Mesenteric Node Lymphocytes Research Articles

Oral glucosamine modulates the response of the liver and lymphocytes of the mesenteric lymph nodes in a papain-induced model of joint damage and repair

To assess whether glucosamine (GlcN), an oral supplement commonly taken to relieve the symptoms of osteoarthritis, modulates the immune and inflammatory responses to joint injury in organs proximal to GlcN absorption; namely, the liver and the gut-draining lymph nodes. Using a papain-injected knee mouse model, standard histological methods were used to validate our model and document the impact of GlcN (100mg/kg/day) on groups of C57BL/6 mice (n=5). Circulating inflammatory cytokines were assessed by Luminex-based immunoassays and the relevance of this cytokine profile on proteoglycan biosynthesis evaluated using a patellar-cartilage assay. Real-time PCR was used to document the role of the liver in cytokine production. Finally, we appraised the activation of mesenteric lymph nodes (MLNs) lymphocytes by flow cytometry. Papain significantly degraded the proteoglycans in the injected knees by 2 days. Cartilage proteoglycan content was significantly higher in GlcN-treated, papain-injected knees at Day 14. The peak concentration of serum pro-inflammatory cytokines occurred earlier and decreased sooner in the injected, GlcN-supplemented mice; this trend was in agreement with the expression of these factors by the liver. GlcN did not alter the percentage of MLN populations but accelerated their activation. Oral GlcN alters the physiology of the liver and MLNs, which in turn, could indirectly alter the biology of the injured joint.

Induced susceptibility of host is associated with an impaired antioxidant system following infection with Cryptosporidium parvum in Se-deficient mice.

BackgroundSusceptibility or resistance to infection with Cryptosporidium parvum (C.parvum) correlates with Selenium (Se) deficiency in response to infection. Both adult Se-adequate and Se-deficient mouse models of cryptosporidiosis were used to study the cell-mediated immune response during the course of C. parvum infection.Methodology/Principal FindingsBlood samples from mouse models were used for Se status. The concentration of MDA, SOD, GPx and CAT in blood has revealed that lower Se level exist in Se-deficient mice. Mesenteric lymph node (MLN) lymphocytes from both mouse models were proliferated after ex vivo re-stimulation with C. parvum sporozoite antigen. The study of the cytokine profiles from the supernatant of proliferated MLN cells revealed that Se-adequate mice produced higher levels of Th1 (IFN-γ and IL-2) and moderate amounts of Th2 (IL-4) cytokines throughout the course of infection. Whereas, MLN cells from Se-deficient mice produced lower levels of IFN-γ, IL-2 and IL-4 cytokines. The counts of total white cell and CD3, CD4, CD8 cell in Se-adequate were higher than that in Se-deficient mice.SignificanceThese results suggest that Cell immunity is affected by Se status after infection with C.parvum from kinetic changes of different white cells and cytokine. In conclusion, induced susceptibility of host is associated with an impaired antioxidant system following infection with C.parvum in C57BL/6 Selenium deficient mice.

Immunoglobulin Secretions in the Mesenteric Lymph Node in Stressed Rats

To study the effects of different types of stressors on intestinal immune function, the lymphocyte subsets and associated immunoglobulin production in stressed rats were observed. Physical (electric foot shock) or psychological (non foot-shock) stress respectively were induced using a communication box. Rats were exposed to stress for 2 h per day, and the treatment was maintained for 14 consecutive days. Lymphocytes were isolated from the mesenteric lymph node (MLN) and spleen using Lympholyte-Rat. There was no change the lymphocyte subsets in MLN or spleen in either group. Foot-shock stress increased immunoglobulin secretions in MLN lymphocytes. These results demonstrated that intestinal immune functions were adaptively regulated under conditions of moderate stress.

