Lymphocyte plasma membranes. V. Specificity and mitogenicity of absorbed anti-lymphocyte sera.

Anti-lymphocyte sera against human thymus [ALS-(THY)] were absorbed serially with cultured human lymphoblasts (CHL) or thymus and residual antigen-binding activity was tested. The absorbed ALS were used to bind 125I-labeled antigens from lymphocytes labeled by the lactoperoxidase catalyzed iodination technique. Absorption of ALS(THY) with CHL led to the absorbed serum having less than 5 to 10% of its original antigen-binding activity against labeled CHL antigens while maintaining from 20 to 40% of its original activity against labeled THY. Serial absorption of ALS(THY) with THY led to an equal decrease in activity against both THY and CHI. When the immunoprecipitates from these experiments were examined on polyacrylamide gels containing SDS it was found that serial absorption of ALS(THY) with THY first removed activity against a component of m.w. similar to 48,000 leaving relatively greater activity against material of apparent high molecular wieght. In contrast, absorption of ALS-(THY) with CHL removed the antibodies against the high molecular weight material while leaving activity against the component of m.w. 48,000. When these absorbed ALS were used to induce in vitro lymphocyte proliferation, it was found that ALS(THY) absorbed with CHL, did not. The retention or loss of mitogenicity seemed to correlate with retention or loss of binding activity against the component(s) of m.w. similar to 48,000.     

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.     

Cell transfer studies in cyclophosphamide-induced tolerance

Thymectomized, irradiated adult CBA mice were restored with various combinations of bone marrow and thymus cells from nontolerant animals and from animals made tolerant to sheep erythrocytes or to hemocyanin with the drug cyclophosphamide. Mice reconstituted with tolerant marrow and thymus responded as well as those that received nontolerant cells. Thus it is concluded that the tolerant state of the transferred marrow and thymus cells is not a significant factor in the tolerant state of the recipient, and that antigenic diversity is restored in the interaction and proliferation of bone marrow and thymus cells that follow transfer.Thymectomized irradiated mice restored with thymocytes, in contrast to unoperated animals, require multiple antigen injections to demonstrate comparable immune response, but develop tolerance normally when treated with cyclophosphamide and antigen. Reconstitution with tolerant marrow and thymus cells resembles the recovery of immune responsiveness seen after lethal irradiation of tolerant mice; in both instances a complete breakdown of immunological tolerance is observed.     

Use of the immune tolerance induction in the production of an antilymphocyte serum (ALS) free of antibodies against serum proteins]

The possibility has been investigated of a direct gain of ALS free of undesirable antibodies against serum proteins by inducing immunologic tolerance in productive animals (pigs). Preliminary experiments made with tolerogenic amounts of 10 and 50 ml of sera and with immunization by the serum alone proved applicability of this method. Electrophoresis showed antibodies against 6 to 7 and 2 to 3 fractions in animals tolerated with 10 and 50 ml respectively, compared to 18 to 20 fractions in the control group, which was not tolerated. This has been confirmed when preparing ALS in practice, where the toleration was carried out with 25 ml of serum or with the same amount of serum with the addition of hemoglobin and immunization by lymphocytes isolated from peripheral blood. Final ALS of untolerated animals contained antibodies against 7 to 8 fractions, whereas that of experimental group tolerated with serum and Hb was free of antibodies against serum protein, hemoglobin included. ALS of the group tolerated with normal serum contained only antibodies against hemoglobin. In vitro tests (i.e. lymphoagglutination t., lymphocytotoxicity t., rosette inhibition t.) proved that by inducing tolerance towards serum protein the activity of ALS was in no was affected. According to the results this method can be employed not only for the preparation of ALS, but also for other purposes, such as preparation of monovalent antisera for immunoelectrophoresis.     

Responses in Xenopus to the thymus independent antigen polyvinylpyrrolidone (PVP) and its haptenated derivative, trinitrophenylated PVP (TNP-PVP)

Xenopus laevis (the South African clawed toad) can respond to thymus dependent (TD) and thymus independent (TI) antigens. However, the response to trinitrophenylated Ficoll (TNP-Ficoll), a TI-2 antigen in mammals, is thymus dependent in Xenopus. Polyvinylpyrrolidone (PVP), classed as a TI antigen in mammals, is also a TI antigen in Xenopus, but responses to PVP and TNP-PVP are thymus regulated. As with TNP-Ficoll, capacity to respond to TNP-PVP diminishes during metamorphosis, and tolerance can be induced via the stimulation of TD suppression with trinitrobenzene sulphonic acid. Animals treated with N-methyl-N-nitrosourea and adult-thymectomised Xenopus, which lack certain TD responses, can nevertheless respond to TNP-PVP. Based on this and other information, it is concluded that TNP-PVP should be classed as a TI-2 antigen in Xenopus.     

