The dietary supplement ephedrine induces beta-adrenergic mediated exacerbation of systemic lupus erythematosus in NZM391 mice.

The dietary supplement and adrenergic receptor agonist ephedrine has been a controversial topic as its safety has been questioned. Beta-adrenergic receptor (beta-AR) activation causes immunomodulation, which may contribute to promotion of autoimmune pathology. This report investigated the ability of ephedrine to exacerbate processes associated with autoimmune disease in a lupus-prone mouse model. To mimic human supplementation, ephedrine was administered to NZM391 (lupus-prone) and BALB/c (nonlupus prone) mice orally twice a day for three months at a dose of 50 and 100 microg/day. Some ephedrine-treated NZM391 mice also were preadministered the beta-AR antagonist propranolol to investigate beta-AR involvement. Mice were bled monthly, and sera were assayed for a variety of lupus manifestations and immunological measurements. In NZM391 males and females, both doses of ephedrine significantly increased lupus manifestations, including IgG production and organ-directed autoantibody titers, and significantly lowered the ratio of IgG2a/IgG1 compared to controls. Ephedrine significantly decreased female lifespan and significantly increased circulating populations of plasma cells (CD38(hi) CD19(lo) cytoplasmic IgG+) and CD40+ B1a cells, while preventing an age-related decrease in the B1a cell population expressing a high level of CD5. While ephedrine induced gender-specific immunomodulation in BALB/c mice, increases in the lupus manifestations of anti-dsDNA titers and serum urea nitrogen were not detected. Preadministration of propranolol decreased lupus manifestations and serum levels of IgG and IgE in ephedrine-treated mice, but did not block the shift towards IgG1 production. These findings indicate that ephedrine via beta-AR can exacerbate lupus symptoms in NZM391 mice and that blockade of the beta-ARs on B cells, and not T cells, apparently was of greater importance as the inhibition of lupus symptoms corresponded to an inhibition of immunoglobulin levels, not a change of Th1/Th2 balance.

TNF-alpha, IL-6, IFN-gamma, and IL-10 gene expression polymorphisms and the IL-4 receptor alpha-chain variant Q576R: effects on renal allograft outcome.

BACKGROUND: There has been much interest recently in the effects of various cytokine gene expression polymorphisms on graft outcome. However, the results of these investigations reveal the outcomes to be organ-specific and center-specific. We sought to confirm and add to some of the earlier findings by studying the impact of tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6) polymorphisms and the interleukin-4 (IL-4) receptor alpha-chain variant on posttransplant renal allograft outcome. METHOD: TNF-alpha, IL-6, IFN-gamma, and IL-10 gene promoter region polymorphisms were assayed genotypically by PCR-SSP on 120 patients transplanted at the Albany Medical Center. These patients were also typed for the IL-4 receptor alpha-chain variant Q576R. RESULTS: Producers of high levels of the proinflammatory cytokine TNF-alpha were found to be at increased risk for acute rejection episodes if the allograft was mismatched for the molecular products of the class II region of the human major histocompatibility complex (HLA-DR). Expression level polymorphisms of the IL-6, IFN-gamma, and IL-10 genes were not associated with acute rejection episodes, nor was the IL-4 receptor alpha-chain variant Q576R. CONCLUSIONS: These data would suggest that the production of high levels of the cytokine TNF-alpha is especially detrimental to graft survival when the recipient's T-helper lymphocytes are being activated by mismatched donor HLA-class II antigens. Typing all potential kidney recipients for TNF-alpha, and providing well-matched organs for high producers of this cytokines, may be expected to increase rejection-free graft survival in these patients.

Promoter-region alleles of the TNF-alpha and IL-10 genes have no effect on pretransplant alloantibody production.

