Flow cytometric analysis of canine colonic mucosal lymphocytes from endoscopically obtained biopsy specimens

OBJECTIVE: To validate use of canine colonic biopsy specimens obtained via endoscopy as a source of mucosal lymphocytes (ML) for flow cytometric analysis. SAMPLE POPULATION: Mucosal biopsy specimens from 10 adult dogs. PROCEDURE: Mucosal lymphocyte subsets obtained from excised colon were compared with ML subsets obtained from biopsy specimens obtained by use of an endoscopic forceps (6 dogs). Endoscopic colonic biopsy specimens from 4 other dogs were used to define whether obtained ML were predominantly of intraepithelial or lamina propria origin. Mucosal lymphocytes were isolated and labeled, using commercially available monoclonal antibodies directed against canine cell surface antigens. Lymphocyte subsets (cytotoxic or helper T cells; B cells) were determined by use of flow cytometric analysis. RESULTS: A large number of viable ML was obtained after dissociation of the colonic epithelium from excised colon (45.5 + 21.5 X 10(6)) and endoscopic (7.2+/-3.4 X 10(6)) biopsy specimens. Lymphocyte subsets obtained with both methods were identical for each dog and consisted predominantly of intraepithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded a large number of viable lymphocytes from the lamina propria (56.7+/-20.4 X 10(6)), but collagenase digestion of endoscopic biopsy specimens was less rewarding. CONCLUSION AND CLINICAL RELEVANCE: A representative sample of viable intraepithelial ML is obtainable from endoscopic biopsy specimens. Flow cytometric analysis, a minimally invasive technique, can be used to study ML of client-owned animals.     

Flow cytometric analysis of ovine peripheral blood lymphocytes.

Leukocyte suspensions were prepared from the peripheral blood of 12 sheep three times at two month intervals beginning at 12 months of age. Monoclonal antibodies and flow cytometric analysis were used to characterize the cells. There were no significant differences over time therefore the data from the three time intervals were pooled. The mean percentages and ranges (minimum to maximum) of the major lymphocyte subtypes were: B-cells (29.6%, 11-50), gamma delta T-cells (36.6%, 22-68), CD4+ T-cells (14.1%, 8-22) and CD8+ T-cells (12.0%, 4-22). Lymphocyte subtype percentages appeared less variable within than between individuals. Two populations of B-cells were noted; one population had more cytoplasm and light scatter characteristics similar to monocytes while a second population of B-cells was more typical of small lymphocytes. The nature of the large B-cells requires further study.     

Flow cytometric analysis of canine umbilical cord blood lymphocytes

Lymphocyte subsets in canine umbilical cord blood were flow cytometrically analyzed and compared with those of the dams' peripheral blood. The proportion of CD3+ T lymphocytes, CD21+CD3- B lymphocytes, and CD3-CD21- non-T non-B lymphocytes in umbilical cord blood was 52.9%, 30.4%, and 16.7%, respectively. T lymphocyte/B lymphocyte ratio was significantly lower in the umbilical cord blood than in the dams' peripheral blood (2.1 +/- 1.4 versus 11.0 +/- 8.1, P < 0.001). In contrast, CD4+ lymphocyte/CD8+ lymphocyte ratio was significantly higher in the umbilical cord blood than in the dams' peripheral blood (7.6 +/- 2.2 versus 1.8 +/- 0.6, P<0.001). These findings clarified the phenotypic characters of canine umbilical cord blood lymphocytes.     

Flow cytometric analysis of lectin-binding to canine blood lymphocytes

The cell surface glycoconjugates of blood lymphocytes from 19 dogs with and without neoplastic disease were quantitated using flow cytofluorometric analysis of the binding characteristics of 3 lectins, namely, wheat germ agglutinin, concanavalin-A, and Lens culinaris agglutinin. The specificity of lectin binding was determined using competitive monosaccharide inhibitors. The results show enhanced binding of concanavalin-A to blood lymphocytes from dogs with lymphosarcoma relative to healthy dogs, or those with a variety of neoplastic and nonneoplastic diseases.     

