Non-tolerant Control Research Articles

Identification of Novel Immunosuppressive Drug Targets for Liver Transplantation Using Genome-Scale Metabolic Modeling

Abstract The global database on donation and transplantation reported 129,681 worldwide organ transplants in 2020, with kidneys and livers being the most commonly transplanted organs. Rejection of the transplanted organ by the host's immune system is a common complication and a major reason for graft failure and patient mortality. To address these limitations, we developed genome-scale metabolic models of liver and CD4+ T cells using the pipeline for MetAbolic Drug Repurposing IDentification-MADRID and integrating our data analysis results from RNA-seq, proteomics, and microarray human sample datasets (step 1). Drug targets obtained from the ConnectivityMap database are mapped to metabolic genes in the model where systematic knockouts are performed on each gene which leads to identifying differential fluxes (step 2). Our human-cell-specific models identified novel immunosuppressive drug targets by integrating differentially expressed genes in post-liver transplanted non-tolerant versus healthy control individuals and differential fluxes (step 3). We identified 193, 217, and 196 potential metabolic gene targets for T-helper 1, 2, and 17, respectively, against patients with a non-tolerant phenotype after liver transplantation. This genome-scale metabolic modeling approach for liver transplantation can assist in personalized treatment by using any individual’s sample data at step 1 and 2. It can also save time and resources in preclinical drug development studies by predicting more precise drug targets and drug repurposing based on patients’ omics data. By using this approach, we aim to improve the success rates of liver transplantations and reduce the complications associated with organ rejection. The work was supported by University of Nebraska collaboration initiatives.

Toxicological and morphological analysis of Africanized Apis mellifera selected for tolerance to the neonicotinoid thiamethoxam

Honeybees are the insect most used for pollination purposes due to its efficient characteristics for this function, which reflects in positive aspects for both nature and man. The expansion of agriculture and the development of agrochemicals to combat pests has had negative impacts on honeybee health, causing its disappearance around the world. This research aimed to evaluate the effects of honeybee exposure to the neonicotinoid insecticide thiamethoxam on its survival rate, as well as on morphological and histological changes in the midgut of adult workers from the F4 generation of Apis mellifera queens tolerant to thiamethoxam and non-tolerant worker honeybees. After the bioassays, the midgut was removed for morphological evaluation. The results showed that F4 bees were more tolerance to thiamethoxam and exhibited less significant morphological changes when compared to the non-tolerant control group.

Naphthalene DNA adduct formation and tolerance in the lung

Naphthalene (NA) is a respiratory toxicant and possible human carcinogen. NA is a ubiquitous combustion product and significant component of jet fuel. The National Toxicology Program found that NA forms tumors in two species, in rats (nose) and mice (lung). However, it has been argued that NA does not pose a cancer risk to humans because NA is bioactivated by cytochrome P450 monooxygenase enzymes that have very high efficiency in the lung tissue of rodents but low efficiency in the lung tissue of humans. It is thought that NA carcinogenesis in rodents is related to repeated cycles of lung epithelial injury and repair, an indirect mechanism. Repeated in vivo exposure to NA leads to development of tolerance, with the emergence of cells more resistant to NA insult. We tested the hypothesis that tolerance involves reduced susceptibility to the formation of NA-DNA adducts. NA-DNA adduct formation in tolerant mice was examined in individual, metabolically-active mouse airways exposed ex vivo to 250 μM 14C-NA. Ex vivo dosing was used since it had been done previously and the act of creating a radioactive aerosol of a potential carcinogen posed too many safety and regulatory obstacles. Following extensive rinsing to remove unbound 14C-NA, DNA was extracted and 14C-NA-DNA adducts were quantified by accelerator mass spectrometry (AMS). The tolerant mice appeared to have slightly lower NA-DNA adduct levels than non-tolerant controls, but intra-group variations were large and the difference was statistically insignificant. It appears the tolerance may be more related to other mechanisms, such as NA-protein interactions in the airway, than DNA-adduct formation.