BACKGROUND: The presence of high levels of alloantibodies are known to be a risk factor in renal graft outcome. Expression level polymorphisms in cytokine genes are also thought to have an effect on allograft outcome, but the studies examining this have been inconsistent. This may be due to center-specific differences in immunosuppressive protocols. Therefore, we studied the effects of these polymorphisms on pretransplant class I alloantibody production in nonexogenously immunosuppressed candidates. METHODS: Tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) gene polymorphisms were assayed genotypically by PCR-SSP on 177 renal transplant candidates. Candidates with a peak goat antihuman immunoglobulin-enhanced T-cell panel reactive antibody (PRA) of >or=10% were considered to be positive for alloantibody (32% of 177 total). RESULTS: Previous transplants, transfusions, or pregnancies were all associated with alloantibody production, but TNF-alpha and IL-10 phenotypes were not. High levels of alloantibody production (peak PRA >50%) were also not effected by cytokine phenotype. CONCLUSIONS: These data suggest that differences in TNF-alpha and IL-10 phenotype do not effect a patient's likelihood of becoming sensitized by transfusions, pregnancies, and prior transplants.

Identification of DRB alleles in rhesus monkeys using polymerase chain reaction-sequence-specific primers (PCR-SSP) amplification.

Major histocompatibility complex (MHC) class In molecules play a vital role in the regulation of T-cell functions in the mammalian immune system. Two key features characterize the polymorphism of MHC haplotypes in humans and non-human primates: the existence of a large number of alleles, and the high degree of genetic diversity between those alleles. Rhesus monkeys and Chimpanzees have been extensively used as relevant models for human diseases and transplantation We have investigated DRB genes in 19 macaques, members of 3 families, using polymerase chain reaction with sequence-specific primers (PCR-SSP) and denaturing gradient gel electrophoresis (DGGE). After amplification PCR products were purified and subjected direct sequencing. Seven animals (Madison #1) were typed by DDGE also. We report that the DRB haplotypes defined by PCR-SSP exhibit a high degree of concordance with the data obtained by DGGE and direct sequening. Our data show prominent variability in the number of DRB1 alleles ranging from 1-4 per genotype within these families. This analysis demonstrated that most of the amplicons were identical to Mamu-DRB alleles that our PCR primers were to amplify. However, 98-99% similarity was noticed in the case of Mamu-DRB1*0303, Mamu-DRB6*0103 and Mamu-DRB*W201 alleles. The observed mismatches were located in non-polymorphic regions. Thus, family studies in rhesus macaques performed by molecular methods confirmed the multiplicity of Mamu-DRB1 alleles per haplotype and the existence of allelic associations published earlier. In addition, we propose 3 more DRB allele associations (haplotypes): Mamu-DRB1*04-DRB5*03; Mamu-DRB1*04-*DRB*W5; Mamu-DRB1*04*W2. The proposed medium-resolution PCR-SSP technique appears to be a highly reproducible and discriminatory typing method for detecting polymorphisms of DRB genes in rhesus monkeys.

Reversal of hydroquinone-mediated suppression of T cell proliferation by transfection of the M2 subunit of ribonucleotide reductase.

Hydroquinone (HQ) is a benzene derivative that is found in large quantities in cigarette tar as a result of the pyrolysis of tobacco flavinoids. HQ is a potent inhibitor of T cell proliferation, causing an immediate and complete cessation of DNA synthesis in IL-2-dependent human T lymphoblasts and Jurkat T cells without loss of cell viability. Previous studies from our laboratory demonstrated that the antiproliferative effects of RQ could be partially reversed by the addition of deoxyribonucleosides, but not by the corresponding ribonucleosides, suggesting that HQ might inhibit ribonucleotide reductase. In the present study, the molecular mechanism behind this observation was further investigated. Jurkat T cells were stably transfected with a pMEP4 expression vector containing the gene for the M2 subunit of ribonucleotide reductase under transcriptional control of the human metallothionein IIA promoter. M2-transfected Jurkat T cells exhibited a greater than three-fold increase in resistance to HQ compared to untransfected cells or cells transfected with the M2 gene in the reverse orientation. HQ resistance was associated with an increased level of M2 protein detected by Western blot. These results suggest that the benzene derivative inhibits lymphocyte proliferation by inhibiting ribonucleotide reductase.

Use of allogeneic bone marrow labeled with neomycin resistance gene to examine bone marrow-derived chimerism in experimental organ transplantation.