Adaptation of Escherichia coli to Elevated Sodium Concentrations Increases Cation Tolerance and Enables Greater Lactic Acid Production

Adaptive evolution was employed to generate sodium (Na⁺)-tolerant mutants of Escherichia coli MG1655. Four mutants with elevated sodium tolerance, designated ALS1184, ALS1185, ALS1186, and ALS1187, were independently isolated after 73 days of serial transfer in medium containing progressively greater Na⁺ concentrations. The isolates also showed increased tolerance of K⁺, although this cation was not used for selective pressure. None of the adapted mutants showed increased tolerance to the nonionic osmolyte sucrose. Several physiological parameters of E. coli MG1655 and ALS1187, the isolate with the greatest Na⁺ tolerance, were calculated and compared using glucose-limited chemostats. Genome sequencing showed that the ALS1187 isolate contained mutations in five genes, emrR, hfq, kil, rpsG, and sspA, all of which could potentially affect the ability of E. coli to tolerate Na⁺. Two of these genes, hfq and sspA, are known to be involved in global regulatory processes that help cells endure a variety of cellular stresses. Pyruvate formate lyase knockouts were constructed in strains MG1655 and ALS1187 to determine whether increased Na⁺ tolerance afforded increased anaerobic generation of lactate. In fed-batch fermentations, E. coli ALS1187 pflB generated 76.2 g/liter lactate compared to MG1655 pflB, which generated only 56.3 g/liter lactate.     

ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis

Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.     

Comparison of increased expression of wild-type and herbicide-resistant acetolactate synthase genes in transgenic plants, and indication of posttranscriptional limitation on enzyme activity

Genes encoding wild type acetolactate synthase (ALS) and a sulfonylurea herbicide-resistant form of the enzyme, isolated from Arabidopsis thaliana, were expressed in transgenic Nicotiana tabacum plants under the control of their native promoters or of the highly active cauliflower mosaic virus 35S promoter. Expression of the wild type coding region from the 35S promoter resulted in a small, threefold increase in sulfonylurea tolerance above the levels measured in tissue expressing the native wild type gene. A much larger, 300-fold increase in herbicide tolerance was conferred by the mutant gene encoding a herbicide-resistant ALS. An additional 10-fold increase in tolerance was attained by expressing this coding region from the 35S promoter. The increase in both wild type and mutant gene expression directed by the 35S promoter resulted in over 25-fold higher levels of ALS messenger RNA in some transformants as compared with those expressing the native genes. However, ALS specific activity increased at most twofold, indicating that the amount of functional enzyme and messenger RNA are not correlated.     

Antigen recognition by T lymphocytes. 3. Evidence for two populations of thymus-dependent rosette-forming cells in spleen and lymph nodes

Rosette-forming cells, present in normal spleen, are composed of 70% theta-positive cells with “high” in vitro sensitivity to Azathioprine (AZ) (1 μg/ml) and 30% theta-negative cells with “low” sensitivity to AZ (500 μg/ml). Theta-positive RFC are also found in the thymus (with “high” sensitivity to AZ) and in lymph nodes and peripheral blood (with “intermediate” sensitivity to AZ:50 μg/ml). RFC with “high” sensitivity to AZ (T1) are eliminated from the spleen by adult thymectomy in less than 6 days; it takes more than 48 hours exposure to antilymphocyte serum (ALS) in vivo to suppress them, whereas RFC with “intermediate” sensitivity to AZ (T2), present in lymph nodes and peripheral blood, disappear 6 hours after in vivo ALS treatment but not after adult thymectomy.     

Tolerogenic ability of thymocytes in organ-cultured thymus lobes.