A relative quantitative Methylation-Sensitive Amplified Polymorphism (MSAP) method for the analysis of abiotic stress

BackgroundWe present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms.MethodsFive Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward’s Agglomeration method whereas MSAP characteristics for ANOVA.ResultsRelative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines.ConclusionsOur relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII–MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control and stressed Al-tolerant and non-tolerant triticale inbred lines. The method could also be used to analyze methylation events affecting CG and CHG contexts, which were differentially methylated under Al stress. We cannot exclude that the methylation changes revealed among lines as well as between Al-tolerant and non-tolerant groups of lines were due to some experimental errors or that the number of lines was too small for ANOVA to prove the influence of Al stress. Nevertheless, we suspect that Al tolerance in triticale could be partly regulated by epigenetic factors acting at the level of DNA methylation. This method provides a valuable tool for studies of abiotic stresses in plants.

Identification of Methylmercury Tolerance Gene Candidates in Drosophila

Methylmercury (MeHg) is a ubiquitous environmental contaminant that preferentially targets the developing nervous system. Variable outcomes of prenatal MeHg exposure within a population point to a genetic component that regulates MeHg toxicity. We therefore sought to identify fundamental MeHg tolerance genes using the Drosophila model for genetic and molecular dissection of a MeHg tolerance trait. We observe autosomal dominance in a MeHg tolerance trait (development on MeHg food) in both wild-derived and laboratory-selected MeHg-tolerant strains of flies. We performed whole-genome transcript profiling of larval brains of tolerant (laboratory selected) and nontolerant (control) strains in the presence and absence of MeHg stress. Pairwise transcriptome comparisons of four conditions (+/-selection and +/-MeHg) identified a "down-down-up" expression signature, whereby MeHg alone and selection alone resulted in a greater number of downregulated transcripts, and the combination of selection + MeHg resulted in a greater number of upregulated transcripts. Functional annotation cluster analyses showed enrichment for monooxygenases/oxidoreductases, which include cytochrome P450 (CYP) family members. Among the 10 CYPs upregulated with selection + MeHg in tolerant strains, CYP6g1, previously identified as the dichlorodiphenyl trichloroethane resistance allele in flies, was the most highly expressed and responsive to MeHg. Among all the genes, Turandot A (TotA), an immune pathway-regulated humoral response gene, showed the greatest upregulation with selection + MeHg. Neural-specific transgenic overexpression of TotA enhanced MeHg tolerance during pupal development. Identification of TotA and CYP genes as MeHg tolerance genes is an inroad to investigating the conserved function of immune signaling and phase I metabolism pathways in MeHg toxicity and tolerance in higher organisms.

Concomitant enhancement of the response to Mls-1a antigens and the induction of post-thymectomy autoimmune gastritis in BALB/c mice.

We examined the role of Mls antigens in the induction of autoimmune gastritis (AIG) in BALB/c and DBA/2 mice subjected to thymectomy. The prevalence of AIG in Mls-1b mice which underwent thymectomy on day 3 after birth (3d-Tx) was 78% (mean), while in Mls-1a DBA/2 mice it was < 6%. Whereas AIG-negative 3d-Tx DBA/2 mice produced 2-mercaptoethanol (2-ME)-sensitive antiparietal cell autoantibody, AIG-positive BALB/c mice made 2-ME-resistant anti-parietal cell autoantibody. In addition, the prevalence of AIG in 3d-Tx BALB/c mice which were rendered tolerant to MIs-1a antigens by injection of bone marrow cells from (BALB/c x DBA/2)F1 mice within 24 h after birth was decreased compared with the non-tolerant control mice; the prevalence being 80% in the controls and 30% in the tolerant animals. Thus, the activation of helper T cells, including T cells responding to Mls-1a antigens and including immunoglobulin class switch, appeared to be closely associated with the induction of AIG. Flowcytometric analysis confirmed that CD4- V beta 6 T cells had increased in the regional lymph nodes of the stomach in AIG mice. However, an increase in the number of V beta 11 T cells, which are known to increase in 3d-Tx mice, occurred in the CD8, but not in the CD4 T cell population. Injection of MoAb to L3T4, but not Lyt2, V beta 6- or V beta 8-TCR, into 3d-Tx BALB/c and syngeneic nude mice which had received spleen cells of 3d-Tx BALB/c mice bearing AIG completely abrogated the development of AIG, despite there being remarkable decreases in T cells expressing relevant markers to the injected antibodies in all the mice. These findings suggest that the increase of V beta 6+ L3T4+ T cells in AIG mice was concomitant with the activation of AIG-inducing V beta 6- L3T4+ T cells.

