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Evaluation of duodenal endoscopic and histologic findings, including counts of forkhead box P3-positive regulatory T cells, in dogs with immunosuppressant-responsive enteropathy

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  • 1 Veterinary Teaching Hospital Mario Modenato, Department of Veterinary Sciences, University of Pisa, Pisa, Italy
  • | 2 Laboratorio di Analisi MyLav La Vallonea, Passirana di Rho (MI), Italy
  • | 3 Endovet Professional Association, Turin, Italy
  • | 4 Department of Veterinary Clinical Sciences, School of Veterinary Medicine, University of Bologna, Ozzano dell'Emilia (BO), Italy

Abstract

OBJECTIVE

To prospectively evaluate the clinical and prognostic importance of duodenal endoscopic and histologic findings, including duodenal mucosal counts of forkhead box P3-positive regulatory T cells (Foxp3+ Tregs), in dogs with immunosuppressant-responsive enteropathy (IRE).

ANIMALS

57 client-owned dogs with IRE.

PROCEDURES

The canine chronic enteropathy clinical activity index (CCECAI) was used to assess each dog when IRE was diagnosed (T0) and 1, 3, 6, and 12 months later. Dogs were grouped on the basis of clinical response (responder group vs nonresponder group) and 12-month long-term outcome (responded to treatment and did not relapse [good outcome group] vs did not respond to treatment or had relapsed [bad outcome group]). At T0, dogs underwent gastrointestinal endoscopy and endoscopic biopsy, with results for variables of duodenal endoscopic and histologic evaluations scored and compared across groups.

RESULTS

At T0, the overall median CCECAI score was 7; CCECAI score was not associated with clinical response or relapse. Dogs had significantly greater odds of being in the bad outcome group (vs the good outcome group) if they had a histologic score of 3 (OR, 3.5; 95% CI, 1.09 to 11.3). No differences in the counts of Foxp3+ Tregs were detected between groups.

CONCLUSIONS AND CLINICAL RELEVANCE

In dogs with IRE, results indicated that evaluation of Foxp3+ Tregs did not have prognostic value, whereas a duodenal histologic score of 3 could be a negative prognostic factor for response and relapse, and higher severity scores for intraepithelial lymphocytes and lamina propria lymphocytes and plasma cells in duodenal biopsy samples may be negatively associated with response.

Abstract

OBJECTIVE

To prospectively evaluate the clinical and prognostic importance of duodenal endoscopic and histologic findings, including duodenal mucosal counts of forkhead box P3-positive regulatory T cells (Foxp3+ Tregs), in dogs with immunosuppressant-responsive enteropathy (IRE).

ANIMALS

57 client-owned dogs with IRE.

PROCEDURES

The canine chronic enteropathy clinical activity index (CCECAI) was used to assess each dog when IRE was diagnosed (T0) and 1, 3, 6, and 12 months later. Dogs were grouped on the basis of clinical response (responder group vs nonresponder group) and 12-month long-term outcome (responded to treatment and did not relapse [good outcome group] vs did not respond to treatment or had relapsed [bad outcome group]). At T0, dogs underwent gastrointestinal endoscopy and endoscopic biopsy, with results for variables of duodenal endoscopic and histologic evaluations scored and compared across groups.

RESULTS

At T0, the overall median CCECAI score was 7; CCECAI score was not associated with clinical response or relapse. Dogs had significantly greater odds of being in the bad outcome group (vs the good outcome group) if they had a histologic score of 3 (OR, 3.5; 95% CI, 1.09 to 11.3). No differences in the counts of Foxp3+ Tregs were detected between groups.

CONCLUSIONS AND CLINICAL RELEVANCE

In dogs with IRE, results indicated that evaluation of Foxp3+ Tregs did not have prognostic value, whereas a duodenal histologic score of 3 could be a negative prognostic factor for response and relapse, and higher severity scores for intraepithelial lymphocytes and lamina propria lymphocytes and plasma cells in duodenal biopsy samples may be negatively associated with response.