Effects of Blocking the Chemokine Receptors, CCR5 and CXCR3, With TAK-779 in a Rat Small Intestinal Transplantation Model

The effect of blocking lymphocyte chemokine receptors with TAK-779 was investigated using a rat intestinal transplantation model. Dark Agouti rat small intestines were heterotopically transplanted into Lewis rats. The recipients were treated with TAK-779 (10 mg/kg per day) by subcutaneous injection. Graft survival, histologic changes, mixed lymphocyte reaction, and antibody production were examined. Furthermore, expression of the chemokine receptors on the graft-infiltrating lymphocytes in the mesenteric lymph node (MLN) and Peyer's patches (PP) were measured using fluorescence-activated cell sorter analysis. Average duration of survival was greater for group T (with TAK-779: 9.8+/-1.4) than group A (without TAK-779: 7.0+/-0.6). Histologic findings and immunohistochemistry of the graft were consistent with the prolonged survival in group T. In the fluorescence-activated cell sorter analysis, several CD4+ and CD8+ cells were significantly suppressed in the blood, spleen, and MLN of the recipient and in the PP of the graft on postoperative day (POD) 6, but recovered in recipient spleen and MLN by POD 9. However, double-staining of graft-infiltrating lymphocytes in MLN and PP showed a significant reduction in the numbers of T cells expressing CCR5 and CXCR3 by POD 9. On the other hand, mixed lymphocyte reaction and cytokine production, and the antidonor antibody titer were suppressed on POD 6 but not on POD 9. TAK779 diminished not only the number of the graft-infiltrating cells and their expression of CCR5 and CXCR3, but also the total number of recipient T cells that play a role in graft rejection. Further exploration of these effects will provide the novel treatment of the intestinal transplantation.

Immunoglobulin and Cytokine Production from Mesenteric Lymph Node Lymphocytes Is Regulated by Extracts ofCordyceps sinensisin C57Bl/6N Mice

Cordyceps sinensis, one of the well-known fungi used in traditional Chinese medicine, is recognized to play a role in the metabolic process of inflammation and immunity. The purpose of this study was to evaluate the effects of water extracts of C. sinensis on the immune function of mesenteric lymph node (MLN) lymphocytes in C57Bl/6N mice. C. sinensis-treated mice were administered the respective extract by oral gavage for 4 weeks. Immunoglobulin E concentrations in serum and MLN lymphocytes were significantly lower in C. sinensis-treated mice than in control mice. In contrast, the immunoglobulin A concentration from the C. sinensis group was higher than that in control mice. C. sinensis increased the proportion of CD4(+) and CD8(+) T cells in MLN lymphocytes. C. sinensis significantly decreased interleukin-4 and interleukin-10 cytokine concentrations. Therefore, water extracts of C. sinensis modulate immune parameters through regulation of immunoglobulin production resulting from decreased T-lymphocyte helper 2 cytokine secretion and reduce cytokine secretion in MLN lymphocytes.

Soy lecithin supplementation alters macrophage phagocytosis and lymphocyte response to concanavalin A: a study in alloxan‐induced diabetic rats

Dietary soy lecithin supplementation decreases hyperlipidemia and influences lipid metabolism. Although this product is used by diabetic patients, there are no data about the effect of soy lecithin supplementation on the immune system. The addition of phosphatidylcholine, the main component of lecithin, to a culture of lymphocytes has been reported to alter their function. If phosphatidylcholine changes lymphocyte functions in vitro as previously shown, then it could also affect immune cells in vivo. In the present study, the effect of dietary soy lecithin on macrophage phagocytic capacity and on lymphocyte number in response to concanavalin A (ConA) stimulation was investigated in non-diabetic and alloxan-induced diabetic rats. Supplementation was carried out daily with 2 g kg(-1) b.w. lecithin during 7 days. After that, blood was drawn from fasting rats and peritoneal macrophages and mesenteric lymph node lymphocytes were collected to determine the phospholipid content. Plasma triacylglycerol (TAG), total and HDL cholesterol and glucose levels were also determined. Lymphocytes were stimulated by ConA. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye reduction method and flow cytometry were employed to evaluate lymphocyte metabolism and cell number, respectively. Soy lecithin supplementation significantly increased both macrophage phagocytic capacity (+29%) in non-diabetic rats and the lymphocyte number in diabetic rats (+92%). It is unlikely that plasma lipid levels indirectly affect immune cells, since plasma cholesterol, TAG, or phospholipid content was not modified by lecithin supplementation. In conclusion, lymphocyte and macrophage function were altered by lecithin supplementation, indicating an immunomodulatory effect of phosphatidylcholine.

