, 9] . Nevertheless, many other potential applications have been proposed [12] . This review will focus on potential new indications for NIV. Although potentially risky, bronchoscopy may be required for some severely hypoxaemic patients [13] . In the past, the American Thoracic Society (ATS) did not recommend flexible bronchoscopy and bronchoalveolar lavage (BAL) in such conditions when supplemental oxygen cannot correct an arterial oxygen tension (PaO 2 ) at least to 75 mmHg or an arterial oxygen saturation (SaO 2 ) to 90 % [14] . On the other hand, non-use of bronchoscopy in these high risk patients may result in less effective, empiric treatment. Until recently, when bronchoscopy wws needed in hypoxaemic conditions, only ETI and mechanical ventilation were available to provide adequate ventilation and oxygenation. Unfortunately, invasive mechanical ventilation is associated with complications related to ETI, baro-or volutrauma, and the loss of airway defense mechanisms. NIV has the potential to avoid these complications while ensuring a similar level of ventilatory efficacy and control of hypoxemia.

Pro/Con long-term NPPV

There is limited evidence to support the provision of NPPV in the home environment after successful treatment of acute hypercapnic respiratory failure in COPD patients. However, those studies supporting this intervention had limitations, including small sample size, retrospective nature, and a lack of control group. Struik et al. [58] evaluated whether home NPPV after successfully treated acute respiratory failure reduces re-hospitalization and improves survival. The investigators randomized patients to home NPPV or standard treatment 48 h after "acute" ventilator support was terminated. The study failed to show a positive effect of home NPPV on time to readmission or death. This was not anticipated and stands in clear contrast to the smaller studies published before. Looking deeper into the study it can be seen that both groups had reductions in PaCO 2 over time. Therefore, one explanation of why this multicenter study was negative was the fact that patients were randomized too early: given the natural course of the disease, patients might have been randomized while they were still recovering from acute hypercapnia. Therefore, home NPPV might have been prescribed to patients not suffering from chronic hypercapnia. This study underscores the importance of carefully selecting patients for home NPPV.
Another study [59] was unable to show an improvement in 2-year survival, despite the demonstration of reductions in day-time PaCO 2 (while breathing oxygen), improvements in health status, and reductions in readmissions. Therefore, it appears unlikely that differences in 1 year survival between the Köhnlein study [52] and others [58, 59] are due only to "high inspiratory pressures" or simply reductions in PaCO 2 [52] . As a matter of fact, the control group of the Köhnlein study suffered from a high mortality rate, which may indicate that severity of disease rather than the correction of hypercapnia or the beneficial effect of "high inspiratory pressures" primarily drives survival in patients treated with NPPV. Furthermore, claim that chronic hypercapnia is associated to worse survival is questionable -at least in those patients receiving long-term oxygen therapy [43] . Furthermore there is growing evidence that mortality in COPD is influenced by several other factors, such as exercise capacity, comorbidities and inflammatory status [60] .