An evaluation of the mitogenic reactivity of intestinal intraepithelial lymphocytes of chickens.

A colorimetric assay employing MTT (3-[4,5-dimethylthiazole-2-yl], 2-5-diphenyltetrazolium bromide) was used to determine the mitogenic response of intestinal intraepithelial lymphocytes (i-IELs) of chickens to T- and B-cell mitogens. Comparisons between mitogenic responses of i-IELs and peripheral blood lymphocytes (PBLs) were made to examine potential relationships. The results from this study indicated that T-cell mitogens, concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) induced mitogenic stimulation in i-IELs. Although stimulation indexes of both i-IELs and PBLs were similar, the optical densities (ODs) of i-IEL cultures containing Con A or PHA-P were 20- to 50-fold lower than the ODs of PBL cultures containing the same mitogen. The lower conversion of MTT to formazan resulting in lower ODs in i-IEL cultures indicated a lower level of cellular activity in the i-IELs than in the PBLs. The mitogenic responses of both i-IELs and PBLs to Con A and PHA-P were dose dependent. The responsive concentration of Con A for i-IELs was within the range of 25-50 micrograms/ml, whereas the responsive concentration of PHA-P for i-IELs was 50 micrograms/ml. Three days of incubation was found to be adequate to induce a significant (P < 0.05) mitogenic response for both T-cell mitogens. Lipopolysaccharide was unable to induce a mitogenic response in i-IELs, which was attributed to the lack of B cells in the i-IEL population. This technique may prove useful in evaluating and studying the role of i-IELs in local cell-mediated immune responses of the gastrointestinal tract.     

3种添加剂对肉鸡小肠上皮内淋巴细胞和杯状细胞分布的影响

抗生素具有促进动物生长、预防病原微生物感染和疾病发生等方面的重要作用而被作为饲料添加剂广泛应用于动物生产中。然而随着饲料抗生素的长期使用,人们逐渐发现使用抗生素存在的诸多负面效应:如动物产品中药物残留、病原菌的耐药性提高等[1,2]。为了禁用或少用抗生素作为饲料     

A colorimetric assay to evaluate the cytotoxic activity of the intestinal intraepithelial lymphocytes of chickens

After the successful use of 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) in cell proliferation assays, its use has been established by different workers in cytotoxicity assays and research on leukaemia. In the present study, a colorimetric assay using MTT was adopted to evaluate the cytotoxic activity of chicken intestinal intraepithelial lymphocytes (iIELs), which constitute an important cellular component of the gut-associated lymphoid tissue (GALT). These iIELs are found to exhibit natural killer (NK) cell-like cytotoxic activity, which is spontaneous, non-MHC-restricted, and does not need to be primed. Hitherto, conventional chromium-release assays have been used to evaluate the cytotoxic activity of iIELs, but these assays have disadvantages such as radiation hazards and loss of the cells in washing steps. The mean percentage cytotoxic activity of chicken iIELs evaluated by the colorimetric assay was 90.37±2.53 in a group of 5-week-old chickens and 80.2±3.45 in a group of 8-week-old chickens. These findings established the successful use of a colorimetric assay using MTT for evaluating the cytotoxic activity of chickens iIELs.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethyl sulphoxide - E effector cells - GALT gut-associated lymphoid tissue - GM growth medium - iIELs intestinal intraepithelial lymphocytes - MTT 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide - NK cell natural killer cell - OD optical density - RPMI Rosewell Park Memorial Institute medium - T target cells     

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。     

A comparative analysis of the mitogenic response of intestinal intraepithelial lymphocytes in various age groups of turkeys.

Mitogenic responsiveness of intestinal intraepithelial lymphocytes (i-IEL) to concanavalin A (Con A), phytohemagglutinin P (PHA-P), and lipopolysaccharide (LPS) from Salmonella typhimurium were evaluated in various age groups of turkeys by a colorimetric blastogenic microassay. Comparisons were made between mitogenic responses of turkey i-IEL and peripheral blood lymphocytes (PBL). The results from this study demonstrated that i-IEL and PBL of turkeys responded to T-cell mitogens, Con A and PHA-P, in every age group examined. The LPS induced a significant mitogenic response in PBL but not in i-IEL of turkeys. The mitogenic responses of turkey i-IEL and PBL to the three mitogens examined were similar to mitogenic responses observed in an earlier study performed by using chicken i-IEL and PBL. The results indicated a difference in mitogenic response between different age groups. An increase was found in mitogenic response of i-IEL to both T-cell mitogens from 3 days of age to 1 wk of age, whereas mitogenic response of PBL to all three mitogens declined significantly from 1 day of age to 3 days of age. The highest mitogenic response of i-IEL to T-cell mitogens was observed at 1 wk of age. The highest mitogenic response of PBL to both T-cell mitogens was observed at 1 day of age and the highest PBL response to LPS was observed at 16 wk of age. The mitogenic response induced by PHA-P provided less variability between age groups than the mitogenic response induced by Con A.     

