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1.
Quantification of surface IL-2R expression on activated lymphocytes by flow cytometry have recently been reported to be useful in measuring cellular immunity against Mycobacterium avium subsp. paratuberculosis in goats (Whist et al., 2000, Vet. Immunol. Immunopathol. 73, 207-218). To characterise the phenotype of the peripheral lymphocytes expressing IL-2R after in vitro stimulation with purified protein derivative (PPD) from M. a. paratuberculosis, cells were processed for dual or triple colour analysis by flow cytometry (CD4 and IL-2R or CD8, gammadelta-TcR and IL-2R). To distinguish the response of antigen-specific T cells from non-specific stimulation, we performed a time-course study of proliferating cells in a group of M. a. paratuberculosis-infected animals and a control group. Following in vitro stimulation with PPD of whole blood for three different periods of time, IL-2R expression was detected mainly not only in gammadelta-T cells, but also in CD4+ and CD8+ T cells. We found a specific response of gammadelta-T cells from infected animals after 24h of stimulation. Following 120h of stimulation, however, gammadelta-T cells from control animals up-regulated IL-2R to the same level as those from infected animals, indicating either a non-specific stimulation or activation due to a first line of defence against Mycobacterium antigens. The CD4+ cells showed a specific response to PPD stimulation at all three time points. A minor population of antigen reactive gammadelta+ cells also expressed CD8. The proliferative responses differed between alphabeta and gammadelta-T cells; the IL-2R+ alphabeta T cell population mainly comprised proliferating cells, while the gammadelta+ population showed less expansion.  相似文献   

2.
It has been established that maternal leukocytes, conditioned by the mammary environment, cross the neonatal gut and circulate in the newborn calf. However, the impact of these cells on the development of neonatal immunity remains to be determined. This study examined the effects of maternal colostral leukocytes on development and maturation of neonatal adaptive immunity by examining the expression of surface markers on neonatal lymphocytes. At birth, neonatal calves were fed whole colostrum, or colostrum that had the maternal cells removed (cell-free colostrum), from their respective dams. Peripheral blood samples were collected at regular intervals over the first 4 weeks of life and lymphocytes were evaluated for surface expression of cellular markers. The results of these studies demonstrated that calves receiving whole colostrum had fewer CD11a positive lymphocytes in circulation during the first 2 weeks of life and this marker was expressed at a lower density than calves receiving cell-free colostrum. In addition, calves receiving whole colostrum also had a higher percentage of lymphocytes expressing the activation markers CD25 and CD26 by 7 days after birth. During the first week of life, lymphocytes from calves receiving whole colostrum had a higher density of MHC class I expression on their surfaces than cells from calves receiving cell-free colostrum. In general, these results indicate that transfer of maternal cells with colostrum allows for more rapid development of lymphocytes and maternal cells appeared to enhance their activation.  相似文献   

3.
Although it has been established that maternal leukocytes traffic from colostrum into the neonatal circulation, the effects of these cells on neonatal immunity are only beginning to be understood. This study examined the effects of maternal colostral leukocytes on development and maturation of neonatal antigen presenting cells. At birth, groups of neonatal calves received whole or cell-free colostrum (CFC) from their respective dams. Peripheral blood samples were obtained over the first 4 weeks of life, and expression of surface markers associated with cellular activation and physiological stress were monitored on monocyte lineage cells. Calves receiving cell-free colostrum at birth expressed elevated levels of CD11a, CD11c, and CD14, compared to calves receiving whole colostrum (C). Calves receiving cell-free colostrum had an elevated number of monocytes in the peripheral blood during the first 2 weeks of life, however, these cells expressed lower levels of expression of CD25 and MHC class I compared to calves receiving whole colostrum. The most significant differences in marker expression occurred within the first 7 days of life.  相似文献   

4.
To examine the effect of recombinant bovine interferon-gamma (rbIFN-gamma) on cattle persistently infected with bovine leukemia virus (BLV), BLV-infected cattle were inoculated intraperitoneally with IFN-gamma. All cattle were febrile after inoculation with IFN-gamma and then recovered within 48 h. Flow cytometric analysis showed that the numbers of CD4+ and CD8+ T cells were decreased for 2-3 days and then their numbers were recovered. The number of gammadelta T cells increased after the fever. In contrast, the number of IgM+ lymphocytes remained low for about 1 week. Moreover, the numbers of syncytia produced by peripheral blood lymphocytes decreased and remained low compared to that before IFN-gamma administration. These results suggest that IFN-gamma induces the up-regulation of gammadelta T cells, decreases the number of IgM+ lymphocytes and suppresses the growth of BLV in BLV-infected cattle in vivo.  相似文献   