Posttransplant infusion of viable donor bone marrow cells (DBMC) has been shown in our previous studies to promote acceptance of incompatible kidney allografts in rhesus monkeys after treatment with polyclonal antithymocyte globulin to deplete peripheral T-lymphocytes. In this nonhuman primate model, the infusion of the DBMC is requisite for the induction of functional graft tolerance and specific MLR and CTLp unresponsiveness, although the relevant role and fate of bone marrow-derived chimeric cells is uncertain. Standard immunological and molecular techniques applied to this monkey model are unable to differentiate between chimeric cells derived from the infused DBMC and those derived from allograft-borne passenger leukocyte emigrants. To distinguish chimerism due to infused DBMC, we transduced DBMC with a functional neomycin resistance gene (Neo(r)) using the retroviral vector pHSG-Neo.Neo(r)-transduced BMC were infused into recipients approximately 2 wk after kidney transplantation and treatment with rabbit antithymocyte globulin. No maintenance immunosuppressive drugs were given. Genomic DNA isolated from peripheral blood leukocytes was used to monitor the presence ofNeo(r)-positive cells. Tissue samples obtained at necropsy also were assessed forNeo(r)-positive chimeric cells. The presence of DBMC-derived chimerism was assessed by polymerase chain reaction usingNeo(r) sequence-specific primers (PCR-SSP). Chimerism was detectable in recipient tissues at various times for up to 6 mo after DBMC infusion. These studies using gene transduction methodology indicate that a stable genetic marker can provide capability to examine DBMC-derived chimerism for prolonged periods in a nonhuman primate model. This approach should facilitate future studies in preclinical models to study the role and type of chimeric cell lineages in relation to functional allograft tolerance.

A gene transfer method for the study of chimerism derived from donor bone marrow in organ transplant recipients.

Transduction of DBMC with the neo gene did not negatively affect the viability of DBMC or the graft-prolonging effect of DBMC infusion and provided a sensitive method to monitor DBMC-derived chimerism at a microchimeric level for at least 6 months by molecular and immunohistochemical techniques. Selection of neo-transduced DBMC with G418 prior to infusion will enable more uniform neo expression and will provide novel opportunities to further investigate chimerism in primates.

A gene transfer method for the study of chimerism derived from donor bone marrow in organ transplant recipients.

Transduction of DBMC with the neo gene did not negatively affect the viability of DBMC or the graft-prolonging effect of DBMC infusion and provided a sensitive method to monitor DBMC-derived chimerism at a microchimeric level for at least 6 months by molecular and immunohistochemical techniques. Selection of neo-transduced DBMC with G418 prior to infusion will enable more uniform neo expression and will provide novel opportunities to further investigate chimerism in primates.

The facilitating effect of one-DR antigen sharing in renal allograft tolerance induced by donor bone marrow in rhesus monkeys.

Infusion of donor bone marrow cells (DBMC), a long-standing, successful strategy for inducing tolerance in experimental rodent transplantation models, can promote long-term acceptance of life-sustaining renal allografts in rhesus monkeys with no maintenance immunosuppression. To investigate the immunological basis for heterogeneity in duration of long-term graft acceptance following infusion of the DR-/dim fraction of DBMC into RATG-treated rhesus monkeys, we examined the relationship of recipient-donor major histo-compatibility class I and II DR matching to the development of antidonor antibody-dependent cellular cytotoxicity (ADCC) and renal allograft survival. The findings indicate a requirement for sharing one DR allele to achieve long-term graft acceptance. The observed immunological consequence of DR sharing that correlated with functional graft tolerance in this model was the suppression of early antidonor ADCC+ IgG antibody responses. Significant associations were observed between graft survival and suppression of ADCC antibody (P < 0.0005), graft survival and DR sharing (P < 0.005), and DR sharing and suppression of ADCC (P < 0.02). Early antidonor ADCC antibody responses associated with failure to maintain graft tolerance and were most consistently directed to donor class I. The required one DR antigen sharing in DBMC-induced suppression of antidonor class I antibody suggests a restriction for recipient DR, implying critical regulation of a response to donor antigen presented on recipient cells. We hypothesize a DBMC tolerogenic mechanism in which presentation of donor class I peptide by a shared DR allele configuration allows a veto effect by DBMC. Thus DR sharing would allow DBMC veto cells to reduce clonal expansion elicited by both the direct and indirect antigen presentation pathways.

Assessment of donor bone marrow cell-derived chimerism in transplantation tolerance using transgenic mice.