Dietary plasma proteins, the intestinal immune system, and the barrier functions of the intestinal mucosa1

ABSTRACTThe intestinal mucosa contributes to homeostasis by preventing the entrance of biological and chemical agents across the epithelium that could alter the stability of the system. This protective function is especially important at the time of weaning, when animals are exposed to infectious agents and to numerous stresses such as the change of environment and diet. Diets supplemented with spray-dried plasma or plasma protein fractions have been shown to improve growth performance of farm animals and have been proposed as an alternative to antibiotics. In this review, we summarize our findings on the mechanism of action of dietary plasma proteins using a rat model of intestinal inflammation, based on the administration of Staphylococcus aureus enterotoxin B (SEB). Staphylococcal enterotoxin B activates the gut-associated lymphoid tissue (GALT), increasing T-lymphocytes in Peyer's patches and the number of activated T lymphocytes in mesenteric lymph nodes (organized GALT). In the lamina propria SEB increased cytotoxic T δγ and natural killer cell populations of the diffuse GALT. Staphyloccocal enterotoxin B significantly increased proinflammatory cytokines in Peyer's patches and mucosa. Plasma protein supplements modulated the mucosal immune response in organized and diffuse GALT, protecting GALT from possible excessive activation by the SEB challenge. These effects are accompanied by a reduction of proinflammatory cytokine production, supporting the view that changes in cytokine production mediate the effects of dietary plasma proteins during intestinal inflammation. The increase in mucosal permeability and intestinal secretion induced by SEB was associated with decreased expression of mucosal tight-junction and adherent-junction proteins. Both plasma and plasma protein fractions prevented the effects of SEB on intestinal permeability, thus reducing the exposure of the host to microbial and food antigens across the interstitial space. These findings indicate that dietary plasma proteins modulate functional and structural properties of the intestinal mucosa.

Deficiency of purine nucleoside phosphorylase activity in thymocytes from the immunodeficient diabetic BB rat

The spontaneously diabetic BB (BBd) rat displays marked T lymphopenia. The present study was designed to investigate whether the immunodeficiency in this animal may be associated with deficiency of purine nucleoside phosphorylase (PNP) and possibly adenosine deaminase (ADA). The activities of these two enzymes were measured in lymphoid and non-lymphoid cells from both non-diabetes-prone (BBn) and BBd rats as well as from streptozotocin-induced diabetic (STZ) BBn rats. There were no significant differences between BBn and BBd rats in ADA activities in thymocytes, skeletal muscle or brain. However, ADA activity was increased (P less than 0.01) by 50% in BBd mesenteric lymph node lymphocytes and splenocytes as compared with BBn cells, but was not altered in cells from STZ-BBn rats. On the other hand, the PNP activity in BBd thymocytes was only 61% (P less than 0.01) of that observed in BBn cells. This PNP deficiency was not the consequence of diabetes per se, as its activity was normal in thymocytes from STZ-BBn rats. There were no significant differences in PNP activities between BBn and BBd rats in all other cell types examined. The diabetic BB rat may be a novel source of PNP-deficient thymocytes (mainly immature T cells) for studying biochemical mechanisms of immunodeficiency in association with decreased PNP activity. The findings also raise the question of whether a causal relationship exists between PNP deficiency and the recently demonstrated abnormality in T cell maturation in the thymus of the BBd rat.

Interleukin-23 Restrains Regulatory T Cell Activity to Drive T Cell-Dependent Colitis

SummaryInterleukin-23 (IL-23) is an inflammatory cytokine that plays a key role in the pathogenesis of several autoimmune and inflammatory diseases. It orchestrates innate and T cell-mediated inflammatory pathways and can promote T helper 17 (Th17) cell responses. Utilizing a T cell transfer model, we showed that IL-23-dependent colitis did not require IL-17 secretion by T cells. Furthermore, IL-23-independent intestinal inflammation could develop if immunosuppressive pathways were reduced. The frequency of naive T cell-derived Foxp3+ cells in the colon increased in the absence of IL-23, indicating a role for IL-23 in controlling regulatory T cell induction. Foxp3-deficient T cells induced colitis when transferred into recipients lacking IL-23p19, showing that IL-23 was not essential for intestinal inflammation in the absence of Foxp3. Taken together, our data indicate that overriding immunosuppressive pathways is an important function of IL-23 in the intestine and could influence not only Th17 cell activity but also other types of immune responses.