A Study of T Cell Tolerance to the Tumor-Associated Antigen MDM2: Cytokines Can Restore Antigen Responsiveness, but Not High Avidity T Cell Function

BackgroundMost tumor-associated antigens (TAA) currently used for immunotherapy of cancer are also expressed in normal tissues, which may induce tolerance and impair T cell-mediated immunity. However, there is limited information about how physiological expression in normal tissues alters the function of TAA-specific T cells.Methodology/Principal FindingsWe used a T cell receptor transgenic model to study how MDM2 expression in normal tissues affects the function of T cells specific for this TAA that is found at high levels in many different types of tumors. We found that some MDM2-specific T cells escaped thymic deletion and persisted in the peripheral T cell pool. When stimulated with antigen, these T cells readily initiated cell division but failed to proliferate and expand, which was associated with a high rate of apoptosis. Both IL-2 and IL-15 efficiently rescued T cell survival and antigen-specific T cell proliferation, while IL-7 and IL-21 were ineffective. Antigen-stimulated T cells showed impaired expression of the effector molecules CD43, granzyme-B and IFN-γ, a defect that was completely restored when T cells were stimulated in the presence of IL-2. In contrast, IL-15 and IL-21 only restored the expression of CD43 and granzyme-B, but not IFN-γ production. Finally, peptide titration experiments with IL-2 rescued T cells indicated that they were of lower avidity than non-tolerant control T cells expressing the same TCR.Conclusions/SignificanceThese data indicate that cytokines can rescue the antigen-specific proliferation and effector function of MDM2-specific T cells, although this does not lead to the recovery of high avidity T cell function. This study sheds light on possible limitations of immunotherapy approaches that target widely expressed TAA, such as MDM2.

Cellular mRNA expression of interferon-gamma (IFN-gamma), IL-4 and transforming growth factor-beta (TGF-beta) in rats nasally tolerized against experimental autoimmune myasthenia gravis (EAMG).

Nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR plus Freund's complete adjuvant (FCA) resulted in prevention or marked decrease of the severity of EAMG, suppression of AChR-specific B cell responses and of AChR-reactive T cell functions. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radio-labelled synthetic oligonucleotide probes was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine IFN-gamma, the B cell stimulating IL-4 and the immune response-down-regulating TGF-beta. Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4 and TGF-beta mRNA-expressing cells compared with control rats receiving PBS nasally and injected with FCA only. Nasal tolerance to EAMG was accompanied by decreased numbers of AChR-reactive IFN-gamma and IL-4 mRNA-expressing cells, and strong up-regulation of TGF-beta mRNA-positive cells in lymphoid organs compared with non-tolerized EAMG control rats. The relative affinity of anti-AChR antibodies was lower, but muscle AChR amounts were higher in nasally tolerized rats compared with non-tolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG, and that TGF-beta plays an important role in tolerance induction to EAMG.

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 (IFNγ) production by activated T cells. To determine the role of IFNγ 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. IFNγ 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 IFNγ 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 IFNγ expression starting on day 6 and had no IL10 expression. Early induction of IFNγ expression by oral antigen may be associated with systemic tolerance in the experimental colitis setting. In contrast, late expression of IFNγ is associated with a pro-inflammatory response in non-tolerized controls.