Introduction

Immunosuppressant-responsive enteropathy in dogs is an idiopathic intestinal inflammatory condition for which there is histologic evidence of inflammation, diet and antimicrobial treatment trials have failed, and immunosuppressant (eg, corticosteroid) treatment is needed.1,2,3 The CIBDAI4 and CCECAI5 are widely accepted scoring systems used to assess the clinical severity of IBD and response to treatment in dogs.2 In addition, a study3 shows that CIBDAI and CCECAI scores for clinical signs in dogs correlate with histologic scores for endoscopic biopsy samples of duodenum and colon assessed by use of a simplified scoring system6 developed on the basis of the WSAVA histopathologic guidelines.1

On a cellular level, Foxp3+ Tregs are a subset of CD4+ T cells necessary for maintaining immunologic peripheral tolerance. The expression of Foxp3 is critical in the development and function of Foxp3+ Tregs, and investigators can distinguish Foxp3+ Tregs from other T-cell subpopulations with the use of antibodies against this nuclear marker. Studies7,8 show that the number of Foxp3+ Tregs identified in sections of duodenal mucosa from dogs with IBD did not correlate with CCECAI score or survival rate but was lower than the number of Foxp3+ Tregs in healthy dogs. Therefore, the aim of the present study was to prospectively evaluate the clinical and prognostic importance of duodenal endoscopic and histologic findings, including duodenal mucosal counts of Foxp3+ Treg, in dogs with IRE.

Materials and Methods

Animals

This multicentric, prospective study was conducted at 2 veterinary teaching hospitals (the University of Pisa and University of Bologna) and a private veterinary clinic (Centro Veterinario Specialistico, Rome) between March 2017 and March 2019. For inclusion, dogs had to have had intestinal disease diagnosed as IRE after the exclusion of extraintestinal diseases and infectious, parasitic, or other underlying causes of intestinal disease (eg, mechanical obstruction from intussusception, foreign body, or intestinal tumors). Dogs were excluded if they had food-responsive enteropathy (eg, responded to a hydrolyzed diet or a novel antigen diet) or antimicrobial-responsive enteropathy (ie, responded to treatment with tylosin [10 mg/kg, PO, q 12 h]). Signed informed consent was obtained from owners before dogs were enrolled, and the study was approved by the University of Pisa Institutional Committee for Animal Care (OPBA No. 31834/2017).

Clinical severity

The CCECAI5 was used to assess the clinical severity of IRE at diagnosis (T0) and 1 (T1), 3 (T3), 6 (T6), and 12 (T12) months after diagnosis for each dog. The CCECAI scoring system assessed 9 categories of clinical signs, including attitude and activity (on a scale from 0 [clinically normal] to 3 [severely decreased]), appetite (on a scale from 0 [clinically normal] to 3 [severely decreased]), vomiting (on a scale from 0 [< 1 event/wk] to 3 [> 3 events/wk]), fecal consistency (on a scale from 0 [clinically normal] to 3 [watery diarrhea]), fecal frequency (on a scale from 0 [< 2 events/d] to 3 [> 5 events/d]), weight loss (on a scale from 0 [none] to 3 [> 10%]), serum albumin concentration (on a scale from 0 [> 20 g/L] to 3 [< 12 g/L]), peripheral edema and ascites (on a scale from 0 [none] to 3 [severe]), and pruritus (on a scale from 0 [none] to 3 [pruritis regularly wakes the dog]). For each dog, their scores for the individual categories were summed to yield their CCECAI, which was used to categorize IRE severity as insignificant (CCECAI, 0 to 3), mild (CCECAI, 4 to 5), moderate (CCECAI, 6 to 8), severe (CCECAI, 9 to 11), or very severe (CCECAI, ≥ 12). Serum albumin concentration was evaluated at T0 and at every subsequent assessment time point. Dogs with a T0 serum albumin concentration < 2.7 g/dL were considered to have had PLE.9

Dogs received immunomodulatory treatment with prednisolone (0.5 to 1 mg/kg, PO, q 12 h) with or without cyclosporine (5 mg/kg, PO, q 24 h) at the discretion of the clinician. Treatment started at T0, after the CCECAI assessment.

After 1 month of treatment, dogs were reassessed and grouped on the basis of whether their T1 CCECAI had decreased by > 25% (responder group) versus ≤ 25% (nonresponder group), compared with their T0 CCECAI.10 Dogs that died ≤ 1 month after diagnosis of IRE were assigned to the nonresponder group.