Concanavalin A-induced suppressor cell activity in intestinal mucosal leukocytes obtained from healthy cows

Leukocytes isolated from the intraepithelium, lamina propria, and aggregated lymphoid follicles (ALF) of the bovine small intestinal mucosa were activated by concanavalin A (conA) to generate suppressor activity against the proliferative response of autologous and allogeneic leukocytes to conA, phytohemagglutinin, and pokeweed mitogen. Freshly obtained intraepithelium, lamina propria, and ALF leukocytes, preincubated with 25 to 50 micrograms/ml of conA for 24 to 48 hours, were able to inhibit the mitogenic responses of autologous and allogeneic lymphocytes when cocultured at a ratio greater than or equal to 1:1 (suppressor cells to responder cells). Depletion of adherent cells (monocytes/macrophages) by sequential plastic and gel adherence completely abrogated the conA-induced suppressor activity of all 3 leukocytes populations. However, reconstitution with autologous or allogeneic monocytes/macrophages during the induction phase restored the inducible suppressor activity. Addition of recombinant human interleukin-2 at a concentration as low as 5 U/ml during the responder phase enabled the responder population to recover the response apparently impaired by the conA-treated ALF leukocytes. At a concentration of 10 U/ml, human interleukin-2 was not only able to restore the responder population's response to phytohemagglutinin stimulation, but highly enhanced its proliferative ability. Although quantitative variations were observed between different cell populations and cells obtained from individual cows, the extent and general pattern of the inducible suppressor activity were similar in cells from cows studied.     

Flow cytometric detection of activated platelets in the dog

Background: Platelet activation appears to play a role in a variety of canine thrombotic disorders. At present, tests for the detection of activated platelets are not used routinely in veterinary clinical laboratories. Objective: The purpose of this study was to develop a clinically applicable method to detect activated canine platelets. Methods: A flow cytometric assay was developed to detect activated platelets, platelet aggregates, and platelet microparticles in the dog. Blood was collected from healthy dogs using EDTA or sodium citrate as the anticoagulant, and platelet‐rich plasma was prepared. Platelets were activated by adding phorbol myristate acetate. In some experiments, platelets were fixed by incubation with 0.5% paraformaldehyde. In other experiments, platelets were stored for 4 or 24 hours at 4°C before analysis. Activated platelets were detected by measuring surface expression of P‐selectin and by determining the percentages of platelet aggregates and microparticles using forward‐angle vs side‐angle light scatter plots. Results were analyzed by using 2‐way ANOVA and the SchefféF‐test. Results: Platelets collected in EDTA had minimal expression of P‐selectin, whereas platelets collected in sodium citrate had greater median fluorescence intensity. Fixation with 0.5% paraformaldehyde before labeling platelets with anti‐P‐selectin did not affect antibody binding or the percentages of platelet aggregates and microparticles. Storage of platelet‐rich plasma at 4°C for 4 hours did not affect antibody binding or the percentages of platelet aggregates or microparticles. Activation of platelets ex vivo by addition of 10 ng/mL phorbol myristate acetate resulted in a large increase in expression of P‐selectin but only slight increases in platelet aggregates and microparticles. Conclusion: Determination of platelet P‐selectin expression and percentages of platelet aggregates and platelet microparticles may provide a clinically applicable means for detection of activated platelets in dogs. The capacity to use EDTA‐anticoagulated blood samples and to fix platelets for evaluation at a later time makes the test attractive as a routine diagnostic tool.     

Effects of medetomidine on intestinal and colonic motility in the dog

The motor responses of the jejunum and colon to stimulation of α₂-adrenoceptors by medetomidine and clonidine were investigated in four dogs. In fasting dogs, medetomidine, at a dose rate of 30 μg/kg i.v., disrupted the migrating myoelectric complex (MMC) pattern of the small intestine for about 2 h. Similar, but shorter-lasting effects were also induced by clonidine (30 μg/kg i.v.) on the jejunum. The administration of α₂-agonists inhibited colonic motility in fasting dogs, although medetomidine-induced inhibition was preceded by a short period of increased muscle tone. All these effects were reversed by the α₂-antagonists atipamezole (0.15 mg/kg i.v.) and yohimbine (0.20 mg/kg i.v.). In fed dogs, medetomidine (30 μg/kg i.v.) induced a strong increase of the tone on the proximal colon, while the activity of the medium and distal colon was completely suppressed. Yohimbine (0.50 mg/kg i.v.) immediately restored the activity of the colon and induced a propagated giant contraction and defaecation by the animal. These data confirm the importance of a₂-adrenergic receptors in the control of intestinal and colonic motility in the dog.     