5.
Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-gamma) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-gamma and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4(+)CD8(+)CD25(+) cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4(-)CD8(+)CD25(+) cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-gamma producing cells detected in this experiment was relatively low, the CD4(+)CD8(+) T cells were major IFN-gamma producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-gamma producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4(-)CD8(+) cells were major IFN-gamma producing cells in vaccinated pigs, while both CD4(+)CD8(+) and CD4(-)CD8(+) populations contributed to the IFN-gamma production in the control group. Interestingly, the enhanced IFN-gamma production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4(-)CD8(+) populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-gamma production were not tightly associated in porcine lymphocytes. In addition, the CD4(-)CD8(+) lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge.  相似文献   

6.
Pairs of newborn calves were exposed to bovine leukemia virus (BLV) when they were given their 1st colostrum feeding. Calves that were given 10(6) BLV-infected lymphocytes in colostrum free of BLV-specific antibody became infected. Calves that were fed 10(7), 10(8), or 10(9) infected lymphocytes in colostrum that contained BLV-specific antibody did not become infected. One of 2 calves inoculated intradermally with 250,000 infected lymphocytes was protected by colostral antibody, but the other was not. Colostral antibody titers in the unprotected calf decreased normally until the calf was 4 months old and then increased markedly; this pattern indicates that the presence of colostral antibody may have prolonged the latent period of the BLV infection.  相似文献   

7.
Mammary secretions contain leucocytes which may be of value to the neonate. The cells obtained from sow colostrum (1 to 2·5 × 106 ml−1) are mainly lymphocytes (10 to 25 per cent) and epithelial cells (more than 20 per cent). In milk, there are few lymphocytes (0·5 to 2 per cent) and mostly alveolar epithelial cells. The study of lymphocytes in the mammary secretions of sows has been made difficult by the high proportion of epithelial cells, which could not be separated from lymphocytes, and by a high background in membrane immunofluorescence labelling. This paper describes a method for the study of the cells in the mammary secretions of sows by flow cytometry. It showed that 70 to 90 per cent of colostral lymphocytes were T lymphocytes, with T8 lymphocytes predominating over T4, and that the ratio T4/T8 was significantly lower in colostrum (0·57) than in blood (0·80). There were no lymphocytes expressing interleukin-2-receptors in the colostrum of sows.  相似文献   

8.
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.  相似文献   

9.
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10.
The relationship between the colostral environment and the function of leukocytes in colostrum is not clearly defined. This study examined the effects of defatted, acellular colostrum (AC) on the phenotype of peripheral blood mononuclear cells (PBMC) and their capacity to enter the circulation of neonatal calves after ingestion as a model of this relationship. Maternal PBMC were exposed to medium alone or medium supplemented with 25% AC. Expression of CD11a, CD11b, CD11c, CD43, CD49d, CD49e, and CD62L was assessed on freshly isolated and treated PBMC. Exposure to AC increased the percentage of cells expressing CD11a, CD11c and CD43, but decreased the percentage of cells expressing CD62L relative to freshly isolated PBMC. The density of expression of CD11b and CD11c was reduced, but increased for CD43 after exposure to AC relative to freshly isolated PBMC. Density of CD62L expression and percentage of cells expressing CD11a and CD43 were significantly different for cells treated with AC relative to medium alone. Further, these changes could not be attributed to occult bacterial contamination of the AC, as treatment of PBMC with LPS in the same medium yielded none of the observed changes. Maternal PBMC (treated as described) were labeled with the fluorescent tracer, PKH26-GL, and fed to neonatal calves within 6 h of birth. The circulation of these cells in the neonate was monitored by flow cytometry. We observed that: (1) cells exposed to AC, but not medium alone, entered the circulation; (2) peak trafficking occurred 12-24 h after ingestion; (3) a large fraction of labeled cells appeared in the neonatal circulation; and (4) labeled cells disappeared from circulation by 36 h after ingestion. This study indicates that exposure to the colostral environment induced phenotypic changes facilitating trafficking of colostral cells into the neonate.  相似文献   