Dogs were monitored for relapse for 12 months after T0. Dogs with a CCECAI ≤ 3 at T3, T6, or T12 were considered to have been in clinical remission at the given time point. For dogs with a CCECAI > 3 at any time point, the difference between their CCECAI at that time point and the immediately previous time point was calculated (eg, T3 CCECAI minus T1 CCECAI), and dogs with a difference in CCECAI ≥ 2 were considered to have relapsed. In addition, dogs that died between T1 and the end of the study were recorded as having relapsed at the next scheduled time point following their death (eg, a dog that died 4 months after diagnosis of IRE was considered to have relapsed at T6). For analyses of long-term outcome, dogs were grouped on the basis of whether they responded to treatment and did not relapse at any time point (good outcome group) versus did not respond to treatment or had relapsed at any time point (bad outcome group).

Endoscopic, histologic, and immunohistochemical assessments

At T0, endoscopic and histologic evaluations were performed in accordance with guidelines1,11 established by the WSAVA. Each dog underwent general anesthesia for endoscopic examination of the gastrointestinal tract and endoscopic biopsy of the stomach, duodenum, and colon, with 8 biopsy samples obtained from each region. For dogs in which the ileum was viewed, biopsy samples from the ileum were also obtained. Diagnosis of IRE in each dog was confirmed with results of histologic examinations of biopsy specimens. We chose to evaluate only duodenal biopsy findings for the statistical analysis. Duodenal biopsy specimens were fixed in neutral-buffered 10% formalin solution, routinely processed, and stained with H&E stain. The duodenal sections were then evaluated for histologic presence and severity of lesions, including CD, LD, MF, IEL, and LMLPL. Endoscopic and histologic results were recorded and scored on a 3-point scale (0 = clinically normal, 1 = mild severity, 2 = moderate severity, and 3 = marked severity) applied on the basis of established criteria.3,6,11

For immunohistochemical analyses, tissue sections with 3-μm slice thicknesses were placed on commercially available glass slidesa to which the tissue sections adhered electrostatically. Antigen retrieval was performed with an immunohistochemical slide processorb that used heat, and staining was performed with an automatic immunostainerc that used a kit with an avidin-horseradish peroxidase detection system. All reagents were dispensed automatically. The primary antibody was an anti-rat IgG antibodyd that was stored at 37°C for 2 hours before use. For each immunohistochemical analysis, a section of lymph node tissue was used as a standard for the positive control. A duodenal tissue section was used as a negative control and tested for Foxp3, and we replaced the primary antibody with antibody diluent.

Sections of duodenum were chosen for immunohistochemical analyses because previous studies7,8 show selective distribution of Foxp3+ Tregs in higher numbers in the duodenum, compared with the jejunum, ileum, or colon. Similarly, the lamina propria of villi and the lamina propria of crypts were considered as separate compartments.7

Cells with lymphoid morphology, distinct nuclear staining for Foxp3, and an inconspicuous, unstained cytoplasm were classified as Foxp3+ Tregs and quantified in areas with the highest numbers of Foxp3+ Tregs. The number of Foxp3+ Tregs/100 cells with lymphocyte morphology was recorded as the percentage of Foxp3+ Tregs. In addition, 5 consecutive hpfs (400×) were evaluated for each compartment (ie, the lamina propria of villi [Foxp3+ Tregs villi] and the lamina propria of crypts [Foxp3+ Tregs crypts]), and the counts were summed and expressed as a proportion of 500 cells for each compartment for each dog and as the number of Foxp3+ Tregs per μm2 area.

Statistical analysis

The d'Agostino-Pearson test was used to assess the normality of distribution for continuous variables (age, CCECAI at any assessment point, count of Foxp3+ Tregs villi, count of Foxp3+ Tregs crypts, and severity of CD, LD, MF, IEL, and LMLPL). The scores for CD, LD, MF, IEL, and LMLPL were summed for each dog, and this composite score was also considered as a continuous variable. Sex, sexual status, histologic and endoscopic scores, and presence (yes or no) of CD, LD, MF, IEL, and LMLPL were analyzed as categorical data.

Response rate was calculated at T1, and relapse rate was calculated at each subsequent assessment point (T3, T6, and T12). The Mann-Whitney U test or unpaired t test was used to compare results for continuous variables across groups, and the Fisher exact test was used to compare results for categorical variables across groups. When results of the Fisher exact tests indicated significant association between 2 variables, the OR was calculated.

