Early expression of interferon gamma following oral antigen administration is associated with peripheral tolerance induction

Oral tolerance is the induction of immune hyporesponsiveness to orally administered antigens. It was described in association with a decrease in interferon gamma (IFNgamma) production by activated T cells. To determine the role of IFNgamma and IL10 in immunemodulation via oral tolerization. Colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid. Treated mice received five oral doses of colitis-extracted proteins (CEPs) every other day, starting immediately after colitis induction. Control mice received similar doses of bovine serum albumin. Colitis was assessed in both groups by standard clinical, micro- and macroscopic scores. IFNgamma and IL10 expression in splenic lymphocytes from both groups was tested by RT-PCR immediately after oral feeding, 1, 4, 8, 12, and 24 h thereafter and then every 24 h for 2 weeks. Feeding of CEPs markedly ameliorated experimental colitis. These mice gained weight and showed markedly improved macro- and microscopic parameters of colitis. Tolerized mice exhibited IFNgamma expression in splenic lymphocytes starting immediately following oral CEP immunization and up to 14 d thereafter. IL10 was expressed starting 1 h after CEP feeding and during the first 48 h thereafter. In contrast, non-tolerized control mice manifested IFNgamma expression starting on day 6 and had no IL10 expression. Early induction of IFNgamma expression by oral antigen may be associated with systemic tolerance in the experimental colitis setting. In contrast, late expression of IFNgamma is associated with a pro-inflammatory response in non-tolerized controls.     

Oral administration of alginic acid oligosaccharide suppresses IgE production and inhibits the induction of oral tolerance

We have found that alginic acid oligosaccharide (ALGO) enhanced Th1 by promoting IL-12 production, suggesting that ALGO can be applied as an anti-allergic food. In this study we examined both positive and negative functions of ALGO. First we investigated the anti-allergic activity of ALGO, as a positive function, when orally administered. IgE production was significantly inhibited in mice fed ALGO as compared to control mice. This result indicates that ALGO had anti-allergic activity even when orally administered. On the other hand, we also found a negative function of ALGO. Oral co-administration of a protein antigen and ALGO inhibited the induction of oral tolerance to the protein. These data indicate the potential of ALGO as an anti-allergic food material and the necessity of further examination to determine a safe method application.     

Splenic dendritic cells induced by oral antigen administration are important for the transfer of oral tolerance in an experimental model of asthma

Peripheral tolerance can be induced after the feeding of Ag, which is referred to as oral tolerance. We demonstrated in this study that the oral administration of OVA induced tolerance in an experimental model of asthma in mice, and investigated which cells function as the regulatory cells in the transfer of this oral tolerance. In OVA-fed mice, the percentage of eosinophils in bronchoalveolar lavage fluid, serum IgE levels, airway hyperresponsiveness, and mRNA levels of IL-13 and eotaxin were significantly lower than found in nonfed mice. Histological examination of lung tissue showed a suppression of the accumulation of inflammatory cells in the peribronchial area of OVA-fed mice. Feeding after the first immunization or between the first and the second immunization suppressed these findings, whereas feeding just before the airway Ag challenge did not. The suppression of disease in OVA-fed mice was successfully transferred by injection of whole spleen cells of OVA-fed mice. When CD11c+ dendritic cells (DCs) were removed from splenocytes, this transfer of suppression was completely abolished. The injection of splenic DCs purified from OVA-fed mice alone transferred the suppression, whereas the injection of splenic DCs from naive mice that were cocultured with OVA in vitro did not. These data suggest that not only CD4+ T cells, but also CD11c+ DCs induced by Ag feeding are important for the active transfer of oral tolerance in this murine experimental model of asthma.     