Effects of diets supplemented with organic acids and nucleotides on growth, immune responses and digestive tract development in weaned pigs

Sixty-eight (Experiment 1, 46 days feeding) and sixteen (Experiment 2, 21 days feeding) 21-days-old weaned pigs were allotted to four dietary treatments including control, 0.6% organic acids (OA), 0.1% nucleotides (NA) and 0.6% OA plus 0.1% NA for determining the dietary effects. In Experiment 1, OA enhanced peripheral blood mononuclear cells proliferation on day 28 and 46. The plasma immunoglobulin (Ig) A level was elevated by OA (p < 0.06) and NA (p < 0.07), respectively. In Experiment 2, NA increased plasma IgM level, and had an interactive effect with OA on ileal Peyer's patches and mesenteric lymph node lymphocyte proliferation, bile and plasma IgA levels, and jejunal crypt depth. NA elevated gastric pepsin and jejunal alkaline phosphatase activities, however, decreased ileal aminopeptidase N, sucrase or maltase activity. These results suggest that OA and NA have synergistically enhanced the gut-associated lymphocyte responses and NA modulates the digestive tract development of weaned pigs.

Paeoniflorin induced immune tolerance of mesenteric lymph node lymphocytes via enhancing beta 2-adrenergic receptor desensitization in rats with adjuvant arthritis

Paeoniflorin (Pae), a monoterpene glucoside, is one of the main bioactive components of total glucosides of paeony (TGP) extracted from the root of Paeonia lactiflora. TGP has anti-inflammatory and immunoregulatory effects. In this study, we investigated the effects of Pae on inflammatory and immune responses to the mesenteric lymph node (MLN) lymphocytes and the mechanisms by which Pae regulates beta 2-adrenergic receptor (beta 2-AR) signal transduction in adjuvant arthritis (AA) rats. The onset of secondary arthritis in rats appeared around day 14 after injection of Freund's complete adjuvant (FCA). Remarkable secondary inflammatory response and lymphocytes proliferation were observed in AA rats, along with the decrease of anti-inflammatory cytokines interleukin (IL)-4 and transforming growth factor-beta 1 (TGF-beta 1) of MLN lymphocytes, and the increase of pro-inflammatory cytokine IL-2. The administration of Pae (50, 100 mg kg −1, days 17–24) significantly diminished the secondary hind paw swelling and arthritis scores, reversed the changes of cytokines as discussed above, and further decreased the lowered proliferation of MLN lymphocytes in AA rats. In vitro, Pae restored the previously increased level of cAMP of MLN lymphocytes at the concentrations of 12.5, 62.5 and 312.5 mg l −1. Meanwhile, Pae increased protein expressions of beta 2-AR and GRK2, and decreased that of beta-arrestin 1, 2 of MLN lymphocytes in AA rats. These results suggested that Pae might induce the Th1 cells immune tolerance, which then shift to Th2, Th3 cells mediated activities to take effect the anti-inflammatory and immunoregulatory effects. The mechanisms of Pae on beta 2-AR desensitization and beta 2-AR-AC-cAMP transmembrane signal transduction of MLN lymphocytes play crucial roles in pathogenesis of this disease.

Phenotype and Effector Function of CC Chemokine Receptor 9-Expressing Lymphocytes in Small Intestinal Crohn’s Disease

CCL25/CCR9 chemokine ligand/receptor pair has been reported to play an important role in small bowel (SB) immunity and inflammation. We have previously reported an aberrant SB expression of CCL25 in Crohn's disease (CD) and an increased frequency of CCR9(+) T cells in the peripheral blood of patients with SB inflammatory diseases such as CD and celiac disease. In this study, we have characterized the phenotype and effector function of CCR9(+) T cells in mucosal lymphoid tissues in CD. We show that CCR9(+) T cells isolated from mesenteric lymph nodes (MLN) draining CD SB express a more activated phenotype compared with MLN draining normal SB. Stimulation of CCR9(+) T cells isolated from CD SB lamina propria produced more IFN-gamma and IL-17 in response to anti-CD3 or IL-12/IL-18 stimulation compared with those isolated from normal SB. The addition of TL1A to the cytokine combination markedly augmented the secretion of IFN-gamma, but not IL-17, by CD lamina propria CCR9(+) T cells. CCL25 incubation of CD SB lamina propria lymphocytes and MLN lymphocytes increased their adhesion to VCAM-1/Fc in vitro. Finally, the TCRVbeta analysis of CCR9(+) T cells revealed a diverse TCRVbeta repertoire among MLN CCR9(+) T cells in patients with SB CD. Our data indicate that CCR9(+) T cells in SB CD are proinflammatory and support the rationale for the use of CCR9 antagonists for the treatment of human SB CD.