11.
The aim of this study was to evaluate the influence of bovine colostrum supplementation on the immune system of weaned piglets in a context of a full ban of in-feed antibiotics. After weaning at 21 days, 24 outbred piglets were fed with a diet supplemented daily for three weeks with 0, 1 or 5 g of colostrum. Feed intake, growth performance, haematological parameters, and serum and local anti-colostrum immunoglobulin levels were examined. Lymphocytes from the blood, spleen, and gut-associated lymphoid were analysed for phenotype as well as for their ability to produce cytokines. The stimulation index (SI) of mononuclear cells from different organs was obtained after colostral or mitogenic stimulation. Feed intake, growth, and haematological parameters were not significantly affected by colostrum. Total serum IgA levels were increased after colostrum supplementation, with a transient decrease in total IgG. Local anti-colostrum immunization was observed in colostrum-fed piglets. The CD21+/CD3+ cells populations of the ileal Peyer's patch (iPP) were markedly affected. The SI of lymphocyte populations changed significantly whereas, naive blood lymphocytes were not stimulated in vitro in the presence of bovine colostrum, suggesting local anti-colostrum immunization and an absence of direct mitogenic effects of the colostrum. Both Th1 and Th2 cytokine production was present in the different organs of colostrum-fed piglets. Bovine colostrum especially stimulated iPP cells.  相似文献   

12.
Interferon gamma (IFN-gamma) is considered as a key mediator of protective cell-mediated immunity against intracellular pathogens in general, and against Ehrlichia ruminantium, the causative agent of tick-borne heartwater disease of ruminants, in particular. However, the source of this important cytokine in animals immunized against E. ruminantium remains largely unknown. We have analyzed in goats protected by vaccination with a killed E. ruminantium vaccine, the potential of individual, genuine (i.e., non-cloned), T cell subsets to produce IFN-gamma after antigenic recall in vitro. In all vaccinated but none control animals, E. ruminantium-induced IFN-gamma secretion was observed in 24 h stimulated blood. Flow cytometric analysis of stimulated peripheral blood mononuclear cells (PBMCs) collected after each vaccine inoculation indicated that immune CD4+ and CD8+ T cells contribute to the same extent to the production of IFN-gamma, while WC1+ T cells are less important. This was confirmed by blocking the secretion of IFN-gamma with anti-classes I and II major histocompatibility complex antibodies. Blocking experiments also suggest that CD8+ need the help of CD4+ T cells in order to produce IFN-gamma. Thus, this work underlines the key role of CD4+ T cells in the production of IFN-gamma by immune goat PBMC. It also describes, for the first time in ruminants, E. ruminantium-specific CD8+ effector T cells. Since CD4+ and CD8+ T cells collectively contribute to the production of IFN-gamma in most vaccinated animals, and since these responses are associated with protection, it may be that a recombinant vaccine will need to incorporate E. ruminantium antigens capable of driving both responses.  相似文献   

13.
We describe the development of three monoclonal antibodies to feline T lymphocytes. Antibody 1.572 stains 93% of feline thymocytes, 49% of lymph node, and 65% of spleen lymphocytes. Two-color analysis shows 1.572 does not stain Ig-bearing cells, and 1.572-positive lymphocytes plus Ig-positive lymphocytes make up approximately 90% of peripheral blood lymphocytes (PBL), suggesting that 1.572 is a pan-T cell marker. The other two monoclonal antibodies, 3.357 and CAT30A, stain a smaller population of thymocytes (59%) of which 40% express both antigens. The 3.357 antigen is found on 23% of lymph node and 47% of spleen lymphocytes, while the CAT30A antigen is found on 29% of lymph node and 19% of spleen lymphocytes. Two-color analysis shows that 3.357 and CAT30A stain mutually exclusive subpopulations of 1.572-positive cells. Using thymocytes as an antigen source, antibody 3.357 precipitated a molecule of 66,000 molecular weight (Mw) under nonreducing conditions and a heterodimer of 32,000 and 34,000 under reducing conditions, suggesting that 3.357 recognizes the feline CD8 homologue. Antibody CAT30A precipitated a molecule of 55,000 Mw under both reducing and nonreducing conditions, which suggests it recognizes the feline CD4 homologue. Analysis of PBL profiles of 35 normal cats using the three monoclonal antibodies indicates that the distribution of feline PBL subpopulations is similar to man, including the CAT30A:3.357 ratio (1.74), which is identical to reported CD4:CD8 ratios in man. Based on these data, the feline CD4 and CD8 homologues are similar to those reported in other species.  相似文献   