Chronic small intestinal disease in the dog

New approaches to the investigation of chronic small intestinal disease have facilitated the identification and characterization of three enteropathies in the dog. The main features of these diseases are presented and diagnostic problems are discussed.     

Flow cytometric and immunophenotypic evaluation of acute lymphocytic leukemia in dog bone marrow

Monoclonal antibodies provide powerful tools for detection of lineage-specific markers on hematopoietic cells. We used the combination of cell morphology, cytochemistry, flow cytometric scatter plot analysis, and labeling of cells with 6 monoclonal antibodies to detect and subclassify lymphocytic leukemia in bone marrow from 5 dogs. Antibodies included anti-CD18 (a panleukocyte marker), anti-MHC class II (detects most B and T lymphocytes and monocyte/macrophages), anti-Thy-1 (a pan-T-lymphocyte and monocyte/macrophage marker), anti-CD3 (a pan-T-lymphocyte marker), anti-CD21 (a B-lymphocyte marker), and anti-CD14 (a monocyte/macrophage marker). Of the 5 dogs evaluated, 2 were categorized as acute T-cell prolymphocytic leukemia, 2 as acute non-T, non-B lymphoblastic leukemia, and 1 as acute B-cell lymphoblastic leukemia. Results of this study indicate marked variation in the morphology and immunophenotype of canine lymphocytic leukemia.     

Irregular intestinal mucosal margination in the dog: normal or abnormal?

The apparently random variability of the margination of the small bowel observed during contrast radiography prompted an investigation to determine the significance and aetiology of such a finding. Upper gastrointestinal tract contrast radiography of twenty-five dogs revealed a marked variation in the smoothness of mucosal margination. Gross and histological examination of the small bowel of these dogs revealed no lesions which could account for the variation in mucosal margination. It was noted that the spatial arrangement of the villi varied in such a way that the degree of irregularity of the mucosa observed radiographically could depend on the ability of the contrast medium to dissect around upright villi.     

仔猪小肠黏膜淋巴细胞的分离及表型鉴定

对小肠不同解剖位点分离得到的淋巴细胞进行表型鉴定,将有助于我们掌握猪黏膜免疫系统区分共生菌和致病菌的机理。我们的研究描述了分离空肠和回肠的上皮内淋巴细胞(IELs)和黏膜固有层淋巴细胞(LPLs)以及派伊氏结(PP)淋巴细胞的方法,并对表达CD3、CD4、CD8的淋巴细胞进行三色流式细胞分析。研究结果表明,分离的淋巴细胞活性高达90%以上。IELs和LPLs大部分是CD3+T细胞(67.90%⁸2.44%),少数是CD3-CD4-CD8+NK细胞(<5%)。IELs以CD4-CD8+细胞毒性T细胞(CTLs)占主导(43.61%82.44%),少数是CD3-CD4-CD8+NK细胞(<5%)。IELs以CD4-CD8+细胞毒性T细胞(CTLs)占主导(43.61%⁴8.49%),CD4+CD8-辅助性T细胞(TH)细胞只占10%左右。相反,LPLs中TH细胞占多数,为40%左右,而CTLs细胞占了16.97%48.49%),CD4+CD8-辅助性T细胞(TH)细胞只占10%左右。相反,LPLs中TH细胞占多数,为40%左右,而CTLs细胞占了16.97%¹9.24%。由于解剖位点不同,淋巴细胞的表型分布也不相同,最明显的是空肠和回肠PP结的淋巴细胞。空肠PP结CD3+T细胞的数量(54.08%)明显要多于回肠的PP结(15.26%)。小肠淋巴细胞中也可检测到CD4+CD8+双阳性细胞(DP)以及CD4-CD8-双阴性细胞(主要为γδT细胞),其中7.85%19.24%。由于解剖位点不同,淋巴细胞的表型分布也不相同,最明显的是空肠和回肠PP结的淋巴细胞。空肠PP结CD3+T细胞的数量(54.08%)明显要多于回肠的PP结(15.26%)。小肠淋巴细胞中也可检测到CD4+CD8+双阳性细胞(DP)以及CD4-CD8-双阴性细胞(主要为γδT细胞),其中7.85%²1.50%的肠黏膜淋巴细胞表达CD4-CD8-,但只有0.51%21.50%的肠黏膜淋巴细胞表达CD4-CD8-,但只有0.51%².06%的细胞表达CD4+CD8+。IELs表达CD4-CD8-的细胞要比LPLs多。本试验为研究黏膜免疫反应提供了细胞分离技术以及相关基础知识。     