Inflammatory response in patients with malignant obstructive jaundice

Objective. Surgery in patients with malignant obstructive jaundice is associated with increased risks for postoperative septic complications. The aim of this study was to investigate the inflammatory and the local cellular immune response in patients accepted for surgery because of tumours in the hepatic-pancreatic-biliary (HPB) tract. Material and methods. Patients with obstructive jaundice (group HPB+) were compared with those without (HPB−). Patients undergoing surgery for benign abdominal disorders served as controls. Obstructive jaundice was present in 18 out of 33 HPB patients. Preoperatively, blood was analysed for bacteria, endotoxins and cytokines (TNF-α, IL-6 and IL-10). At operation, mesenteric lymph nodes (MLNs) were excised for bacterial cultures using standard microbiological techniques, and immunohistochemistry, using antibodies CD4 and CD8 (mainly staining T lymphocytes), CD68 (macrophages), and anti-caspase-3 (to determine the rate of apoptosis). Results. Bacterial translocation was not demonstrated in any of the patients. Increased preoperative concentrations of endotoxins were found in group HPB+. The number of macrophages and the rate of apoptosis in MLNs were increased in jaundiced patients, while the number of T lymphocytes was decreased. Conclusions. Malignant obstructive jaundice causes increased blood concentrations of endotoxins and cytokines, an increased number of macrophages in MLNs, a higher rate of apoptosis in MLNs, but a decreased number of T lymphocytes in MLNs. The lymphocyte depletion is probably due to the increased rate of apoptosis, and might reduce the ability of jaundiced patients to eradicate infection.

Induction of apoptosis in rat lymphocytes by starvation

The aim of the present study was to investigate whether fasting for 24 and 48 h induces apoptosis of rat mesenteric lymph node lymphocytes similar to that observed previously in diabetic patients and alloxan-induced diabetic rats. Several features of lymphocyte death were evaluated by flow cytometry. Plasma levels of glucose, NEFAs (non-esterified fatty acids) and ketone bodies (acetoacetate and beta-hydroxybutyrate) were determined in rats fasted for 24 and 48 h. Lymphocytes obtained from fasted rats had an increase in DNA fragmentation and phosphatidylserine externalization after 48 h of culture, although there was no loss of membrane integrity in lymphocytes even after 48 h of culture. Cytochrome c release from the mitochondrial intermembrane space into the cytosol was increased significantly in lymphocytes from fasted rats cultured for 24 h, whereas the levels of bcl-2 and bax proteins were not affected. Activities of caspases 3, 6, 8 and 9 were increased significantly in lymphocytes from rats fasted for 24 h, whereas only an increase in caspase 3 and 9 activities were observed in rats fasted for 48 h. In conclusion, fasting for 24 and 48 h caused a significant increase in the proportion of lymphocytes undergoing apoptosis. The occurrence of apoptosis was observed by DNA fragmentation, phosphatidylserine externalization, cytochrome c release from the mitochondria and activation of the caspase cascade. These findings support the hypothesis that conditions that raise plasma fatty acids levels (e.g. diabetes and starvation) may impair immune function by causing lymphocyte death.

High Efficiency Cross‐Reactive Monoclonal Antibody Production by Oral Immunization with Recombinant Norwalk Virus‐Like Particles

Monoclonal antibodies (MAbs) are important for in-depth antigenic characterization and diagnosis of infections with human caliciviruses that cause almost all outbreaks of nonbacterial gastroenteritis. We compared different routes of immunization with nonreplicating virus-like particles (VLPs) from recombinant Norwalk virus (rNV) and recombinant Mexico virus (rMX) administered to BALB/c mice to determine the efficiency of hybridoma production. Oral immunization with VLPs without adjuvant resulted in high yields of MAb-secreting hybridomas (90%) to these VLPs of IgG (61%), IgM (29%) and IgA (10%) isotypes. Fusions with mesenteric lymph node lymphocytes yielded MAbs of various subclasses including IgG2a, IgG3, IgM and IgA. These results suggest that an immunization route that mimics the natural route of viral infection pathway may facilitate MAb technology by increasing the yields of antibody secreting hybridoma cells.