Oral administration of specific antigens to allergy-prone infant dogs induces IL-10 and TGF-β expression and prevents allergy in adult life

Background: Oral administration of allergens can induce immune tolerance to specific allergens in rodents and hence might be a possibility to prevent and treat allergic diseases in human subjects. However, the gastrointestinal tract of mice is different from that of human subjects. The absorption of specific antigens and subsequent antigen presentation to intestinal T cells is different in both species, making it difficult to extrapolate results. Objective: We investigated primary oral tolerance to ovalbumin (OVA) in an IgE high-responder dog model, which is more predictive for human allergic diseases than corresponding rodent models. Methods: Oral tolerance was induced by means of a 28-day treatment with OVA dissolved in cow's milk. Results: We observed reduced OVA-specific IgE and IgG production in response to ensuing subcutaneous challenges. Allergic conjunctivitis induced by means of ocular and airway provocation was significantly reduced in tolerized animals compared with that seen in nontolerized control animals. In addition, eosinophilia and neutrophilia in bronchoalveolar lavage fluid and bronchoconstriction after airway allergen challenge were significantly suppressed in tolerized animals. Cytokine analysis by means of real-time PCR on bronchoalveloar fluid cells after allergen challenge revealed a high-level expression of IL-10 and transforming growth factor β, predominantly in the CD14+ population. Conclusion: Feeding infant beagles with OVA for 4 weeks is sufficient to prevent hallmark manifestations of asthma and allergy in adult life. The mechanism of oral tolerance involved an increased expression of IL-10 and transforming growth factor β cytokines. (J Allergy Clin Immunol 2003;111:1069-75.)

Oral administration of specific antigens to allergy-prone infant dogs induces IL-10 and TGF-beta expression and prevents allergy in adult life.

Oral administration of allergens can induce immune tolerance to specific allergens in rodents and hence might be a possibility to prevent and treat allergic diseases in human subjects. However, the gastrointestinal tract of mice is different from that of human subjects. The absorption of specific antigens and subsequent antigen presentation to intestinal T cells is different in both species, making it difficult to extrapolate results. We investigated primary oral tolerance to ovalbumin (OVA) in an IgE high-responder dog model, which is more predictive for human allergic diseases than corresponding rodent models. Oral tolerance was induced by means of a 28-day treatment with OVA dissolved in cow's milk. We observed reduced OVA-specific IgE and IgG production in response to ensuing subcutaneous challenges. Allergic conjunctivitis induced by means of ocular and airway provocation was significantly reduced in tolerized animals compared with that seen in nontolerized control animals. In addition, eosinophilia and neutrophilia in bronchoalveolar lavage fluid and bronchoconstriction after airway allergen challenge were significantly suppressed in tolerized animals. Cytokine analysis by means of real-time PCR on bronchoalveloar fluid cells after allergen challenge revealed a high-level expression of IL-10 and transforming growth factor beta, predominantly in the CD14(+) population. Feeding infant beagles with OVA for 4 weeks is sufficient to prevent hallmark manifestations of asthma and allergy in adult life. The mechanism of oral tolerance involved an increased expression of IL-10 and transforming growth factor beta cytokines.

Active suppression in orally tolerized rats coincides with in situ transforming growth factor-beta (TGF-beta) expression in the draining lymph nodes.

Adult rats were fed pellets containing ovalbumin (OvA) during 4 weeks, and were 2 weeks thereafter immunized subcutaneously with a mixture of OvA and human serum albumin (HSA) in Freund's complete adjuvant (day 0). As a result of the immunization, the draining lymph nodes of the nontolerized (control) rats were heavily enlarged from day 10 to day 18; however, this size increase was absent in the OvA-fed rats. This manifestation of active suppression in the tolerized rats was preceded by the appearance of scattered CD4+ TGF-beta-expressing T cells in the T cell area of their lymph nodes (days 5-8); correspondingly, the levels of TGF-beta mRNA in the nodes were elevated in the tolerant rats compared with the control rats. The anti-OvA antibody levels in sera from the rats revealed that there was an initial B cell priming in the OvA-fed group, with levels higher than in the control group during the first week. Thereafter, suppression governed the response, and from day 10 onwards the anti-OvA levels were considerably lower than in the controls. When other groups of animals were pretreated with neutralizing anti-TGF-beta antibodies 1 day before the immunization, the anti-OvA response of the OvA-fed rats was restored to the levels of the control group, demonstrating the importance of TGF-beta in the maintenance of suppression. In conclusion, we demonstrate that TGF-beta-producing cells appear in the draining lymph nodes shortly after immunization in rats made orally tolerant using a relatively high-dose feeding regime; these cells are probably responsible for the down-regulation of the immune response observed in the OvA-fed rats.