Use of atropine to reduce mucosal eversion during intestinal resection and anastomosis in the dog

OBJECTIVE: To determine whether atropine altered the degree of mucosal eversion during jejunal resection and anastomosis in the dog. STUDY DESIGN: Part I: Prospective, blinded, randomized, controlled study using a therapeutic dose (0.04 mg/kg systemic) of atropine. Part II: Prospective, unblinded, assigned, controlled study using a pharmacologic (0.04 mg/kg local arterial) dose of atropine. ANIMALS: Part I: Twenty-two young adult female Beagle dogs used during a nonsurvival third-year veterinary student surgical laboratory (small intestinal resection and anastomosis). Part II: Ten young adult female Beagle dogs used immediately after completion of a nonsurvival third-year veterinary student orthopedic surgical laboratory. METHODS: Part I: Dogs were randomly assigned to receive either atropine (0.04 mg/kg), or an equal volume of saline, given intramuscularly (premedication) and again intravenously prior to intestinal resection. Part II: In each dog, atropine (0.04 mg/kg)/saline was alternately given in the proximal/distal jejunum. RESULTS: Part I: There was no clinically or statistically significant difference between systemic atropine and saline solution on the degree of jejunal mucosal eversion after resection. Part II: There was a statistically significant decrease in jejunal mucosal eversion with atropine compared with saline solution when injected into a local jejunal artery. CONCLUSION: Systemic atropine (0.04 mg/kg) does not alter the degree of jejunal mucosal eversion during resection and anastomosis. Jejunal intraarterial atropine (0.04 mg/kg) reduced jejunal mucosal eversion during resection and anastomosis. CLINICAL RELEVANCE: The clinical usefulness and consequences of jejunal arterial atropine administration to reduce mucosal eversion remain to be determined.     

Invasion of enterocytes in cultured porcine small intestinal mucosal explants by Salmonella choleraesuis.

Porcine small intestinal explants maintained in vitro were inoculated with Salmonella choleraesuis to study the characteristics of its invasion of enterocytes. The explants were fixed at selected intervals for up to 12 hours after inoculation and examined by conventional light microscopy, immunoperoxidase staining, and transmission electron microscopy. Although there was diffuse loss of villous enterocytes during the first hour of incubation, the villi were reepithelialized by the end of 2 hours of culture, and the mucosal epithelium remained intact and appeared to be viable through 12 hours of culture. Intraepithelial S choleraesuis were not detected before 6 hours after inoculation, but after 12 hours of incubation, bacteria were numerous within enterocytes. Ultrastructurally, penetration of the brush border by S choleraesuis resulted in focal loss of microvilli. Bacteria were endocytosed into membrane-bound vacuoles where most remained, but a few were free within the cytoplasm of enterocytes. Invasion of the explants closely resembled that described for live animal and cell culture models of Salmonella spp invasion.     

Flow cytometric analysis of T-lymphocyte subsets in cats.

We report a rapid, reliable method for the immunophenotype analysis of feline lymphocytes. Fluorescein isothiocyanate (FITC) conjugated to murine monoclonal antibodies f43, Fel 7 and fCD8 was used to identify phenotypes corresponding to feline T-cells, CD4+ T cells and CD8+ T cells. For isolation of white blood cells, whole blood lysis was faster, less variable and required much less sample than density gradient separation. To identify feline CD4+ and CD8+ cells simultaneously, directly conjugated FITC-fCD8 and phycoerythrin (PE) fCD4 (Fel 7) were used in two-color analysis. The two T cell sub-populations were non-overlapping. Dual-label and single-label values were not significantly different. Mean lymphocyte subset percentages in conventional and specific-pathogen-free (SPF) cats did not differ significantly. These values were: pan T lymphocytes (f43), 54.8%, CD4+ cells (Fel 7), 33.9%, and CD8+ cells (fCD8), 19.1%. Mean CD4/CD8 ratio was 1.9 in normal cats; the range was 1.2-2.6.     