Manipulation of CD45 antigen in transplantation tolerance

CD45 is known to have tyrosine phosphatase activity for signal transduction of T cells. Immunomodulation of CD45 has been tried to prevent T cell-mediated graft rejection in organ transplantation. In vitro study showed that blockade of CD45RB, an alternative splicing isoform of CD45, inhibited proliferative response of T cells after allogeneic stimulation. Treatment with a monoclonal antibody (mAb) against CD45RB induced long-term allograft acceptance in some mouse organ transplantation models. In a rat heart allograft model, a single injection of anti-rat CD45 (RT7) mAb which bound to allomorphic region of RT7 also induced allograft acceptance. CD45/RT7 is also a useful tool of targeting hematopoietic cells, because of the selective expression on all hematopoietic cells. There are two allomorphic forms of CD45 (RT7a and RT7b) in the rat. Using RT7 system, a rat heart allograft model from RT7a donors to RT7b recipients was designed to test functional relevance of graft-associated hematopoietic cells (microchimerism) to allograft acceptance. Then donor-derived hematopoietic cells were selectively depleted using anti-RT7a mAb in vivo. Depletion on day 0 prevented allograft acceptance and was associated with severe acute or chronic graft rejection, while depletion on day 18 after transplantation showed no effect. This experimental study showed a crucial role of microchimerism in induction phase of allograft acceptance. In conclusion, the CD45/RT7 system is not only a target molecule for tolerance induction, but also an useful tool for experimental models in transplantation immunology. In this review, we introduce basic properties of CD45 and recent results with manipulation of CD45.     

Abrogation of oral tolerance by feeding encapsulated antigen

Results from previous studies have indicated that suppression of immune responses cannot be abrogated once oral tolerance has been established. Using a new methodology, OVA was encapsulated and fed to tolerized BDF1 mice. Results from these studies indicated that although IgG2a titers remained low, total IgG and IgG1 antibody titers were no longer suppressed compared to controls. Furthermore, in vitro splenocyte proliferation was not significantly suppressed in tolerized mice fed encapsulated OVA. To determine whether oral tolerance could be abrogated in other strains of mice, BALB/c mice were tolerized and fed encapsulated OVA. Results from these studies indicated that IgG2a as well as IgG1 and total anti-OVA antibody titers were no longer suppressed. Although splenocyte proliferation did remain significantly suppressed in these mice, IFN-gamma and IL-4 levels were no longer decreased. To the best of our knowledge, this is the first time that abrogation of an established oral tolerance has been demonstrated.     

Persistence of physiological self antigen is required for the regulation of self tolerance

Endogenous Ag requirement for induction and maintenance of T cell tolerance has been extensively investigated in mice that express a transgenic Ag and/or its cognate transgenic TCR. In contrast, studies on tolerance for physiologically expressed self Ag and normal T cells are limited. Herein, we showed that the murine ovarian-specific ZP3 Ag is detectable from birth. Tolerance to ZP3 is detected in female relative to male mice. In comparison to males, 100-fold more ovarian peptide (pZP3) is required to elicit a comparable pathogenic response in females. Female tolerance to pZP3 was dependent on the presence of endogenous ovarian Ag, because neonatal ovariectomy converted the female response to that of males. Moreover, in female mice that were ovariectomized from the ages of 1-6 wk, the pZP3 responses were enhanced to the male level if ovaries were removed up to 7 days, but not 3 days, before adult challenge with pZP3. Thus, the physiologically expressed ZP3 Ag induces tolerance to pZP3, and the maintenance of tolerance is critically dependent on the continuous presence of the endogenous ovarian Ag. In contrast, exposure to endogenous ovarian Ag confined to the neonatal period is insufficient for the induction and maintenance of tolerance to ZP3.     

Route of administration of pentobarbital affects activity of liver glycogen phosphorylase

Liver phosphorylase a activity in intact animals is mostly determined during anesthesia. The aim of this study was to investigate the effect of administering pentobarbital by different routes on activity of liver phosphorylase a. Rats had chronically implanted venous catheters and received pentobarbital (5 mg/100 g body wt) either intraperitoneally, as a slow intravenous infusion, or as an intravenous or intracardial bolus. Times from administration of barbiturate to sampling of the liver were 10 min, 10 min, 85 +/- 32 s (mean +/- SE), and 53 +/- 10 s, respectively. Phosphorylase a activity in % of total phosphorylase activity was 40 +/- 2, 56 +/- 4, 82 +/- 3, and 92 +/- 2, respectively, all significantly different. Thus the route of administration of pentobarbital affects the phosphorylase a activity and should be considered when evaluating this activity. This fact can only be partially explained by differences in duration before the drug takes effect. It is proposed that intraperitoneal injection of pentobarbital may anesthetize hepatic sympathetic nerves or have a direct inhibiting effect on phosphorylase a activity.     

Repeated administration of naltrexone produces tolerance to naloxene-induced hyperalgesia

Repeated administration (i.p.) of the narcotic antagonist naltrexone produced tolerance in mice to naloxone-induced hyperalgesia in the hot plate test. Chronic naltrexone treatment had no effect on pawlick latencies but produced significantly increased jump-reaction times in animals receiving an acute injection of saline. The findings are interpreted as indicating development of supersensitivity in endorphinergic systems mediating affective components in nociception.     

