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.     

Aminoguanidine administered during the induction of oral tolerance alters the systemic response of the tolerised rats

Herewith we investigated the role of nitric oxide synthase (NOS)-II in the establishment of oral tolerance induced by low antigen dose. To accomplish this, we used a rat model of oral tolerance induced by intragastric administration of low doses of ovalbumin (OVA). NOS-II was inhibited in vivo during the onset of tolerance by intraperitoneal (i.p.) treatment with aminoguanidine (AMG), a selective NOS-II inhibitor. Four experimental groups were generated: (TOL), tolerised rats, receiving OVA but no AMG; (TAG), rats tolerised with OVA and simultaneously receiving AMG i.p.; (CAG), controls treated with AMG but no oral antigen; and (CONT), controls receiving neither OVA nor AMG treatment. The state of oral tolerance was evaluated in all groups by analysing several immune parameters upon subcutaneous administration of OVA in Freund’s complete adjuvant. First, we were able to determine that NOS-II inhibition altered the TH1/TH2 balance in tolerised rats, driving the TH2 anti-OVA response in TOL rats towards TH1 in TAG animals, which showed enhanced delayed hypersensitivity responses. Second, splenocyte cultures from TAG rats showed lower levels of IL-10 production compared to TOL samples as determined by ELISA analysis. Last, we detected the presence of a functional distinct Tr1 regulatory T cell population in spleen samples recovered from TAG animals. Contrary to what happened with TOL Tr1 cells, the levels of Tr1 cells in TAG samples were modified by in vitro stimulation with OVA.All together, these data indicate a preponderant role for NOS-II in the process of oral tolerance induced by low antigen dose.     

Cellular aspects of tolerance. VII. Inheritance of the resistance to tolerance induction

The half-lives of elimination ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of ¹³¹I-RGG from the body of normal A or Balb/c animals was much longer than the $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of SJL mice. At all ages, the $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of normal hybrids (A × SJL, SJL × A, Balb/c × SJL) was similar to or longer than that of the A or Balb/c parents. Thus, in terms of the $\text{T}\text{1}\text{2}$ of normal animals, the SJL responsiveness to ¹³¹I-RGG appeared to be a recessive trait. Tolerance could be induced in newborn animals and, in terms of $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, the degree of unresponsiveness at the age of 6 weeks, was the same in A, Balb/c, A × SJL, and Balb/c × SJL animals but was much shorter in SJL mice. Thus, in neonatally induced tolerance, the duration of tolerance was recessive for the SJL type. The average $\text{T}{}_{\mathrm{<mtext>built1</mtext><mtext>2</mtext>}}$ after tolerance induction in 3-week-old hybrids (A × SJL, SJL × A, Balb/c × SJL) was similar to that of the A or Balb/c parent, but by the 8th and 12th week it approached the average $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of the SJL parent. Comparing 8-week-old hybrids, the average $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was longest in A × SJL hybrids and identical in SJL × A and Balb/c × SJL mice. An examination of $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ distribution in various 8- and 12-week-old crosses and backcrosses revealed a fairly large proportion of individuals with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ which was intermediate between SJL and the other parent. There was a tendency for this number to decrease in 12 weeks as compared to 8-week-old mice. In 8-week-old mice, the number of animals with intermediate $\text{T}{}_{\mathrm{<mtext>built1</mtext><mtext>2</mtext>}}$ was smallest when SJL was the maternal animal [(SJL × A); SJL × (A × SJL); SJL × (SJL × A)]. There was no link between $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of tolerant animals and either the immunoglobulin allotype (MuAl/MuA2) or the C5 eniotype (MuB1 positive/MuB1 negative).     

Analysis of early events occurring during neonatal induction of tolerance to histoincompatible cells in mice

Mice have been analyzed at different times post neonatal inoculation of F₁ hybrid lymphoid cells for their ability to respond to foreign MHC antigens expressed on the tolerizing cell population, and to impart that response phenotype to normal adult spleen cells. At early times following neonatal challenge with F₁ spleen cells all mice produced serum immunoglobulins of F₁ donor origin which inhibited the response of normal adult cells. The antigenic specificity of the inhibitory factors suggests a role for an antireceptor antibody in the inhibition seen, and the presence of such serum-mediated inhibition is indicative of (and may be causally related to) the late appearance of a cell-mediated suppressor mechanism in these mice.     