Potential correlations among age; severity of CD, LD, MF, IEL, or LMLPL; composite sum of the severity scores for CD, LD, MF, IEL, and LMLPL; and T0 CCECAI were assessed with parametric or nonparametric correlation tests on the basis of data distribution. Analyses were performed with available software,e and values of P < 0.05 were considered significant.

Results

Animals

There were 57 client-owned dogs included in the study. Of these, 36 (63%) were males (1 castrated and 35 sexually intact) and 21 (37%) were females (10 spayed and 11 sexually intact). Median age was 4 years (range, 1 to 15 years). There were 18 mixed-breed dogs, 11 German Shepherd Dogs, 3 Maltese, 2 Doberman Pinschers, 2 Boxers, 2 Yorkshire Terriers, and 1 each of 19 other breeds.

Clinical severity

At T0, the median CCECAI was 7 (range, 2 to 15), with the clinical severity of IRE categorized as very severe in 14 (25%) dogs, severe in 11 (19%), moderate in 22 (39%), mild in 6 (11%), and insignificant in 4 (7%). Further, 27 dogs were determined to have had PLE, and their median CCECAI was 6 (range, 5 to 13).

One month later, results for T1 CCECAI, compared with T0 CCECAI, had reduced by > 25% for 49 of the 57 (86%) dogs (responder group) versus ≤ 25% for 8 (14%) dogs (nonresponder group; Table 1). Results for T0 CCECAI were not associated with response. Of the 49 dogs in the responder group, 18 (37%) had relapsed as determined from results at T3 (6/49 [12%]), T6 (7/43 [16%]), or T12 (5/36 [14%]).

Table 1

Findings for duodenal endoscopic and histologic evaluations and the presence or absence of PLE in 57 client-owned dogs with IRE grouped on the basis of whether their results for T1 CCECAI indicated clinical response (responder group [n = 49] vs nonresponder group [8]) and on the basis of long-term outcome (ie, responded to treatment and did not relapse [good outcome group; 31] vs did not respond to treatment or had relapsed [bad outcome group; 26]).

Clinical response at TILong-term outcome
VariableNo. (%) of dogs in the responder groupNo. (%) of dogs in the nonresponder groupP valueNo. (%) of dogs in the good outcome groupNo. (%) of dogs in the bad outcome groupP value
PLE0.13*0.18*
 Absent28 (57)2 (25)19 (61)11 (42)
 Present21 (43)6 (75)12 (39)15 (58)
Endoscopic score (scale of 0–3)0.11*0.14*
 222 (48)1 (13)15 (52)8 (32)
 324 (52)7 (88)14 (48)17 (68)
Histologic score (scale of 0–3)0.03*0.03*
 234 (72)2 (29)23 (79)13 (52)
 313 (28)5 (71)6 (21)12 (48)
CD0.90*0.60*
 Absent16 (33)3 (38)9 (29)10 (38)
 Present33 (67)5 (63)22 (71)16 (62)
CD score (scale of 0–3)0.25§0.60§
 016 (33)3 (38)9 (29)6 (23)
 117 (35)1 (13)10 (32)6 (23)
 215 (31)2 (25)11 (35)3 (12)
 31 (2)2 (25)1 (3)0 (0)
LD0.12*0.80*
 Absent23 (47)1 (13)14 (45)10 (38)
 Present26 (53)7 (88)17 (55)16 (62)
LD score (scale of 0–3)0.02§0.17§
 023 (47)1 (13)14 (45)10 (38)
 114 (29)1 (13)12 (39)3 (12)
 210 (20)6 (75)3 (10)13 (50)
 32 (4)0 (0)2 (6)0 (0)
MF0.70*0.80*
 Absent31 (63)4 (50)20 (65)15 (58)
 Present18 (37)4 (50)11 (35)11 (42)
MF score (scale of 0–3)0.60§0.29§
 031 (63)4 (50)20 (65)15 (58)
 111 (22)2 (25)8 (26)5 (19)
 24 (8)2 (25)2 (6)4 (15)
 33 (6)0 (0)1 (3)2 (8)
IEL0.25*0.30*
 Absent24 (49)1 (13)16 (52)9 (35)
 Present25 (51)7 (88)15 (48)17 (65)
IEL score (scale of 0–3)0.02§0.07§
 024 (49)1 (13)16 (52)9 (35)
 122 (45)5 (63)14 (45)13 (50)
 23 (6)1 (13)1 (3)3 (12)
 30 (0)1 (13)0 (0)1 (4)
LMLPL
 Absent0 (0)0 (0)0 (0)0 (0)
 Present49 (100)8 (100)31 (100)26 (100)
LMLPL score (scale of 0–3)0.03§0.09§
 00 (0)0 (0)0 (0)0 (0)
 13 (6)0 (0)3 (10)0 (0)
 234 (69)3 (38)21 (68)16 (62)
 312 (24)5 (63)7 (23)10 (38)
SUM5 (2–13)8 (5–10)0.002§5 (2–13)6 (3–10)0.04§
Foxp3+ Tregs villi (cells/μm2)13 (1–68)9 (3–26)0.18§13 (1–68)11 (1–54)0.50§
Foxp3+ Tregs crypts (cells/μm2)1 (0–37)0 (0–3)0.25§1 (0–37)2 (0–20)0.61§