Sub-clinical infection with Salmonella in chickens differentially affects behaviour and welfare in three inbred strains

1. Much evidence exists detailing how animals respond to pathogen challenge, yet information explaining how the various behavioural, immunological, and physiological systems in chickens interplay during such challenges remains limited. 2. To gain an understanding of this interplay while controlling for genetic variation, the current study collected a variety of behavioural, physiological and immunological measures from three inbred lines (P, O and N) of laying hens before and after a sub-clinical infection with Salmonella enterica Typhimurium at 56 d of age. For comparison, an equal number of control birds were inoculated with a Salmonella-free broth. To identify an underlying profile, which might result in reduced susceptibility to infection, data were also collected in the pre-infection period. Post-infection blood and faeces were collected at 1-d post infection (dpi) and faeces again at 8 dpi. Animals were killed 15 d after infection and faeces, caecal contents, and spleen were examined for the presence of Salmonella. 3. Statistical analysis was performed to identify pre- and post-infection differences between genetic lines, changes in bird behavioural patterns between the two periods, and associations between a positive test for Salmonella and the various response measures. 4. Tissues from Line P birds were more often negative for Salmonella than those from birds of other lines, though this was inconsistent and tissue-dependent. The P line was also characterised by relatively greater serum concentrations of immunoglobulins at 1 dpi and α(1)-acid glycoprotein at 15 dpi. In addition, P line birds were more timid and their growth was reduced during the pre-infection period suggesting the possibility of a profile with reduced susceptibility to the bacterial challenge. 5. The current work has identified correlations between attributes of chicken strains and improved clearance. Future work using hypothesis-based testing will be required to determine whether the identified correlations are causally related.     

Comparative pathogenicity of three genetically distinct Trypanosoma congolense-types in inbred Balb/c mice

Inbred Balb/c mice were infected with three clones of Trypanosoma congolense (Sam.28.1, Dind.3.1 and K60.1A) corresponding, respectively, to the three genetically distinct types (savannah, forest and kilifi) defined within this species, for the purpose of comparing their pathogenicity for a better understanding of the epidemiology of African trypanosomosis. Another clone of savannah type, IL 3000, was also tested simultaneously to study a probable strain variation. Both the clones of savannah type were found of extreme virulence with loss of appetite, rough hair, rapid respiration, lethargy, and all mice died within a week. Parasitaemias evolved rapidly to the first peak by day 3-5 post-inoculation without any remission and the course of disease was correlated positively with the prepatent period. The clones of the forest type and the kilifi type were of low virulence with chronic infection and symptoms progressively less patent throughout the infection; only one mouse died in each experimental group.     

Increase of intestinal intraepithelial lymphocytes expressing CD8 antigen following challenge infection with Eimeria acervulina.

Eimerian infection-induced changes in the intestinal intraepithelial lymphocyte (IEL) subpopulations expressing CD8 antigen (cytotoxic/suppressor T cells) or antigen-specific T cell receptor (TCR) heterodimer alpha beta (TCR2) or gamma delta (TCR1) were investigated in F2 crosses of 15I5 B-congenic chickens differing for the major histocompatibility complex (MHC). Duodenum TCR2+ IEL were increased in B2 B2 and B5 B5 chickens 7 days following secondary infection. Two-color immunofluorescence revealed that the majority of CD8+ cells in the duodenum intraepithelium of immune chickens expressed TCR2. A significant increase in the duodenum TCR2+CD8+ and TCR1+CD8+ IEL occurred in B2 B2 chickens, which developed significantly less oocyst production than the B5 B5 chickens following challenge infection. The results suggest that a significant increase in the duodenum CD8+ IEL may reflect an enhanced acquired immunity of B2 B2 chickens.     

Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice

Inbred laboratory mouse strains are highly divergent in their immune response patterns as a result of genetic mutations and polymorphisms. The generation of genetically engineered mice (GEM) has, in the past, used embryonic stem (ES) cells for gene targeting from various 129 substrains followed by backcrossing into more fecund mouse strains. Although common inbred mice are considered "immune competent," many have variations in their immune system-some of which have been described-that may affect the phenotype. Recognition of these immune variations among commonly used inbred mouse strains is essential for the accurate interpretation of expected phenotypes or those that may arise unexpectedly. In GEM developed to study specific components of the immune system, accurate evaluation of immune responses must take into consideration not only the gene of interest but also how the background strain and microbial milieu contribute to the manifestation of findings in these mice. This article discusses points to consider regarding immunological differences between the common inbred laboratory mouse strains, particularly in their use as background strains in GEM.     