Special aspects of the curve of the oral glucose tolerance test]

The insulin response of 10 lean and 23 obese subjects with lag-type and borderline O.G.T.T. has been studied. The O.G.T.T. was interpreted according to the criteria of Fajans and Conn. The maximum increase and the area of increase were examined both for blood glucose and plasma I.R.I., and the corresponding I.R.I./glucose ratios calculated. The shape of the insulin response curve is similar to that of glucose curve. The I.R.I./glucose ratios are decreased in the lag-type curves as compared to borderline in the lean subjects while we observed opposite results in obese ones. A possible physiopathological interpretation of this curves is proposed.     

B7.2 (CD86) but not B7.1 (CD80) costimulation is required for the induction of low dose oral tolerance.

Oral administration of Ag leads to systemic unresponsiveness (oral tolerance) to the fed Ag. Oral tolerance is mediated through active suppression by Th2 or TGF-beta-secreting cells or clonal anergy/deletion, depending on the Ag dose used, with low dose favoring active suppression and high dose favoring anergy/deletion. The nature of APC and inductive events leading to the generation of oral tolerance have not been well defined. To determine the role of costimulatory molecules in the induction of oral tolerance, we have tested the effect of anti-B7.1 or anti-B7.2 mAb on the induction of tolerance by both high and low dose Ag feeding regimens. Our results show that the B7.2 molecule is critical for the induction of low-dose oral tolerance. Injection of anti-B7.2 but not anti-B7.1 intact Ab or Fab fragments inhibited the oral tolerance induced by low-dose (0.5 mg) but not high-dose OVA (25 mg) feeding. In addition, anti-B7.2, but not anti-B7.1, inhibited secretion of TGF-beta, one of the primary cytokines that mediates low-dose oral tolerance. Finally, in the in vivo model of experimental allergic encephalomyelitis, anti-B7.2 mAb treatment abrogated protection offered against disease by low-dose myelin basic protein feeding, while anti-B7.1 had no effect. Anti B7.2 had no effect on disease suppression by high-dose oral Ag. These data demonstrate that B7.2 costimulatory molecules play an essential role in the induction of low-dose oral tolerance.     

Adjuvant potential of vitamin E in the induction of experimental allergic encephalomyelitis: histological aspects

In this study we report the effect of Vit. E, as an immunostimulating factor, on the induction of Experimental Allergic Encephalomyelitis in Lewis rat. The animals were inoculated intracutaneously in the plantar areas with emulsion of isologous spinal cord suspensed in Incomplete Freund's Adjuvant (IFI) and Vit. E. Histologic examination revealed the basic lesion of a perivenous cuff of mononuclear cells and small areas of demyelinated axons. The clinical signes are graded in order to the time of induction. It is possible an action of "adjuvanticity" of Vit. E on the various cells involved in the immune response by cell transformation and moltiplication.     

Thymus dependency of neonatal tolerance induction in the absence of detectable suppressor cells

Neonatal thymectomy prevents tolerance induction with bovine serum albumin (BSA) in Wistar Furth (WF) rats whose thymus-derived (T) cell deficit is reconstituted with adult nonadherent peripheral blood lymphocytes (PBL). Sham-thymectomized (STx) rats given PBL become tolerant. To establish whether the adult T cells become tolerant in STx rats, their carrier-reactivity was studied in a cooperative immune response following challenge with methylated BSA (mBSA). The results indicate that carrier-reactive cells, derived from PBL, do become tolerant of BSA in the presence, but not in the absence, of the thymus. To determine whether thymic function during tolerance induction is mediated by suppressor T cells, attempts were made to replace the thymus with various populations of thymocytes or lymphoid cells from neonatal or adult normal rats or neonatal BSA-injected rats. No cell population tried could substitute for the thymus during tolerance induction. In addition, it was found that BSA-tolerant rats with intact thymi do not contain either nonspecific suppressor cells whose activity can be boosted with mBSA or specific suppressor activity demonstrable on transfer to normal rats. Timed thymectomy experiments showed that the thymus is required for more than 2, but less than 5 to 7 days after tolerogen injection for significant tolerance induction. These results imply that the thymus itself is necessary for tolerance induction in a peripheral T-cell population and that its effect is not mediated by suppressor cells. It is suggested that peripheral T helper cells may periodically recirculate through the thymus, at least in young rats, and become tolerant of antigen complexed with Ia antigens in the thymic epithelium. Such a mechanism may be of great importance in the development of self-recognition.     