Data are reported as the number (percentage) of dogs, except where indicated otherwise.

P value obtained with the Fisher exact test.

Three dogs had an endoscopic score of 1 (mild severity) and were not included in the analysis of this variable because of the low number.

Three dogs had a histologic score of 1 (mild severity) and were not included in the analysis of this variable because of the low number.

P value obtained with an unpaired t test or Mann-Whitney U test.

Data reported as median and range.

— = Not applicable. SUM = Composite sum of the severity scores for the variables of CD, LD, MF, IEL, and LMLPL.

Overall, of the 57 dogs, 31 (54%) were in the good outcome group (responded to treatment and did not relapse), and 26 (46%) were in the bad outcome group (relapsed or did not respond to treatment). The proportion of dogs with PLE did not meaningfully differ between the good outcome group and the bad outcome group Table 1). In addition, sex and sexual status did not meaningfully differ for dogs that did versus did not relapse, and results for the CCECAI were not associated with relapse at any time point. Furthermore, the presence of PLE was not associated with response or relapse in the long-term (12 months) outcome.

Endoscopic, histologic, and immunohistochemical results

The endoscopic score for disease severity was 1 (mild) in 3 of the 57 (5%) dogs, 2 (moderate) in 23 (40%) dogs, and 3 (marked) in 31 (54%) dogs. The endoscopic score was not associated with whether dogs were in the responder group (vs nonresponder group; P = 0.11) or in the good outcome group (vs bad outcome group; P = 0.14; Table 1).

The duodenal histologic score for disease severity was 1 (mild) in 3 of the 57 (5%) dogs, 2 (moderate) in 36 (63%) dogs, and 3 (marked) in 18 (32%) dogs Table 1). Results for dogs with a histologic score of 1, endoscopic score of 0, or both were excluded from statistical analyses of these 2 variables because of the low number of affected dogs (n = 5). Dogs with a histologic score of 2 (moderate severity) had significantly (P = 0.03; OR, 6.5; 95% CI, 1.1 to 38) greater odds of being in the responder group than did dogs with a histologic score of 3 (marked severity). Further, dogs with PLE (vs without PLE) at T0 had significantly (P = 0.034; OR, 4.2; 95% CI, 1.01 to 14.02) greater odds of having a histologic score of 3 (marked severity).

Histologic examination of duodenal biopsy specimens revealed that there were LMLPL in all 57 dogs, CD in 38 (67%) dogs, IELs in 32 (56%) dogs, LD in 33 (58%) dogs, and MF in 22 (39%) dogs. The median result for the composite sum of the severity scores for CD, LD, MF, IEL, and LMLPL combined was significantly higher for dogs in the nonresponder group (median, 8) versus the responder group (median, 5; P = 0.002) and for the bad outcome group (median, 6) versus the good outcome group (median, 5; P = 0.049). Furthermore, the median severity scores for LD, IEL, and LMLPL separately were significantly (P = 0.02, 0.02, and 0.03, respectively) higher for dogs in the nonresponder group (median, 2, 1, and 3, respectively), compared with those in the responder group (median, 1, 1, and 2, respectively). Dogs with a histologic score of 3 had significantly (P = 0.03; OR, 3.5; 95% CI, 1.07 to 11.7) greater odds of being in the bad outcome group (vs the good outcome group).