Signs of abdominal pain caused by disruption of the small intestinal mesentery in three postparturient cows

Three Holstein cows were admitted on postpartum day 3 with signs of abdominal pain, inappetence, and decreased milk production. All 3 were depressed, tachycardic, and dehydrated. Right paralumbar fossa laparotomy was performed on each cow. At surgery, a rent in the proximal portion of the jejunal mesentery, with perforation of the jejunum adjacent to the mesenteric rent, was found in all 3 cows. All had evidence of severe peritonitis and gross contamination of the abdomen with digesta. Because of severe peritonitis and inability to exteriorize the affected segment of jejunum, all 3 cows were euthanatized. We postulate that the mesenteric rent occurred at the time of parturition, resulting in ischemia and jejunal perforation in the segment of jejunum adjacent to the rent.     

The presence and distribution of beta-galactosidases in the small intestinal mucosa of the neonatal dog.

The beta-galactosidase activity of the small intestinal mucosa of the neonatal dog has been studied over the pH range 3-0 - 5-8 using different substrates and in the presence and absence of p-chloromercuribenzoate. Partial separation of two beta-galactosidases has been achieved by gel filtration. The results suggest that three beta-galactosidases, comparable with those in other mammals, exist in canine small intestinal mucosa.     

Flow cytometric analysis of colonic and small intestinal mucosal lymphocytes obtained by endoscopic biopsy in the healthy dog

Flow cytometric analysis of the lymphocyte population of the gut could provide useful information on the immune cells present in the gut that would not be easily obtained in tissue sections. However, little is known of the normal lymphocyte population in the canine gut as determined by flow cytometry, which allows for simultaneous staining of multiple cell surface antigens and identification of specific lymphocytic subsets. Therefore, intraepithelial lymphocytes were obtained from biopsies of the healthy canine proximal small intestine and colon taken with an endoscope, and flow cytometric analysis was used to characterize the lymphocyte subsets present. Endoscopic biopsy of the intestine is a minimally invasive technique commonly used for diagnostic purposes. Although CD3+ lymphocytes were the most abundant subset in both colon and small intestine, CD3+/CD8- lymphocytes predominated in the proximal small intestine, whereas CD3+/CD8+ lymphocytes did in the colon. Canine CD8+ intraepithelial lymphocytes were predominantly CD8alphabeta+ in both small intestine and colon. CD4+ intraepithelial lymphocytes were always much less numerous than CD8+ intraepithelial lymphocytes. As in man, a majority of intraepithelial lymphocytes expressed the T-cell receptor, TCRalphabeta, but TCRgammadelta was expressed by a third of intraepithelial T-cells in the proximal small intestine, and approximately 15% of those in the colon. Very few CD21+ lymphocytes were detected in samples of healthy canine colon and small intestinal intraepithelial cells. We have showed that canine intraepithelial lymphocytes are regionally specialized, and that those from the small intestine are unique in comparison to those of other species such as man and rodents due to the large numbers of CD3+/CD8- intraepithelial lymphocytes. This study provides a baseline for comparison with intraepithelial lymphocytes obtained from canine patients with intestinal disease.     

三种布鲁氏菌病疫苗株的毒力比较

为系统比较我国现有布鲁氏菌病疫苗株A19、M5和S2的毒力,分别用上述3种疫苗株以1×105CFU/只免疫Balb/c小鼠,免疫后每隔2周采集小鼠脾脏,分离细菌,测定各疫苗株在小鼠脾脏中的存留时间。结果 A19、M5、S2在小鼠体内存活时间依次为14周、大于16周、6周。将以上3种疫苗株分别以1×109CFU/只免疫Hartley豚鼠,15日后测定豚鼠脾脏含菌量,结果 A19、M5、S2免疫后每克脾脏含菌量分别为2.8×104CFU、大于6.7×105CFU、3.8×103CFU。研究结果表明,我国目前使用的布鲁氏菌疫苗中,S2毒力最弱,A19其次,M5最强。     

Regional distribution and variation of gamma-globulin absorption from the small intestine of the neonatal calf

