Inflammatory bowel disease is a term referring to a clinical condition characterized by recurrent/persistent gastrointestinal signs, which cannot be diagnosed only through histological findings, being an elimination diagnosis and needing therefore the previous exclusion of all other possible causes of gastrointestinal signs. The aim of the present study was to compare the expression of different cytokines on endoscopic biopsy samples of the small and large intestine of cats suffering from inflammatory bowel disease and healthy controls, by immunohistochemistry evaluation. Nine cats referred for chronic gastrointestinal signs to the Gastroenterology Service, Teaching Hospital, Faculty of Veterinary Sciences, University of Buenos Aires, were included. After being administered with antiparasitic drugs and after running complete laboratory exams, abdominal ultrasonography, etc., upper and lower gastrointestinal endoscopy with biopsy samplings for histopathology and immunohistochemistry was then performed. Controls were represented by archived samples from healthy cats (University of Camerino, Italy, Veterinary Pathology Unit Archive). On biopsy samples, the immunohistochemistry expression of the following antibodies was evaluated: TGF-
Inflammatory bowel disease (IBD) is a term referred in both dogs and cats to a clinical condition characterized by persistent or recurrent gastrointestinal (GI) signs (e.g., vomiting and diarrhea), chronic evolution (3-4 weeks), and histologic evidence of inflammation without any answer to empirical treatments, such as diet changes, or evidence of any other possible known cause [
For the abovementioned reasons, the diagnosis of IBD still remain a difficult achievement and the diagnostic path has been an object of interest worldwide in the last decades [
The aim of the present study was to compare, by immunohistochemistry (IHC) evaluation, the expression of different cytokines on endoscopic biopsy samples of the small and large intestine of cats diagnosed with inflammatory bowel disease and healthy controls, looking for possible markers for monitoring IBD.
The present one is a prospective study including nine cats referred for chronic gastrointestinal signs to the Gastroenterology Service, Teaching Hospital, Faculty of Veterinary Sciences, University of Buenos Aires, and diagnosed with IBD. All patients presented vomiting and/or diarrhea lasting for more than 3 weeks. All patients underwent fecal exams on three consecutive days and were treated with fenbendazole 50 mg/kg every 24 hrs,
Biopsies were preserved in 10% buffered formalin, and after 24 h of fixation, they were dehydrated through a complete alcohol series, then subsequently clarified in xylene and embedded in paraffin using an automatic processor. Paraffin blocks were cut in 3-4-micron-thick sections, then placed on slides and used for morphological evaluation (by hematoxylin and eosin stain) and immunohistochemistry. In all cats included in the study, an inflammatory infiltrate with morphological changes was found. All cats received immunosuppressants (i.e., prednisolone 1 mg/kg every 12 hours, PO, for one week, then tapered to 1 mg/kg every 24 hours, PO, for two-three weeks followed by 0.5 mg/kg every 24 hrs, PO, for two-three weeks, therefore continuing with the minimum effective dose) and hypoallergenic diet that was maintained for the whole study. All cats diminished clinical signs after treatment. Only the 9 cats complying with inclusion criteria and whose owners authorized inclusion were enrolled in the study.
Exclusion criterion was the necessity of performing a full-thickness biopsy of the GI tract, as occurred in patients with ultrasonographic findings of wall alteration involving all layers or submucosal layers and the presence of other causes justifying clinical signs of haemopathogens.
To obtain control tissue from healthy cats for this analysis, archived formalin-fixed and paraffin-embedded GI tissue samples from three cats (two males and one female) with no clinical signs of intestinal disease were retrieved from the University of Camerino, Italy, Veterinary Pathology Unit Archives. These samples had been obtained immediately postmortem from cats that were presented for euthanasia (euthanized cats (EC)) for severe injury due to car accidents. Ages ranged from 5 years to 13 years (mean 9.2 years), and histopathological examination of GI biopsies was normal in all cases.