A novel costimulation pathway via the 4C8 antigen for the induction of CD4+ regulatory T cells

CD4(+)CD25(+) regulatory T (Treg) cells naturally occur in mice and humans, and similar Treg cells can be induced in vivo and in vitro. However, the molecular mechanisms that mediate the generation of these Treg cell populations remain unknown. We previously described anti-4C8 mAbs that inhibit the postadhesive transendothelial migration of T cells through human endothelial cell monolayers. We demonstrate in this work that Treg cells are induced by costimulation of CD4(+) T cells with anti-CD3 plus anti-4C8. The costimulation induced full activation of CD4(+) T cells with high levels of IL-2 production and cellular expansion that were comparable to those obtained on costimulation by CD28. However, upon restimulation, 4C8-costimulated cells produced high levels of IL-10 but no IL-2 or IL-4, and maintained high expression levels of CD25 and intracellular CD152, as compared to CD28-costimulated cells. The former cells showed hyporesponsiveness to anti-CD3 stimulation and suppressed the activation of bystander T cells depending on cell contact but not IL-10 or TGF-beta. The suppressor cells developed from CD4(+)CD25(-)CD45RO(+) cells. The results suggest that 4C8 costimulation induces the generation of Treg cells that share phenotypic and functional features with CD4(+)CD25(+) T cells, and that CD25(-) memory T cells may differentiate into certain Treg cell subsets in the periphery.     

Limiting the amount and duration of antigen exposure during priming increases memory T cell requirement for costimulation during recall

Donor-reactive memory T cells (Tmem) can play an important role in mediating graft rejection after transplantation. Transplant recipients acquire donor-reactive Tmem not only through prior sensitization with alloantigens but also through previous exposure to environmental pathogens that are cross-reactive with allogeneic peptide-MHC complexes. Current dogma suggests that most, if not all, Tmem responses are independent of the requirement for CD28 and/or CD154/CD40-mediated costimulation to mount a recall response. However, heterogeneity among Tmem is increasingly being appreciated, and one important factor known to impact the function and phenotype of Ag-specific T cell responses is the amount/duration of Ag exposure. Importantly, the impact of Ag exposure on development of costimulation independence is currently unknown. In this study, we interrogated the effect of decreased Ag amount/duration during priming on the ability of donor-reactive Tmem to mediate costimulation blockade-resistant rejection during a recall response after transplantation in a murine model. Recipients possessing donor-reactive Tmem responses that were generated under conditions of reduced Ag exposure exhibited similar frequencies of Ag-specific T cells at day 30 postinfection, but, strikingly, failed to mediate costimulation blockade-resistant rejection after challenge with an OVA-expressing skin graft. Thus, these data demonstrate the amount/duration of Ag exposure is a critical factor in determining Tmem's relative requirement for costimulation during the recall response after transplantation.     

Dietary melibiose regulates th cell response and enhances the induction of oral tolerance

We examined how dietary melibiose affected the T-helper (Th) cell responses induced by an orally fed antigen in ovalbumin (OVA)-specific T cell receptor transgenic mice (OVA 23-3). Dietary melibiose markedly decreased the Th2 type responses as shown by a significant decrease in the interleukin (IL)-4 production and T cell proliferative response induced by sensitization from the 7-d oral administration of OVA. It was additionally observed that the Th1 type responses tended to decrease. We therefore examined the effect of melibiose feeding on the induction of immunological tolerance induced by the oral administration of an antigen (oral tolerance). The Th cell responses induced in BALB/c mice by subcutaneous immunization with OVA were suppressed by the prior oral administration of OVA. Such responses in the OVA-fed and immunized mice were further diminished by dietary melibiose. These results suggest that dietary melibiose strongly affected the Th cell responses to an ingested antigen, and further demonstrate the potential of melibiose to enhance the induction of oral tolerance.     