Allospecific skin graft prolongation can be induced in mice using antithymocyte globulin and allospecific donor bone marrow cells (DBMC). This enhancement may be due to the persistence of chimeric cells of donor origin in the host. In this study, we systematically investigated DBMC-derived chimerism in various lymphoid and nonlymphoid tissues over time. To do this, transgenic mice were used as a source of DBMC to clearly distinguish chimerism due only to the injected DBMC. Chimerism in various tissues was assessed at several times points after DBMC infusion by polymerase chain reaction amplification of tissue DNA using transgene specific primers. A cDNA probe specific for the transgene was used to demonstrate DBMC-derived chimerism in polymerase chain reaction products by the method of Southern. Although chimerism was initially detectable in most tissues tested 1 day after DBMC infusion, the presence of chimeric cells generally diminished over time. At 4 weeks or longer, chimerism was consistently confined to recipient skin. Furthermore, the chimeric cells in recipient skin persisted even after the allograft was rejected. In contrast to chimerism in recipient skin, chimerism became undetectable in donor skin as early as 2 weeks after DBMC infusion. The loss of chimerism in donor skin showed a temporal correlation with a reduction of chimerism in host bone marrow and lymphoid tissues that preceded rejection in all experiments by a range of 7-14 days. The use of DBMC from transgenic mice allowed a unique opportunity to monitor the kinetics of DBMC-derived chimerism. The presence of chimerism in the skin of mice in temporal association with chronic allograft rejection suggests that chimerism per se is not a reliable index of allogeneic unresponsiveness.

Veto cells in transplantation tolerance.

Advances in immunosuppressive management for transplantation have improved graft survival. However, lasting success will probably depend on the induction of donor-specific unresponsiveness, avoiding chronic immunosuppressive drug therapy and its debilitating side effects. Tolerance strategies have been developed in rodents, but applicability to human organ transplantation has not been achieved. We have established a preclinical allogeneic kidney transplant model in unrelated outbred rhesus monkeys and have investigated a tolerance-inducing strategy in which posttransplant administration of rabbit antithymocyte globulin and infusion of a subpopulation of donor bone marrow cells yields long-term graft acceptance in the absence of chronic immunosuppressive drugs. Recent studies of the immunological mechanisms by which induction and maintenance of transplant tolerance is achieved in this model are presented within the framework of a veto hypothesis.

Further studies of veto activity in rhesus monkey bone marrow in relation to allograft tolerance and chimerism.

Infusing the DR-/dim fraction of bone marrow cells (BMC) from an allogeneic kidney donor into rabbit antithymocyte globulin-treated transplant recipients delivers a tolerogenic signal, leading to functional allograft tolerance in rhesus monkeys without additional drug therapy. Our updated results in an expanded series show a median 131-day graft survival of recipients given DR-/dim donor BMC with a 23% 1-year survival (P < 0.00001 vs. rabbit antithymocyte globulin controls). Removing DRbright cells from donor BMC appeared to have a significant effect (P < 0.05). We have further investigated the tolerogenic mechanism within the experimental framework of the veto hypothesis in this preclinical model. In limiting dilution assays, we demonstrated the donor specificity of clonal inactivation of CTL precursors (CTLp) after in vitro or in vivo exposure to DR-/dim donor BMC, confirming specific tolerance. Additionally, in vitro studies confirmed the allogeneic specificity of CTLp inactivation in 3-cell MLR assays; minimal bystander effects were seen on normal CTLp responses to third party stimulator cells, while CTLp responses to the BMC donor's cells were abrogated in the same cultures. BMC mediating the veto effect were found to be resistant to L-leucyl-L-leucine methyl ester (Leu-leu-OMe), which excluded BMC-mediated cytotoxicity by NK or lymphokine-activated killer cells, CTL, or activated macrophages. In contrast, veto activity was abolished if the BMC were pretreated with either high dose UV-B light irradiation, mitomycin, or gamma-irradiation, indicating that BMC contained a UV-B-sensitive precursor of the veto effector, and that a proliferative step separated the two. Irradiation of DR-/dim donor BMC or administration of cyclophosphamide after infusion of nonirradiated BMC prevented the tolerogenic effect. Only recipients given nonirradiated DR-/dim donor BMC demonstrated PBL chimerism, which associated with functional deletion of antidonor CTLp and duration of graft survival. The Leu-leu-OMe resistance and the other properties of the allogeneic monkey CD3- CD2+ CD8+ BMC subpopulation that exhibits tolerance-promoting activity in vitro and in vivo lead us to postulate that a donor BMC-derived precursor population, possibly a dendritic cell population, may induce allogeneic unresponsiveness in this model.