ENDOTOXIN TOLERANCE IN RATS: EXPRESSION OF TNF-α, IL-6, IL-10, VCAM-1 AND HSP 70 IN LUNG AND LIVER DURING ENDOTOXIN SHOCK

Endotoxin can induce a state of tolerance against its own pathological effects, commonly referred to as endotoxin tolerance. This phenomenon has been found to be associated with reduced serum levels of cytokines such as TNF-α, IL-1, IL-6 and IL-10. In the present study the expression of TNF-α, IL-6, IL-10, the adhesion molecule VCAM-1 and the heat shock protein 70 was determined in vivo in lung and liver of LPS-tolerant and naive rats by means of semiquantitative RT-PCR after i.v. LPS injection. TNFα, IL-6, IL-10, HSP70 and VCAM-1 were induced in lung and liver after LPS injection. In liver and lung of endotoxin-tolerant rats TNF-α and IL-6 were induced to a lower degree after LPS treatment when compared to non-tolerant controls. The LPS-induced IL-10 expression was also slightly attenuated in the lung of tolerant rats, but in the liver no differences between tolerant and non-tolerant animals were observed. HSP70 and VCAM-1 were expressed after systemic LPS treatment in liver and lung. The degree of induction, however, was the same in tolerant and untreated controls. The presented data show that endotoxin tolerance is reflected by a reduced cytokine expression in lung and liver in vivo. On the other hand, levels of expression of the adhesion molecule VCAM-1 and the stress protein HSP70 do not appear to be changed by endotoxin tolerance.

CLONAL DELETION AND TOLERANCE AFTER IN UTERO BONE MARROW TRANSPLANTATION IN MICE

112 Purpose: Clonal deletion of self reactive T-cells has been shown to be a major mechanism of self tolerance during normal immunologic ontogeny. We hypothesized that clonal deletion would also be involved in the induction of donor specific tolerance following in utero bone marrow transplantation (BMT) To test this hypothesis, we transplanted Mlsa- fetal recipients with Mlsa+ donor cells and tested for skin graft tolerance and TCR-Vβ usage after birth. Methods: DBA/2 mice (Mlsa+, Mtv-7+, I-E+) were used as donors of adult bone marrow and Balb/c mice (Mlsa-, Mtv-7-, I-E+) were used as fetal recipients. 106 light density mononuclear cells were injected intraperitoneally into each fetus at 14 days gestation. Surviving animals received donor specific skin grafts at 5 weeks of age. Tolerance was defined as graft acceptance for >8 weeks. CD4+ and CD8+ splenocytes in both tolerant and non-tolerant animals were compared for Vβ4, Vβ6, and Vβ9 TCR usage by dual color flow cytometry. DBA/2 mice normally delete Vβ6+ and Vβ9+ T-cells based on presentation of the Mtv-7 superantigen in the context of I-E. Results were analyzed by two-tailed T-test with p<0.05 considered significant. Results: We injected 22 fetuses and 11 survived to testing. Four of the 11 rejected donor specific skin grafts with a mean survival time of 16.5 days (range 11-24). The remaining 7 animals accepted donor specific skin grafts for >8 weeks. When these animals were tested for Vβ usage, we found that there was a significant decrease in both Vβ6+ and Vβ9+ T-cells in tolerant animals compared with rejecter controls (Table 1). There was no change in the irrelevant Vβ4 subset.Table 1: - TCR Vβ UsageConclusions: Tolerance following Mls-disparate in utero BMT is associated with a significant decrease in Vβ6 and Vβ9 TCR usage among both CD4+ and CD8+ splenic T-cells compared with non-tolerant controls. This is the first direct evidence for a clonal deletion mechanism of tolerance following in utero BMT. Clonal deletion in this model suggests homing of donor cells to the recipient thymus with subsequent antigen presentation at an immature stage of T-cell development, resulting in deletion of both CD4+ and CD8+ T-cells. However, deletion was not complete in this model, suggesting that other mechanisms including anergy and suppression may also play a role in fetal tolerance induction.