Induction of apoptosis in rat lymphocytes by starvation

The aim of this study was to investigate if 24 and 48 h fasting induces apoptosis of rat mesenteric lymph node lymphocytes similarly to what was previously observed in diabetic patients and alloxan-induced diabetic rats. Plasma levels of glucose, free fatty acids and ketone bodies (acetoacetate and β-hydroxybutyrate) were determined in rats fasted for 24 and 48 h. The following features of cell death were evaluated by flow cytometry: loss of membrane integrity, DNA fragmentation, phosphatidylserine exposure, and mitochondrial depolarization. Accumulation of neutral lipids was determined using Nile red. Activities of caspases-3, -6 and -8 were evaluated by using a spectrofluorometric assays. The cytochrome c release from mitochondrial intermembrane space into cytosol was investigated by Western blot analysis. Lymphocytes obtained from 24 and 48 h fasted rats presented an increase in DNA fragmentation and phosphatidylserine externalization. These apoptotic features were more pronounced after 48 h fasting when blood glucose levels were low, and the plasma levels of free fatty acids and ketone bodies were increased. There was no loss of membrane integrity in lymphocytes even after 48 h culture. Cytochrome c release from mitochondria was significantly increased in lymphocytes from fasted rats. Activities of caspases-3,-6 and -8 were significantly increased in lymphocytes from 24h fasted rats. The cells from 48h fasted animals showed an increase of caspase-3 activity only. In conclusion, 24 and 48 h fasting caused a significant increase in the proportion of lymphocytes in apoptosis. Financial Support: FAPESP, CNPq, CAPES.

Phytohaemagglutinin-stimulated transformation of peripheral and lymph-node lymphocytes in patients with gastro-intestinal cancer.

Abstract Phytohaemagglutinin-induced lymphocyte transformation has been studied in 10 patients with gastro-intestinal cancer and in 8 control subjects. When cultured in autologous serum, peripheral lymphocytes from cancer patients exhibited lower transformation indices (L.T.I.) than did those of control subjects. Culture in AB serum resulted in an elevation of the L.T.I. of peripheral lymphocytes from cancer patients whilst those of the controls were essentially unchanged. Of 6 control subjects, 5 showed reduced L.T.I.'s when their circulating lymphocytes were cultured in serum from cancer patients. Mesenteric lymph-node lymphocytes from patients with cancer also showed lower indices compared with the control group and these were elevated by culture in AB serum.

Altered cytokine expression in mesenteric lymph nodes in a rat strain (Matsumoto Eosinophilic Shinshu) that spontaneously develops hypereosinophilia

The Matsumoto Eosinophilic Shinshu (MES) rat is an inbred mutant strain that spontaneously develops systemic hypereosinophilia with eosinophilic inflammatory lesions similar to those associated with hypereosinophilic syndrome in humans and other mammals. To elucidate the pathogenic mechanisms that underlie these features of MES rats, we examined the pattern of cytokine gene expression in mesenteric lymph nodes (MLNs), the thymus, and peripheral blood mononuclear cells as well as the blood clinicopathology and MLN lymphocytic subsets of these animals. MES rats exhibited both leucocytosis, attributable in large part to hypereosinophilia and neutrophilia, and immunoglobulin M (IgM) and IgA gammaglobulinaemia, with increased titres of IgM autoantibodies to nuclear antigens. Reverse transcription and polymerase chain reaction analysis revealed that the amounts of interleukin (IL)-5, IL-4, eotaxin, and interferon-gamma mRNAs were increased in the MLN lymphocytes of MES rats compared with the corresponding values for Sprague-Dawley rats. Intraperitoneal administration of a monoclonal antibody specific for IL-5 resulted in an immediate suppression of hypereosinophilia and a delayed suppression of neutrophilia in MES rats. Flow cytometry revealed an increased percentage of CD3+ CD4- CD8- T lymphocytes in MLNs of MES rats. Our results suggest that the hypereosinophilia of MES rats results from an increased production of IL-5, and that the eosinophilic inflammatory lesions of these animals, which are largely restricted to the gut, may be related both to cytokine overexpression in MLNs and to T helper 1 and 2 immunological responses.