Ketonic bile acids in urine of infants during the neonatal period

Ketonic bile acids have been found to be quantitatively important in urine of healthy infants during the neonatal period. In order to determine their structures, the bile acids in urine from 11 healthy infants were analyzed by gas-liquid chromatography-mass spectrometry (GLC-MS) and three samples with particularly high levels of ketonic bile acids were selected for detailed studies by ion exchange chromatography, fast atom bombardment mass spectrometry, microchemical reactions, and GLC-MS. The major ketonic bile acid was identified as 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-chol-1-enoic acid, not previously described as a naturally occurring bile acid. The positional isomer 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid, recently described as a major urinary bile acid in infants with severe liver diseases, was also excreted by most infants. Three acids related to cholic acid were identified: 7 alpha, 12 alpha-dihydroxy-3-oxo-, 3 alpha, 12 alpha-dihydroxy-7-oxo-, and 3 alpha, 7 alpha-dihydroxy-12-oxo-5 beta-cholanoic acids. Five bile acids having one oxo and three hydroxy groups were also present. Based on mass spectra and biological considerations two of these were tentatively given the structures 1 beta, 7 alpha, 12 alpha-trihydroxy-3-oxo- and 1 beta, 3 alpha, 12 alpha-trihydroxy-7-oxo-5 beta-cholanoic acids. Some of the others had a hydroxy group at C-4 or C-2. The levels of ketonic bile acids were higher on the third than on the first day of life, and lower after 1 month. The formation and excretion especially of 3-oxo bile acids is proposed to result from changes of the redox state in the liver in connection with birth.     

Beta-endorphin and met-enkephalin in plasma of cattle during pregnancy, parturition and the neonatal period

Plasma concentrations of beta-endorphin and met-enkephalin were measured, with appropriate radioimmunoassays, in cows during gestation and at parturition and in newborn calves. During pregnancy beta-endorphin immunoreactivity (IR) concentration increased, but values during the last month of gestation were not different from those at parturition. Highest met-enkephalin IR levels were obtained in cows during calving. A term Caesarean section caused an increase in plasma beta-endorphin and met-enkephalin IR concentrations, but no such increase occurred in cases of a preterm Caesarean section. In calves beta-endorphin IR values were lower before umbilical cord rupture than immediately after birth. Values decreased continuously thereafter. This was also the case for met-enkephalin IR concentrations in calves born at term. In preterm calves met-enkephalin IR values were low immediately after delivery and increased during the first hour of life. A significant correlation existed between the degree of acidosis and plasma levels of both opioid peptides in the calves. We conclude that a direct stimulation of peripheral beta-endorphin release by the pain or stress associated with calving does not seem to exist in cattle, whereas met-enkephalin seems to be more directly related to parturition. In calves the change to the extrauterine environment causes an immediate, increased release of both opioids.     

The estrogenic effects of clomiphene during the neonatal period in the rat

The ability of clomiphene and its isomers to cause estrogenic responses during the neonatal period in the rat was examined. Rats were injected s.c. with clomiphene (CL), zuclomiphene (ZUC) or enclomiphene (ENC) on days 1,3, and 5 of life and the stimulation of the reproductive tract and estrogen receptor binding was observed. Uterine weight and DNA content were increased significantly by day 7 in animals treated with clomiphene or zuclomiphene. Uterine epithelial hypertrophy was present in all groups by day 10 and hyperplasia was present in the animals treated with ZUC and CL. The time of vaginal opening was greatly accelerated in all drug treated groups with the earliest day of opening occurring on day 7. Ovarian hemorrhage and blood in the periovarian sac occurred between days 12-14 and continued to be present through day 25. Drug treatment caused the estrogen receptor to accumulate in the nuclear fraction of the uterus and to be depleted from the cytosol fraction. We conclude that clomiphene administered to neonatal rats causes estrogenic stimulation of the reproductive tract in a fashion similar to other estrogens. This stimulation may account for the reproductive tract abnormalities which develop in rats treated with those drugs during the neonatal period.     

Improvement of digestibility, reduction in allergenicity, and induction of oral tolerance of wheat gliadin by deamidation

Wheat gliadin was deamidated by using a cation-exchange resin in the presence or absence of added cysteine, with the change in digestibility being measured. The allergenicity of the gliadin was evaluated by using sera from patients RAST-positive to wheat. Gliadin-specific IgE was measured after the gliadin had been orally administered to rats. The addition of cysteine before the treatment with a cation exchanger effectively increased the deamidation level of gliadin. Deamidated gliadin showed higher solubility than the undeamidated form. There was no difference in the peptic digestibility of the gliadin, whereas deamidation enhanced the pancreatic digestibility in vitro and the digestibility in the mouse stomach in vivo. Deamidation of gliadin reduced its reactivity toward the sera of patients with wheat allergy. Rats administered with deamidated gliadin showed suppressed elevation of the gliadin-specific IgE level.