14.
The aim of this study was to characterise the morphological and histochemical features of equine nasopharyngeal tonsillar tissue. Nasal and oropharyngeal tonsillar tissue has been described as the gatekeeper to mucosal immunity because of its strategic location at the entrance to the respiratory and alimentary tracts. A combination of light, scanning and transmission electron microscopy has revealed the presence of follicle-associated epithelium (FAE) overlying lymphoid tissue of the equine nasopharyngeal tonsil caudal to the pharyngeal opening of the guttural pouch. Membranous microvillus (M) cells were identified in the FAE on the basis of short microvilli, an intimate association with lymphocytes, cytoplasmic vimentin filaments and epitopes on the apical surface reactive with lectin GS I-B4 specific for alpha-linked galactose. CD4-positive lymphocytes were scattered throughout the lamina propria mucosae as well as forming dense aggregates in the subepithelial part. The central follicular area was heavily populated with B lymphocytes and the dome and parafollicular areas contained both CD4- and CD8-positive lymphocytes. CD8-positive lymphocytes were also present in the epithelium and, together with B lymphocytes, in small numbers in the lamina propria mucosae. These observations indicate that the nasopharyngeal tonsil is potentially an important mucosal immune induction site in the horse and an appropriate target for intranasally administered vaccines.  相似文献   

15.
To better understand the interaction between Mycoplasma bovis and its bovine host, we have characterized the immune response generated during an experimental lung infection with M. bovis. Proliferation ([3H]-thymidine blastogenesis) and Th1/Th2 cytokine production were used to monitor peripheral cellular immune responses. Flow cytometry analysis was used to determine T-cell subset activity by CD25 expression. Humoral immune response was monitored by the identification of antigen-specific IgG1 and IgG2 isotypes over time. Herein, we show that M. bovis antigen stimulates activation of CD4+ and CD8+ cells in vitro in a manner consistent with memory, and that gammadelta-T cells are activated by antigen in a manner consistent with innate immunity. In addition, the percentage of cells producing IFN-gamma during recall response is equal to that of IL-4 producing cells. Serological analysis shows M. bovis stimulates increased production of antigen-specific IgG1 while very little IgG2 is produced. We therefore submit that experimental lung infection of cattle with M. bovis results in a Th2-skewed immune response.  相似文献   

16.
Serpulina hyodysenteriae infection of pigs, swine dysentery, causes a mucohemorrhagic diarrhoea resulting in significant economic losses to swine producers. The pathogenesis of this disease is poorly understood. Regardless, commercial vaccines have been developed and are in use. Thus, the present study was designed to examine cellular immune responses induced by parenteral S. hyodysenteriae vaccination. Significant antigen-specific interferon-gamma (IFN-gamma) and blastogenic responses were detected from peripheral blood lymphocytes isolated from vaccinated pigs. However, poor IFN-gamma responses were detected from colonic lymph node lymphocytes from these same pigs despite significant antigen-specific blastogenic responses. In addition, peripheral blood IFN-gamma responses were diminished by either in vitro depletion of CD4 expressing cells or by in vitro treatment with porcine IL-10. Colonic lymph node IFN-gamma responses were not inhibited by treatment with porcine IL-10. Vaccination also resulted in increased percentages of both mucosal and peripheral blood CD8 single positive cells with concurrent decreases in percentages of CD4 single positive cells as compared to percentages of these same populations from non-vaccinated pigs. In conclusion, these studies show that parenteral S. hyodysenteriae vaccination results in cellular immune responses detectable both peripherally (systemic immunity) as well as at the site of infection (mucosal immunity). However, it appears that regulatory mechanisms affecting IFN-gamma production in response to S. hyodysenteriae antigen differ between peripheral and colonic compartments.  相似文献   