The project was approved by the CICUAL (
Statistical analysis was performed with GraphPad 5 program. For quantitative variable evaluation, the nonparametric Mann-Whitney test was used, while for the correlation between two quantitative variables, the Spearman test was used. Significant differences were those presenting
This technique was used to evaluate the expression of the following antibodies (Abs): TGF-
For antigenic highlighting, the slides were preincubated with different retrieval solutions according to the suggestions indicated by the Ab producer: citrate buffer (pH 6.0) for TGF-
Immunoreaction with streptavidin-immunoperoxidase (streptavidin-immunoperoxidase, Black & Decker, Towson, MD, USA) was visualized with 3,3-diaminobenzidine substrate (3,3
To perform the intestinal score of CD3+ T lymphocytes, FoxP3+, and Th17+ cells and to score all gastrointestinal cells producing TGF-
The quantity of CD3+, FoxP3+, and Th17+ T cells and of cells producing TGF-
Color images were analyzed with Image-Pro Plus software (Media Cybernetics). Five transversal sections for each cat were recorded as well as the whole amount of immunoreactive T lymphocytes.
Then, for each cat, the total cellular amount, for cellular typology and for biopsy of the entire area, was calculated as the sum of all the examined areas of the specimen, in the consecutive 5 sections of a single slide. The results are expressed as immunohistochemistry- (IHC-) positive cells per 62,500
Considering clinical history, results of the complete work-up, histopathology, and response to anti-inflammatory treatment, nine cats were included in the study. Four were European common cats, three Siamese cats, and two Oriental cats; seven were males and two were females. At the time of biopsy samplings, one cat was 4 years old, three were 6, one was 8, one was 13, and three were 14 years old. Patients presented different clinical signs; the most frequent were vomiting and/or diarrhea, followed by hematochezia and presence of mucus; only one cat had weight loss. Four patients had a single clinical sign, four a combination of two signs, while the remaining one, three clinical signs. During the follow-up evaluation, signs of malnutrition, coat opacity, and worsening of the general condition were noticed.
No patients presented leukocytosis or alteration of the leukocyte formula; only one presented anemia (29% hematocrit). Six cats did not present any alteration of the blood chemistry profile; the remaining three showed variable alterations of hepatic enzymes, however not indicative of the disease. Clinical biochemistry results are presented in Table
Clinical biochemistry results in the nine cats enrolled for the study: abnormal results are highlighted in bold.
Alb | Total prot | Urea | Creatinine | ALT | AST | ALP | Phosphorus | |
---|---|---|---|---|---|---|---|---|
Cat 1 | 2.6 | 30 | 1.47 | 39 | 97 | 3.9 | ||
Cat 2 | 53 | 1.7 | 73 | 50 | ||||
Cat 3 | 2.5 | 55 | 1.56 | 66 | 4.4 | |||
Cat 4 | 2.5 | 7.5 | 60 | 1.84 | 52 | 23 | 93 | 3.8 |
Cat 5 | 2.5 | 7.5 | 38 | 1.08 | 69 | 27 | 70 | 4.3 |
Cat 6 | 2.5 | 7.4 | 64 | 1.71 | 50 | 39 | 33 | 3.7 |
Cat 7 | 2.7 | 8 | 32 | 1.14 | 67 | 30 | 67 | 4.5 |
Cat 8 | 2.8 | 7.7 | 45 | 1.25 | 70 | 62 | 75 | 4.1 |
Cat 9 | 2.5 | 7.3 | 52 | 1.6 | 36 | 35 | 51 | 3.8 |
Alb: albumin (mg/dl); Total prot: total protein (mg/dl); urea (mg/dl); creatinine (mg/dl); ALT: alanine aminotransferase (UI/l); AST: aspartate aminotransferase (mg/dl); ALP: alkaline phosphatase (mg/dl); phosphorus (mg/dl).