The median Foxp3+ Tregs villi count was 3 Foxp3+ Tregs/μm2 (range, 0 to 37 Foxp3+ Tregs/μm2), and the median FoxP3+ Tregs crypts count was 12 Foxp3+ Tregs/μm2 (range, 1 to 68 Foxp3+ Tregs/μm2). Neither count was associated with response, relapse, T0 CCECAI results, or endoscopic or histologic scores.

When results were analyzed for correlations between variables, the severity of CD correlated positively and significantly (P < 0.001; r = 0.45) with the severity of LMLPL. Additionally, the severity of LD positively and significantly correlated with the severities of MF (P = 0.004; r = 0.28) and LMLPL (P = 0.049; r = 0.26).

Discussion

Immunosuppressant-responsive enteropathy in dogs is a chronic intestinal disease that waxes and wanes and is often difficult to manage,12 and previous studies5,9,13 of CE, PLE, and IRE show inconsistent findings regarding prognostic factors useful in monitoring the clinical course of disease. In contrast to a previous study3 that used the CCECAI and CIBDAI for scoring clinical disease, we used only the CCECAI in the present study. We believe that the use of the CCECAI can better allow clinicians to characterize the clinical course of IRE in dogs because the CCECAI incorporates evaluation of serum albumin concentration.

Our findings that only 14% (8/57) of the dogs in the present study were nonresponders and that 37% (18/49) of the responders relapsed were consistent with findings in a previous study14 that shows 13% (10/80) of dogs with IBD did not respond to treatment. However, unlike the present study, that previous study14 did not use clinical scores to evaluate response and classified dogs during the follow-up period as having been in remission, having had intermittent abnormal clinical signs, or having uncontrolled disease. In addition, our results further supported findings from a recent prospective study15 on CE that included 2 dogs with IRE and showed that these dogs were considered to have had good outcomes (long-term follow-up period, 33.5 months).

In dogs of the present study, results for T0 CCECAI were not associated with clinical response at T1 nor with relapse at T3, T6, or T12. Our findings agreed with results of a previous study16 that shows the results for CCECAI at diagnosis did not differ between dogs that died from PLE-related causes within 4 months after diagnosis (n = 22) and dogs alive 1 year after diagnosis or that died of non–PLE-related causes (21). In contrast, a different study13 shows that a CCECAI > 5 at 1 month after diagnosis of PLE in dogs was the best predictor of a poor outcome. This difference may have been because the previous study13 only included dogs with PLE, whereas not all dogs in our study had PLE. Despite this difference, the mean concentration of serum albumin was similar for dogs that died versus dogs that were alive within 6 months after diagnosis of PLE in the previous study,13 consistent with our findings that serum concentration of albumin did not differ substantially for dogs in the good outcome group versus the bad outcome group.

Our results indicated that dogs with a histologic score of 3, high severity scores for LMLPL or IEL, or a high composite sum of the severity scores for CD, LD, MF, IEL, and LMLPL had greater odds of not responding to treatment in the present study. Although previous studies4,5,17,18 fail to show a convincing association between mucosal histologic changes and clinical disease severity either at diagnosis or in response to various treatments, a recent study3 indicates a significant relationship between histologic changes (CD, LMLPL, neutrophilic infiltrate, MF, LD, and villus stunting) and clinical score only at presentation. In contrast, our results indicated that morphological alterations identified on histologic examination of biopsy specimens were not associated with results for CCECAI at diagnosis of IRE but were associated with clinical response. This difference could have resulted from differences in study design in that the previous study3 was retrospective with clinical scores extrapolated from medical records of 2 different veterinary facilities, whereas the present study was prospective with the clinical scores obtained from direct patient assessment at each follow-up examination.

Findings in the present study indicated that dogs with PLE had higher histologic scores than did dogs without PLE; however, we did not detect associations between PLE and response, relapse, or specific histologic results, other than overall score. In contrast, a previous study19 shows that dogs with CE and hypoalbuminemia are more likely to have various intestinal histologic alterations (eg, villous stunting, epithelial injury, CD, LD, IEL, and lamina propria neutrophils) than are dogs with CE but not hypoalbuminemia. However, that study19 included dogs with CE, whereas the present study only included dogs with IRE. In addition, we assigned scores for histologic alterations, whereas the previous study19 evaluated only whether various histopathologic lesions were present or absent.

Our findings indicated a significant association between histologic score and relapse in dogs of the present study. To our knowledge, this is the first investigation into this particular aspect of IRE in dogs. Previous studies show associations between histologic changes and clinical scores3 or hypoalbuminemia19 but not data regarding clinical follow-up evaluations to assess characteristics of dogs that relapsed.