Respiratory tolerance is inhibited by the administration of corticosteroids

Corticosteroids constitute the most effective current anti-inflammatory therapy for acute and chronic forms of allergic diseases and asthma. Corticosteroids are highly effective in inhibiting the effector function of Th2 cells, eosinophils, and epithelial cells. However, treatment with corticosteroids may also limit beneficial T cell responses, including respiratory tolerance and the development of regulatory T cells (T(Reg)), which actively suppress inflammation in allergic diseases. To examine this possibility, we investigated the effects of corticosteroid administration on the development of respiratory tolerance. Respiratory exposure to Ag-induced T cell tolerance and prevented the subsequent development of allergen-induced airway hyperreactivity. However, treatment with dexamethasone during the delivery of respiratory Ag prevented tolerance, such that allergen sensitization and severe airway hyperreactivity subsequently occurred. Treatment with dexamethasone during respiratory exposure to allergen eliminated the development of IL-10-secreting dendritic cells, which was required for the induction of IL-10-producing allergen-specific T(Reg) cells. Therefore, because allergen-specific T(Reg) cells normally develop to prevent allergic disease and asthma, our results suggest that treatment with corticosteroids, which limit the development of T(Reg) cells and tolerance to allergens, could enhance subsequent Th2 responses and aggravate the long-term course of allergic diseases and asthma.     

A strategy for production of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B.

Serum antibody responses to sheep hydatid cyst fluid (SHCF) and a purified Antigen 5 (Ag5) were examined in ELISA, immunoelectrophoresis (IEP) and immunoprecipitation (IP) to facilitate production of monoclonal antibodies (MAb) to E. granulosus Ag5 and Antigen B (AgB). Although sera from mice immunized with SHCF contained antibodies of various classes, the fusions using these donor mice resulted in mainly anti-AgB MAb, possibly due to the preferential selection of MAb to AgB by the SHCF-based ELISA screening system. Donor mice immunized with Ag5 also produced several classes of antibodies, and the resultant fusions enabled selection of IgG MAb to Ag5.     

Induction of tolerance against the Mls antigen in mice no need for Mls-bearing cells

Infusion of CBA mice with spleen cells from the H-2-compatible, but Mis antigen-incompatible, strain C3H leads to a specific reduction of the MLC reactivity of the host's lymphocytes. One explanation of the reduced reactivity could be that the specifically Mls antigen-responsive CBA T cells become exhausted by intense antigen stimulation or that the infused cells actively neutralize specifically responsive cells. In this investigation, we have shown that depletion of membrane Ig-positive cells from C3H × CBA spleen cell preparations strongly reduces their capacity to stimulate CBA lymphocytes in the MLC, indicating that the Mls antigen is expressed on B but not on T cells. However, such B-cell depleted cell preparations were fully capable of reducing the MLC response of CBA hosts. Cell preparations of spleen and lymph nodes exhibited high stimulatory and inhibitory effects. Thymic cells lacked both these characteristics, whereas bone marrow cells were weak stimulators but relatively strong inhibitors. The results support the proposal that the observed reduction of MLC reactivity is due to an active process of the injected cells. The cell type which is working as an inhibitor has not been clearly defined yet.     

Recombinant antigen production for assays of intradermoreaction for diagnosis and surveillance of tuberculosis

The goal of the present work was to develop reagents with potential for tuberculosis diagnosis. Genetic sequences of Mycobacterium tuberculosis secretion antigens were amplified by PCR, cloned into the Gateway^® system, and expressed in Escherichia coli. The recombinant M. tuberculosis proteins were purified by metal affinity chromatography and preparative gel SDS-PAGE electrophoresis followed by electroelution and removal of endotoxins using Triton X-114. In total, seven recombinant proteins were obtained (ESAT-6, CFP10, TB10.3, TB10.4, MTSP11, MPT70, and MPT83). Delayed hypersensitivity reactions (DHR) was evaluated in Cavia porcellus and compared to the response using a standard purified protein derivative (PPD). All seven recombinant proteins produced a positive induration reaction in an intradermal test in guinea pigs previously sensitized with M. tuberculosis. When applied together, at a concentration of each recombinant protein 0.04 mg/mL, the intradermoreaction in C. porcellus was significantly higher than that obtained by standard PPD (p-value = 0.00386).