125I-labeled immunoglobulin (Ig)G1 in colostral whey was used to determine the region of maximum absorption of Ig from the small intestine of the neonatal calf and the variation in Ig absorption among calves at the intestinal level. In experiment 1, 5 segments (approx 5%, 35%, 60%, 80%, and 95% of the duodenocecal length) were formed in the small intestine of 9 colostrum-deprived calves shortly after birth. These segments were injected with colostral whey containing 125I-IgG1 4 hours after birth, and uptake, transfer, and absorption (defined as uptake plus transfer) were determined for each segment 2 hours later. Raw data were adjusted for the milligrams of IgG1 injected per gram of intestinal tissue to obtain the least squares mean (LSM) value. The LSM values for absorption of IgG1 from distal segments 3, 4, and 5 were significantly greater (P less than 0.05) than those values for proximal segments 1 and 2. The region of the maximum IgG1 absorption was the lower small intestine, 60% to 80% of the duodenocecal length. There was also an indication of independence between uptake and transfer in each of the segments. Significant differences (P less than 0.05) were present among calves in the LSM values for uptake and absorption, but not for transfer. In experiment 2, thoracic ducts of 8 newborn calves were cannulated 4 to 5 hours after birth. At 6 hours after birth, colostral whey with 125I-IgG1 was injected into an intestinal segment (approx 60% to 80% of the duodenocecal length).(ABSTRACT TRUNCATED AT 250 WORDS)     

6-羟多巴胺对小鼠体重及其小肠黏膜结构的影响

采用组织学和图像分析技术，研究了小鼠经腹腔注射交感神经损毁剂6-羟多巴胺后小鼠体重和小肠绒毛长度、V／C比值、绒毛宽度以及黏膜厚度的变化。结果表明，注射6-羟多巴胺组与对照组小鼠相比，体重下降（约3．4％）；小肠绒毛变短（5．6％～19．4％），V／C比值减少（0．88％～24．1％），绒毛变窄（8．8％～24．7％）、黏膜厚度变薄（4．4％～16．9％），其中空肠的绒毛长度和黏膜厚度减少较明显，而十二指肠和回肠的V／C比值和绒毛宽度减少较明显。提示交感神经的活动可影响小肠的黏膜结构，从而影响肠道的消化吸收功能。     

近交系基因敲除小鼠微卫星不稳定性分析

前期研究中选取了198个与基因突变和疾病相关的小鼠微卫星不稳定性位点,经对转基因和基因突变小鼠研究,发现有41个位点具有不稳定性.为了进一步确认这些位点与基因改变的相关性,本试验应用这41个微卫星位点对29种C57BL/6J基因敲除小鼠进行了检测,并将野生型C57BL/6J和129品系小鼠(ES供体)作为对照.通过PCR扩增,STR扫描和直接测序的方法检测微卫星的不稳定性.在41个微卫星位点中有10个位点在11种基因敲除小鼠中呈现不稳定性(24.4％,10/41).核心序列为三核苷酸的D3Mit22在9号基因敲除小鼠中显示不稳定性,其余40个位点的核心序列均为二核苷酸,有9个位点呈现不稳定性(22.5％,9/40);另一方面,29种基因敲除小鼠有11种出现了不稳定性(37.9％,11/29);(TG)n为核心序列的二核苷酸表现不稳定性的比率最大(50％,2/4),依次为(GT)n(27.27％,3/11)和(AG)n(25％,1/4),重复序列(CA)n、(CT)n分别为23.08％(3/13)和20％(1/5).克隆测序的结果显示,6种基因敲除小鼠呈现核心序列片段的插入,1种基因敲除小鼠则为缺失.有2个位点(D13Mit3和D14Mit102)在2种基因敲除小鼠中出现不稳定性,9号基因敲除小鼠在2个位点(D3Mit22和D13Mit3)呈现不稳定性.结果显示基因敲除小鼠出现了微卫星不稳定性.     

DNA指纹技术在近交系动物遗传检测中的研究

采用生物素标记的(GGAT)4寡核苷酸探针对DBA1、DBA2两种近交系小鼠及DBA2×DBA1 F1代小鼠进行了DNA指纹图分析,并与常规生化位点标记分析法进行了比较.结果显示,DNA指纹图具有良好的多态性,不仅可以分辨生化位点标记分析法能够区分的不同品系的近交系动物,而且也能够分辨生化位点标记分析法不能区分的不同品系的近交系动物.研究还表明,用同一方法重复同一基因组DNA的指纹图时,获得相同的实验结果.因此,DNA指纹图法不仅比传统的生化位点检测分析法有更好的分辨力,也具有良好的稳定性.     

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.