Results of a study20 of dogs with lymphocytic-plasmacytic enteropathy indicate that dogs with high counts of IELs have higher (worse) CIBDAIs and that hypoalbuminemia correlates with high IEL counts. Intraepithelial lymphocytes are a component of the first-line defense in the gastrointestinal immune system and have pro− and anti-inflammatory roles.21 In the present study, the severity of IEL infiltration was only associated with response at T1 but not with the CCECAI nor the serum albumin concentration at T0. This difference between our findings and those of the previous study20 could have been attributable again to the fact that we assigned scores for histologic alterations, whereas the previous study considered whether histologic lesions of interest were absent or present.

Recent studies7,8 show that dogs with IRE have fewer Foxp3+ Tregs in their duodenal mucosa than do healthy dogs and that the number of Foxp3+ Tregs did not relate to the WSAVA histopathologic score or patient prognosis for dogs with IRE. Similarly, our results indicated that Foxp3 expression was not associated with results for CCECAI, response, relapse, or endoscopic or histologic scores. Thus, in our opinion, the evaluation of Foxp3+ Tregs does not seem to have prognostic value for dogs with IRE.

Interestingly, a study18 of 20 dogs with IBD shows higher counts of Foxp3+ Tregs in dogs treated with (n = 10) versus without (10) probiotics for 1 month. However, in the present study, repeating endoscopic and histologic examination at T1 was not feasible, mainly for ethical reasons. It would be interesting to evaluate histologic and immunohistochemical findings relative to medical management during the follow-up period.

The present study had several limitations. We used duodenal biopsy specimens alone for histologic and immunohistochemical evaluations. In addition, although all dogs in the present study underwent immunosuppressant treatment, specific treatments (eg, drugs, dosages, and durations) varied and could have influenced outcomes. In addition, differences among dogs could have influenced outcomes.

To our knowledge, the present study was the first prospective study to evaluate potential associations between variables for histologic and immunohistochemical findings and variables for clinical response and relapse during a 12-month follow-up period in dogs with IRE. Our findings indicated that a histologic score of 3 and a high composite sum of the severity scores for CD, LD, MF, IEL, and LMLPL (ie, ≥ 6) could be negative prognostic factors for dogs with IRE in that these factors were associated with nonresponse and relapse in dogs of the present study. Also, higher severity scores for IEL and LMLPL could have negative prognostic value for response to treatment in dogs with IRE because dogs in the nonresponder group had significantly higher scores for IEL, LMLPL, or both, compared with dogs in the responder group. Additional research is warranted.

Acknowledgments

No third-party funding or support was received in association with the present study or the writing or publication of the manuscript. The authors declare that there were no conflicts of interest.

Presented as a poster at the 2019 European College of Veterinary Internal Medicine Congress, Milan, Italy, September 2019.

The authors thank Dr. Maria Massaro from the Laboratorio di Analisi MyLav La Vallonea, Passirana di Rho, Italy, for assistance with histologic evaluations.

Abbreviations

CCECAI

Canine chronic enteropathy clinical activity index

CD

Crypt distension

CE

Chronic enteropathy

CIBDAI

Canine inflammatory bowel disease activity index

Foxp3

Forkhead box P3 transcription factor

IBD

Inflammatory bowel disease

IEL

Intraepithelial lymphocyte

IRE

Immunosuppressant-responsive enteropathy

LD

Lacteal dilation

LMLPL

Lamina propria lymphocytes and plasma cells

MF

Mucosal fibrosis

PLE

Protein-losing enteropathy

Treg

Regulatory T cell

WSAVA

World Small Animal Veterinary Association

Footnotes

a.

Superfrost Plus, Fisher Scientific Italia, Rodano, Italy.

b.

Lab Vision PT Module, Thermo Scientific, Fremont, Calif.

c.

Lab Vision Autostainer 480S, Thermo Scientific, Fremont, Calif.

d.

Monoclonal Antibody Clone FJK-16s, Thermo Fisher-Invitrogen-eBioscience, San Diego, Calif.

e.

GraphPad Prism 6, GraphPad Software, La Jolla, Calif.

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Contributor Notes

Address correspondence to Dr. Pierini (pierini.alessio2004@gmail.com).