Regional oral tolerance in transgenic 2C mice

Antigen injected subcutaneously (SQ) results in a strong systemic immune response, whereas antigen infused orally or by the portal vein tends to induce tolerance. Nontransgenic C57BL/6J (H-2(b)), or B6, mice and transgenic 2C mice (that express a cytotoxic T cell receptor against major histocompatibility complex Class I L(d)) were used to investigate the regional and systemic responses to oral antigen. B6 (H-2(b)) and 2C (H-2(b)) mice were given either saline or BALB/cByJ (H-2(d), L(d+)) (BALB/c) spleen cells (SCs) (25 x 10(6)) by oral gavage (PO) on day 0. Injection of 10 x 10(6) BALB/c SCs SQ was performed after 7 days, followed by a footpad injection of 10 x 10(6) BALB/c SC on day 14. Specific footpad swelling was measured 24 hours later by means of a micrometer. Mixed lymphocyte culture was performed on splenic and mesenteric lymph node (MLN) lymphocytes. The percentage of splenic and MLN CD4+ and CD8+ T cells was quantitated by fluorescence activated cell sorter. Cytokine messenger RNA (mRNA) and protein was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. Whereas B6 mice were shown to have specifically decreased responsiveness to Balb/c cells in vivo and in vitro after oral Balb/c, 2C mice did not demonstrate any downregulation in delayed-type hypersensitivity responsiveness or splenic lymphocyte proliferation. However, MLN lymphocytes from 2C mice did demonstrate decreased proliferation against Balb/c after oral Balb/c gavage (P < .01). Subset percentages of naïve B6 (n=8) spleen and MLN lymphocytes were 50% to 52% for CD4+ and 46% to 47% for CD8+ T cells. These percentages were unchanged in B6 mice after PO Balb/c. Subset percentages of naïve 2C (n=6) spleen and MLN lymphocytes were 2% to 5% for CD4+ and 61% to 64% for CD8+ T cells. After PO Balb/c, MLN CD4+ and CD8+ T cells were unchanged, whereas splenic CD8+ T cells decreased to 44% to 48% and CD4+ T cells increased to 32% to 33% (P < .05, P < .05 vs naïve 2C spleen). After PO Balb/c, inflammatory interleukin (IL)-2 and interferon gamma mRNA and protein were increased in 2C spleen cells, whereas they were decreased in 2C MLN. Anti-inflammatory cytokine IL-4 and IL-10 mRNA and protein were increased in 2C MLN after PO Balb/c, whereas they were not detectable in 2C spleen cells. Systemic oral tolerance can be induced in B6 mice, whereas only regional mesenteric lymph node tolerance develops in transgenic 2C mice. The inability to induce systemic oral tolerance in 2C mice appears to be due to a significant increase in peripheral (splenic) CD4+ T cells.

Kinetics of Cryptosporidium parvum-specific cytokine responses in healing and nonhealing murine models of C. parvum infection

Susceptibility or resistance to infection with Cryptosporidium parvum correlates with the ability of mice to produce characteristic panels of cytokines in response to infection. Both adult healing and nonhealing mouse models of cryptosporidiosis were used to study the cell-mediated immune response during the course of C. parvum infection. Mesenteric lymph node (MLN) lymphocytes from both mouse models were proliferated after ex vivo re-stimulation with C. parvum sporozoite antigen. Study of the cytokine profile from the supernatant of proliferated MLN cells revealed that healing mice produced greater levels of Th1 (IFN-gamma and IL-2) and moderate amounts of Th2 (IL-4, IL-5, IL-6, and IL-10) cytokines throughout the course of infection. Whereas, MLN cells from nonhealing mice produced no IFN-gamma, low levels of IL-2 and IL-4, and higher levels of IL-5, IL-6, and IL-10 cytokines. These results suggest that the capacity to produce both Th1 and Th2 cytokines, rather than the presence of Th2 cytokines alone, determines the effective immune response against C. parvum infection.