Synthetic peptides fail to induce nasal tolerance to experimental autoimmune myasthenia gravis

Nasal administration of Torpedo acetylcholine receptor (AChR) to Lewis rats prior to induction of experimental autoimmune myasthenia gravis (EAMG) is highly efficient in prevention of clinical weakness, and suppression of AChR-specific T and B cell responses. To identify possible antigenic determinants within the receptor which can modulate EAMG and anti-AChR response, we evaluated the effects of nasal administration of α 61–76, α 100–116, α 146–162, δ 354–367, and α 261–277 of Torpedo AChR at different doses on the tolerance induction against EAMG irrespective if given at lower, the same or higher doses than whole Torpedo AChR protein, that was confirmed to be highly efficient as tolerogen to EAMG. None of these peptides, neither administrated alone nor in combination, induced tolerance to EAMG. Peptide administration did not affect the levels or affinities of anti-AChR antibodies when compared with non-tolerized control EAMG rats, while administration of whole AChR protein affected both variables. The results may indicate that the T and B cell heterogeneity of AChR epitopes makes it difficult to induce tolerance using synthetic peptide.

Skin graft tolerance across a discordant xenogeneic barrier.

Specific T-cell tolerance may be essential for successful xenotransplantation in humans. Grafting of thymectomized, T cell-depleted normal mice with xenogeneic fetal pig thymus and liver (FP THY/LIV) tissue results in the recovery of functional CD4 antigen-positive cells. We have tested T-cell tolerance by skin grafting. Donor-matched pig skin survived permanently (> 200 days), whereas allogeneic mouse skin was rapidly rejected. Nontolerant control mice rejected pig skin within 26 days. Both porcine and murine histocompatibility class IIhigh cells were detected in long-term thymus grafts, and T-cell repertoire analyses suggested that tolerance to both donors and recipients developed, at least in part, by intragraft clonal deletion. This study demonstrates the principle that tolerance, measured by the stringent criterion of skin grafting, can be induced across a widely disparate species barrier.

Mucosal tolerance to experimental autoimmune myasthenia gravis is associated with down-regulation of AChR-specific IFN-gamma-expressing Th1-like cells and up-regulation of TGF-beta mRNA in mononuclear cells.

Oral and nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freund's adjuvant (CFA) resulted in prevention or marked decrease of the severity of experimental autoimmune myasthenia gravis (EAMG) and suppression of AChR-specific B-cell responses and of AChR-reactive T-cell function. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radiolabeled cDNA oligonucleotide proves was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine interferon-gamma (IFN-gamma), the B cell-stimulating interleukin-4 (IL-4), and the immunosuppressive transforming growth factor-beta (TGF-beta). Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells, compared to control rats receiving PBS orally or nasally and injected with CFA only. Oral and nasal tolerance was accompanied by decreased numbers of AChR-reactive IFN-gamma and IL-4 mRNA-expressing cells and strong up-regulation of TGF-beta mRNA-positive cells in lymphoid organs when compared to nontolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG and that TGF-beta plays an important role in tolerance induction to EAMG.

Thermal hyperalgesia in association with the development of morphine tolerance in rats: roles of excitatory amino acid receptors and protein kinase C