It is generally believed that macrophages and dendritic cells are the major cell populations that present tolerogenic self antigens to developing thymocytes. However, it is still controversial whether self antigens expressed on thymocytes themselves work as tolerogens in the thymus. To evaluate this possibility, Thy-1 bright cells were sorted out from fetal thymus cells on the 15th gestation day, and were colonized into 2'-deoxyguanosine-treated allogeneic thymus lobes. The repopulated thymus lobes were organ-cultured, and the allo-specific killer activity of thymocytes recovered from the lobes was examined. These cells were tolerant to class I but not to class II-MHC of the donor haplotype, indicating that class I molecules expressed on the thymocytes worked as tolerogen. Tolerogenic ability of Thy-1+ cells was also demonstrated in another system. Upon intimate contact with allogeneic thymus lobes on a polycarbonate filter, thymus lobes fused with each other and Thy-1+ cells co-migrated (Eur. J. Immunol. 19:1525-1530, 1989). In thymus lobes rendered parabiotic from day 5, CTL tolerance was achieved against class I but not to class II MHC. These data indicate that thymocyte-thymocyte interaction is sufficient to induce class I CTL tolerance in developing thymocytes.     

Exendin-4 ameliorates motor neuron degeneration in cellular and animal models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS.     

Effect of antilymphocyte serum on animals experimentally infected with Histoplasma capsulatum or Cryptococcus neoformans

The anti-mouse and anti-guinea pig antilymphocyte sera (ALS) prepared for this study were shown to contain cytoxic and leucoagglutinating antibodies, and were capable of producing severe lymphopenia in these animals. Guinea pigs treated weekly with ALS were more susceptible to development of fatal infection when inoculated with Histoplasma capsulatum. No fatalities occurred in guinea pigs infected with equal doses of H. capsulatum but treated with normal rabbit serum (NRS) or saline. The time necessary to reach 50% fatality in mice infected with Cryptococcus neoformans was greatly reduced by pretreatment with ALS in comparison with infected controls treated with NRS or saline. When low dosages were used (0.1 ld(50)), the effect was even more pronounced. Spleen homogenates from mice infected with equal dosages of H. capsulatum and treated with ALS or NRS were cultured. More than 150 times as many organisms were present in the spleens of the ALS-treated group. Similar results were obtained from culturing the lungs and liver. Delayed hypersensitive skin reactions were radically decreased or abrogated in H. capsulatum-infected guinea pigs inoculated intraperitoneally with ALS 12 hr before skin testing with histoplasmin. When ALS was given weekly, the influence on skin reactivity was less notable. Given intradermally, ALS was shown to inhibit the delayed reaction to histoplasmin within a radius of 40 mm.     

Antibody-induced transplantation tolerance that is dependent on thymus-derived regulatory T cells

Targeting of the CD45RB isoform by mAb (anti-CD45RB) effectively induces donor-specific tolerance to allografts. The immunological mechanisms underlying the tolerant state remain unclear although some studies have suggested the involvement of regulatory T cells (T-regs). Although their generative pathway remains undefined, tolerance promoting T-regs induced by systemic anti-CD45RB treatment have been assumed to originate in the peripheral immune system. We demonstrate herein that separable effects on the peripheral and central immune compartments mediate graft survival induced by anti-CD45RB administration. In the absence of the thymus, anti-CD45RB therapy is not tolerogenic though it retains peripheral immunosuppressive activity. The thymus is required for anti-CD45RB to produce indefinite graft survival and donor-specific tolerance, and this effect is accomplished through thymic production of donor-specific T-regs. These data reveal for the first time an Ab-based tolerance regimen that relies on the central tolerance pathway.     

Amino acid residues conferring herbicide tolerance in tobacco acetolactate synthase

Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to valine, leucine, and isoleucine in plants and microorganisms. ALS is the target site of several classes of structurally unrelated herbicides including sulfonylureas, imidazolinones, and triazolopyrimidines. To identify the residues conferring herbicide tolerance in tobacco ALS, site-directed mutagenesis for three residues, Ala121, Pro187 and Ser652, was performed. Mutant A121T showed strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), while mutant S652T was resistant only to Cadre. The S652N mutation abolished the binding affinity of FAD, and inactivated the enzyme. Double mutation of Ala121 and Ser652 with Thr yielded a mutant highly tolerant to Londax, Cadre, and TP (a triazolopyrimidine sulfonamide), but has enzymatic properties similar to those of wild-type. Substitution of Pro187 with Ser resulted in the enzyme highly susceptible to oxidation and fragmentation. These results suggest that two residues Ala121 and Ser652 are potent residues conferring herbicide resistance in tobacco ALS, and that double mutation of Ala121 and Ser652 by Thr can confer stronger tolerance to Londax, Cadre, and TP.     