All immunohistochemical evaluations, in healthy controls and IBD cats, were performed both in duodenal and colonic samples, and relative results are reported in Tables
Duodenal immunohistochemistry—controls vs. diseased cats: distribution of each marker investigated in the duodenum of both healthy (
IHC | Controls ( | Diseased cats ( |
---|---|---|
TGF- | 6 (2-24) | 5 (0-32) |
TNF- | 0 (0-5) | 12 (0-321) |
IL-1 | 0 (0-5) | 11 (0-215) |
IL-12 | 0 (0-2) | 19 (1-241) |
IL-10 | 3 (0-11) | 3 (0-51) |
Th-17+ | 0 (0-0) | 8 (1-121)N/C |
CD3+ | 5 (0-11) | 124.5 (26-507) |
FOXP3+ | 1 (0-4) | 2 (0-9) |
Colonic immunohistochemistry—controls vs. diseased cats: distribution of each marker investigated in the colon of both healthy (
IHC | Control ( | Diseased cats ( |
---|---|---|
TGF- | 4 (1-19) | 3 (0-38) |
TNF- | 1 (0-7) | 15 (0-371) |
IL-1 | 1 (0-7) | 9 (0-246) |
IL-12 | 1 (0-4) | 14 (2-324) |
IL-10 | 2 (0-13) | 7 (0-33) |
Th-17+ | 0 (0-0) | 7 (0-310)N/C |
CD3+ | 2 (0-9) | 161 (32-495) |
FOXP3+ | 1 (0-2) | 1 (0-9) |
Correlation among different IHC markers investigated in the duodenum: the table reports the “
IHC | TGF- | TNF- | IL-1 | IL-12 | IL-10 | Th-17+ | CD3+ | FOXP3+ |
---|---|---|---|---|---|---|---|---|
TGF- | — | — | — | — | — | — | — | — |
TNF- | -0.47 | — | — | — | — | — | — | — |
IL-1 | -0.3 | 0.78 | — | — | — | — | — | — |
IL-12 | -0.32 | 0.79 | 0.84 | — | — | — | — | — |
IL-10 | 0.52 | -0.33 | -0.11 | -0.07 | — | — | — | — |
Th-17+ | -0.6 | 0.75 | 0.67 | 0.73 | -0.4 | — | — | — |
CD3+ | -0.26 | 0.67 | 0.71 | 0.79 | 0.79 | 0.69 | — | — |
FOXP3+ | 0.3 | 0.23 | 0.24 | 0.21 | 0.21 | -0.13 | -0.13 | — |
In the present study, European common cat represented the majority (4 out of 9 cats) of our patients. The possible higher presence of this disease in such group of animals had previously been noticed in patients of Teaching Hospital, Faculty of Veterinary Sciences, University of Buenos Aires [
Clinically, always in accordance with previous reports [
The distribution of the different interleukins investigated furnished interesting data regarding their duodenal and colonic location in cats with IBD. Interleukins and mediators’ profiles have been more deeply investigated in dogs, and therefore, in literature, only little information is reported for cats. In studies performed in both dogs and cats, techniques usually used are fluorescence
Interestingly, other papers described increases of mRNA for IL-10 and TGF-
The missing mRNA analysis might thereby be seen as a limitation of our work, but it is known that mRNA levels do not necessarily correlate with protein levels. Particularly, IL-10 is known to underlie posttranscriptional regulation processes articulately influencing later protein levels [
The present manuscript showed a highly significant positive correlation among proinflammatory cytokines, accordingly to what was previously described by other authors. A moderate positive correlation was also evidenced between proinflammatory cytokines and lymphocytes and between IL-10 and TGF-
In the present study, both proinflammatory cytokines (TNF-
The data used to support the findings of this study are included within the article.
The authors declare no conflict of interest regarding the present manuscript.
The present study was realized with the subsidy of the University of Buenos Aires, code UBACyT: 20020100100002.