Morphology of the small intestinal mucosal surface of broilers in relation to age, diet formulation, small intestinal microflora and performance

1. Three experiments were performed to relate morphological characteristics of the small intestinal mucosal surface to age, dietary factors, small intenstinal microflora and performance of broilers. Characterisation of the small intestinal mucosal surface using a dissecting microscope was based on the orientation of the villi, villus shape and the presence of convoluted villi. 2. In Trial 1, the morphological changes of the mucosal surface were studied weekly in the period from 7 to 28 d of age. At d 7 mainly tongue- and leaf-shaped villi together with some ridge-shaped ones were observed in the middle section of the small intestine, displaying a regular zigzag pattern on 53% of the mucosal surface. During the period from d 7 to 14, the area with ridge-shaped villi increased from 7 to 63% and did not change significantly over the next 2 weeks. 3. In Trial 2, three protein sources, soy isolate (SI), wheat gluten (WG), hydrolysed wheat gluten (HWG) and SI with added L-glutamine (SI + Gln), were studied with respect to their effect as dietary components on villus morphology in the mid-small intestine and performance. Diets were fed with (0 to 14 d) and without pectin (14 to 21 d). Feed conversion ratio on the HWG diet improved in comparison to the native WG diet. During the period 0 to 14 d of age the mucosal area with zigzag-oriented villi increased when the pectin diet was supplemented with Gln. Moreover, weight gain of birds fed the SI + Gln diet increased in the period 41 to 21 d. 4. In Trial 3, a study was made of the morphological response of the villi to a stimulation of microbial activity in the digesta after addition of highly methylated pectin to the soybean meal (SBM) diet. This was performed with and without inoculation of a non-virulent Salmonella typhimurium on d 7. By d 21 the birds fed the pectin diet showed impaired weight gain and higher feed conversion. The pectin affected the mucosal surface by decreasing the area with the zigzag pattern and increasing the area with convoluted, mainly ridge-shaped villi. The Salmonella typhimurium infection increased the effects of pectin on performance and mucosal morphology.     

Normal and abnormal ultrasonographic findings that mimic small intestinal intussusception in the dog

On ultrasonographic examination of the abdomen, the appearance of healthy intestine, enteritis secondary to an intestinal foreign body, and postpartum involution of the uterus may be visualized in some imaging planes as a target-like structure that is subsequently misinterpreted as intestinal intussusception. To avoid misdiagnosis, the ultrasonographer should ensure multi-plane scanning of the lesion, paying particular attention to the completeness of the lesion's peripheral ring structure and the overall width of the concentric rings of the target-like lesion. The presence of a semilunar or G-shaped hyperechoic center and the visualization of the inner intussusceptum (extending into the intussusception lumen) can be useful ultrasonographic findings that distinguish an intussusception from other lesions or from healthy tissues. These principles are illustrated through the following case presentations.     

Flow cytometric analysis of bone marrow leukocytes in neonatal dogs

Dogs represent both an important veterinary species and a convenient model for allogeneic hematopoietic stem cell transplantation. Even though anti-canine CD34 antibodies have recently become available, little is known about hematopoietic lineages in dogs, partially because CD34- cells have been ignored in all analyses performed so far. In this study, we have focused on the bone marrow mononuclear compartment to provide an additional piece of information on the phenotype of CD34+ progenitors and to identify the dominant CD34- population. We have shown that, in contrast to the adults, mature lymphocytes are scarce in neonatal dog bone marrow. Using cross-reactive antibodies against CD79alpha we have shown that the B lineage of hematopoiesis strongly prevails. CD34+ cells were shown to be positive for MHC class II and SWC3, a member of the signal regulatory protein family.