The cat hemoglobin polymorphism: Southern blot analysis of the beta-globin gene region from cats of various Hb A/Hb B phenotypes.

The molecular basis for the genetic control of variable proportions of the two cat hemoglobins Hb A(alpha 2 beta A2) and Hb B (alpha 2 beta B2) was investigated. Ratios of Hb A/Hb B vary between 50/50 and 90/10 among members of the mongrel cat population, with clusters around 50, 35, and 10% Hb B. Genomic DNA from cats of 50/50, 70/30, and 90/10 phenotypes were cut by restriction endonucleases HindIII, EcoRI, BamHI, BglII, and Pstl and hybridized to a fragment of the human beta-globin gene. The results of the Southern blots suggested a pattern of homozygote, heterozygote, homozygote for the respective cat phenotypes, 50/50, 70/30, and 90/10. Therefore, the cat hemoglobin polymorphism seems to result from the possible combinations of an allelic gene pair.

Human red cell acetyltransferase.

Acetyltransferase was isolated by histone-Sepharose affinity chromatography from human cord blood red cells. The enzyme was detected only in very young red cells. The semipurified enzyme and [14C]acetyl-CoA were used to acetylate isolated Hb F tetramer and alpha and gamma subunits. The in vitro acetylated products were characterized by globin chain separation by CM-cellulose chromatography and tryptic peptide analysis by reverse-phase HPLC. Acetylation of both the gamma-chains and the alpha-chains could occur within the Hb F tetramer. Acetylation also could take place on intact subunits. It appears that some Hb FIC could be formed in the cells by utilizing Hb F or free gamma-chains as acetylation substrate.

Influence of gamma-chain amino-terminal acetylation on subunit assembly of human fetal hemoglobin.

A human hemoglobin F subunit recombination study was performed to determine the relative efficiency of recombination of amino-terminally acetylated gamma-chains and non-acetylated chains with alpha-chains. The results of this work suggested that the acetylated gamma Ic-chains combined more readily with the alpha-chains than the non-acetylated gamma o-chains. An important factor in the function and assembly of multi-subunit macromolecules is the interaction of the unlike subunits. A model system for the study of such interactions has been the protein hemoglobin. With respect to the hemoglobin molecule, it has been noted that relative affinities of normal and mutant subunits for the unlike subunits can have a significant influence on hemoglobin synthesis at the post-translation level, i.e., subunit assembly [1,2]. A similar mechanism may control the formation of human Hb FIc, a hemoglobin with NH2-terminally acetylated gamma- (gamma Ic)-chains. In this instance acetylation may occur on the ribosomes before subunit assembly. A previous report from our laboratory showed a slight increase in the relative proportion of Hb FIc in cord blood samples with over 5% Hb Bart's [3]. The data were interpreted to be due to an influence of alpha-chain deficiency (alpha-thalassemia) on the formation of Hb FIc and Hb Fo tetramers by a preference of alpha-chains for gamma Ic-chains over gamma o- (non-acetylated gamma)-chains. The present study involves an examination of the relative affinity of the gamma Ic- and gamma o-chains for the normal alpha-chains in an in vitro recombination system.

Minor components of Hb Bart's.

The minor components of Hb Bart's were separated by CM-cellulose chromatography, reverse-phase HPLC, and DEAE-cellulose chromatography. These were characterized by amino acid analysis, tryptic peptide analysis by HPLC, electrophoresis, and carbohydrate and phosphate analysis. Acetylated and glycated components of Hb Bart's were present in cord blood red cells. The ratio of gamma Ic/total gamma in Hb Bart's was nearly the same as that of Hb FIc/Hb F which may indicate that the gamma chain after its release from the ribosome is not a better acetylation substrate than Hb Fo. Glycation of the free gamma-chains seems to take place in vivo as readily as the major Hb components.