In a rat model of morphine tolerance, we examined the hypotheses that thermal hyperalgesia to radiant heat develops in association with the development of morphine tolerance and that both the development and expression of thermal hyperalgesia in morphine-tolerant rats are mediated by central NMDA and non-NMDA receptors and subsequent protein kinase C (PKC) activation. Tolerance to the analgesic effect of morphine was developed in rats utilizing an intrathecal repeated treatment regimen. The development of morphine tolerance and thermal hyperalgesia was examined by employing the tail-flick test and paw-withdrawal test, respectively. Intrathecal MK 801 (an NMDA receptor antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; a non-NMDA receptor antagonist), or GM1 ganglioside (an intracellular PKC inhibitor) treatment was given to examine the effects of these agents on the development and expression of thermal hyperalgesia in morphine-tolerant rats. Tolerance to the analgesic effect of morphine was reliably developed in rats following once daily intrathecal (onto the lumbosacral spinal cord) injection of 10 micrograms of morphine sulfate for 8 consecutive days as demonstrated by the decreased analgesia following morphine administration on day 8 as compared to that on day 1. In association with the development of morphine tolerance, thermal hyperalgesia to radiant heat developed in these same rats. Paw-withdrawal latencies were reliably decreased in morphine-tolerant rats as compared to nontolerant (saline) controls when tested on day 8 before the last morphine treatment and on day 10 (i.e., 48 hr after the last morphine treatment). The coincident development of morphine tolerance and thermal hyperalgesia was potently prevented by intrathecal coadministration of morphine with MK 801 (10 nmol) or GM1 (160 nmol), and partially by CNQX (80 nmol). MK 801 (5, 10 nmol, not 2.5 nmol) and CNQX (80, 160 nmol, not 40 nmol), but not GM1 (160 nmol), also reliably reversed thermal hyperalgesia in rats rendered tolerant to morphine when tested 30 min after each drug treatment on day 10 (48 hr after the last morphine treatment). The data indicate that thermal hyperalgesia develops in association with the development of morphine tolerance and that the coactivation of central NMDA and non-NMDA receptors is crucial for both the development and expression of thermal hyperalgesia in morphine-tolerant rats. Furthermore, intracellular PKC activation plays a critical role in the development of thermal hyperalgesia in morphine-tolerant rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Cross-tolerance between carbamazepine and valproate on amygdala-kindled seizures

Carbamazepine and valproate are two clinically used anticonvulsants which are also effective in the treatment of manic-depressive illness. Although the biochemical profiles of these drugs are markedly different, some mechanisms in common may be implied by their partially overlapping spectrum of therapeutic efficacy in seizure and affective disorders. Further evaluation of common biological targets of these agents was attempted by determining whether cross-tolerance would occur to the anticonvulsant effects of carbamazepine and valproate on amygdala-kindled seizures. It had previously been shown that tolerance to carbamazepine's anticonvulsant effects on amygdala-kindled seizures occurs only with contingent drug administration i.e., it occurs only when the drug is injected before the kindling stimulation, and not when the drug is given after the seizure. In the current studies, rats that were made tolerant to carbamazepine showed cross-tolerance to valproate. Kindled rats given carbamazepine after each seizure stimulation (i.e., non-tolerant controls) did not show tolerance to valproate's anticonvulsant effects, indicating that the cross-tolerance between carbamazepine and valproate was also contingent. The clinical implications and potential common biochemical target mechanisms of the cross-tolerance between carbamazepine and valproate deserve further investigation.

ABSENCE OF TOLERANCE TO NONINHERITED MATERNAL TRANSPLANTATION ANTIGENS EXPRESSED ON SKIN AND CORNEA1

Tolerance to noninherited maternal antigens (NIMAs) of the MHC was investigated in a rat model involving both skin and corneal transplants. Recipient animals were obtained by backcrossing F1 hybrids to parental strain animals. In one group of experiments, crosses were (DA[RT1a] x LEW[RT1(1)]) female-to-LEW male and, in a second group, (DAxPVG[RT1c]) female-to-PVG male. Homozygous backcross offspring (RT1(1/1) or RT1c/c) were putatively tolerant to DA NIMAs if the mother was a hybrid animal, having been exposed to these antigens in utero. The equivalent offspring of hybrid fathers, i.e., LEW female x (DAxLEW) male or PVG female x (DAxPVG) male, served as putatively nontolerant controls. Hemagglutinating antibody levels were measured against the class I RT1Aa antigen on days 7 and 14 after up to three consecutive subcutaneous DA strain skin grafts. Significantly lower titers were found in the putatively tolerant group 7 days after the first skin graft in the RT1a-to-RT1(1) combination (P < 0.05). Levels were not significantly different at any other time point, or at any time point in the RT1a-to-RT1c combination. Tolerance to a corneal graft was not demonstrated in either the strongly rejecting RT1a-to-RT1(1) combination or weakly rejecting RT1a-to-RT1c, whether or not animals were presensitized to RT1a antigens with DA skin grafts. We conclude that tolerance to NIMAs is unimportant in this clinical rat model of transplantation.