17.
Extrathymic CD4/CD8 double positive T cells   总被引：6，自引：0，他引：6  
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18.
Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in supernatants of mitogen stimulated equine peripheral blood mononuclear cells (PBMC). The sensitivity of the ELISA for detecting secreted IL-10 was found to be around 200pg/ml. The production of intracellular IL-10 was measured in equine PBMC by flow cytometry. PBMC were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of the secretion blocker Brefeldin A. All three anti-IL-10 mAbs detected a positive population in PMA stimulated lymphocytes which was absent in the medium controls. Around 80% of the IL-10(+) cells were CD4(+). Another 15% were CD8(+) cells. Double staining with IL-4 or interferon-gamma (IFN-gamma) indicated that PMA and ionomycin stimulation induced 80% IL-10(+)/IFN-gamma(+) lymphocytes, while only 5% IL-10(+)/IL-4(+) cells were observed. By calculation, at least 60% of the IL-10(+)/IFN-gamma(+) cells were CD4(+) lymphocytes. This expression profile corresponds to the recently described T regulatory 1 (T(R)1) cell phenotype. In summary, the new mAbs to equine IL-10 detected native equine IL-10 by ELISA and flow cytometry and can be used for further characterization of this important regulatory cytokine in horses.  相似文献   

19.
The influence of colostral leukocytes on lymphocyte counts in the blood of calves and on lymphocyte responses, in particular the Concanavalin A-induced blastogenic response in vitro and the formation of antibodies against sheep erythrocytes, was investigated for four weeks postnatum using four experimental groups. The calves received either complete colostrum (COL+, n = 16), cell-depleted colostrum (COL-, n = 16), colostral cell-supplemented milk substitute (MS+, n = 7) or pure milk substitute (MS-, n = 6) during their first three days of life. In contrast to the calves fed with cell-depleted colostrum (COL-) the calves fed with complete colostrum (COL+) showed no decrease of lymphocyte numbers in the blood on the second day of life, uniform blastogenic responses to two different Concanavalin A concentrations, slightly enhanced antibody formation against sheep erythrocytes and a high spontaneous proliferation of mononuclear cells during the first week of life. In the calves fed with milk-substitute supplemented with colostral cells (MS+) a higher blastogenic response to Concanavalin A and an intensified formation of antibodies against sheep erythrocytes was observed as compared to the MS- calves. A passage of vital colostral lymphocytes through the intestinal wall is postulated. They seem to stimulate and regulate the blastogenic response and enhance the T-helper cell-dependent formation of antibodies against sheep erythrocytes in calves.  相似文献   

20.
Postweaning multisystemic wasting syndrome (PMWS) is an economically important disease in pigs caused by porcine circovirus type 2 (PCV2). Development of this disease is presumably associated with an impairment of the immune system. We, therefore, investigated the systemic expression of relevant cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, TNF-alpha, IFN-gamma) and IL-2Ralpha at mRNA (semiquantitative RT-PCR) and at protein level (flow cytometric intracellular cytokine detection after short-time stimulation of peripheral blood mononuclear cells) in 10 feeder pigs aged 14 weeks suffering from natural PMWS and in 10 clinically healthy pen-mates. Hematological examination revealed a significant (p < 0.001) relative lymphopenia in the diseased animals when compared to reference pigs. IL-1alpha and IL-10 mRNA levels were notably increased in the affected pigs, whereas IL-2 and IL-2Ralpha (CD25) mRNA levels tended to be down-regulated. IL-8, TNF-alpha and IFN-gamma mRNA expressions appeared to be slightly increased. Intracellular cytokine levels as measured by flow cytometry revealed an increase of IL-1beta, IL-2, and IL-6, whereas IL-12 and TNF-alpha expressions were not affected. IFN-gamma was slightly decreased in the diseased animals. In conclusion, despite the assumption, that the cellular immune response to PMWS as a virus-induced disease should be characterized by either a Th1 driven cytokine profile or a cytokine profile indicative of T cell immunosuppression, our results did not support that hypothesis. Nevertheless, data from intracellular cytokine detection suggest an even increased percentage of the remaining lymphocytes capable to produce IL-2 upon in vitro stimulation, which is in contrast to the slightly diminished IL-2 mRNA levels reflecting the in vivo situation at least at the mRNA level.  相似文献   

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