Studies on the termination of immunological tolerance in the mouse thymus cell population after irradiation

Immunological tolerance in the mouse thymus cell population induced by the intravenous injection of deaggregated bovine gamma globulin was terminated by whole body irradiation. After irradiation, the weight of the thymus recovered biphasically, and the termination of tolerance occurred as early as in the first phase. Both Thy-1 antigen expression and helper activity of the thymus cell population in irradiated mice recovered in parallel with the recovery of the thymus weight. Sensitivity of the regenerating thymus cell to the tolerogen was not different from that of the normal thymus cell. The first phase of thymus regeneration may be caused by the proliferation and differentiation of relatively radioresistant and tolerogen insensitive precursors residing in the thymus. Tolerogen and/or immunogen reactive thymus cells may originate from the precursor.     

The thymus and central tolerance

T-cell differentiation in the thymus generates a peripheral repertoire of mature T cells that mounts strong responses to foreign antigens but is largely unresponsive to self-antigens. This state of specific immunological tolerance to self-components involves both central and peripheral mechanisms. Here we review the process whereby many T cells with potential reactivity for self-antigens are eliminated in the thymus during early T-cell differentiation. This process of central tolerance (negative selection) reflects apoptosis and is a consequence of immature T cells receiving strong intracellular signalling through T-cell receptor (TCR) recognition of peptides bound to major histocompatibility complex (MHC) molecules. Central tolerance occurs mainly in the medullary region of the thymus and depends upon contact with peptide-MHC complexes expressed on bone-marrow-derived antigen-presenting cells (APCs); whether tolerance also occurs in the cortex is still controversial. Tolerance induction requires a combination of TCR ligation and co-stimulatory signals. Co-stimulation reflects interaction between complementary molecules on T cells and APCs and probably involves multiple molecules acting in consort, which may account for why deletion of individual molecules with known or potential co-stimulatory function has little or no effect on central tolerance. The range of self-antigens that induce central tolerance is considerable and, via low-level expression in the thymus, may also include tissue-specific antigens; central tolerance to these latter antigens, however, is likely to be limited to high-affinity T cells, leaving low-affinity cells to escape. Tolerance to alloantigens and the possibility of using central tolerance to promote acceptance of allografts are discussed.     

Induction of central deletional T cell tolerance by gene therapy

Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag K(b) were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2K(b) resulted in lifelong expression of K(b) on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2K(b) transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with K(b) transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to K(b). Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.     

Distinct non-target site mechanisms endow resistance to glyphosate,ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant <Emphasis Type="Italic">Lolium rigidum</Emphasis>

This study investigates mechanisms of multiple resistance to glyphosate, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS)-inhibiting herbicides in two Lolium rigidum populations from Australia. When treated with glyphosate, susceptible (S) plants accumulated 4- to 6-fold more shikimic acid than resistant (R) plants. The resistant plants did not have the known glyphosate resistance endowing mutation of 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) at Pro-106, nor was there over-expression of EPSPS in either of the R populations. However, [¹⁴C]-glyphosate translocation experiments showed that the R plants in both populations have altered glyphosate translocation patterns compared to the S plants. The R plants showed much less glyphosate translocation to untreated young leaves, but more to the treated leaf tip, than did the S plants. Sequencing of the carboxyl transferase domain of the plastidic ACCase gene revealed no resistance endowing amino acid substitutions in the two R populations, and the ALS in vitro inhibition assay demonstrated herbicide-sensitive ALS in the ALS R population (WALR70). By using the cytochrome P450 inhibitor malathion and amitrole with ALS and ACCase herbicides, respectively, we showed that malathion reverses chlorsulfuron resistance and amitrole reverses diclofop resistance in the R population examined. Therefore, we conclude that multiple glyphosate, ACCase and ALS herbicide resistance in the two R populations is due to the presence of distinct non-target site based resistance mechanisms for each herbicide. Glyphosate resistance is due to reduced rates of glyphosate translocation, and resistance to ACCase and ALS herbicides is likely due to enhanced herbicide metabolism involving different cytochrome P450 enzymes.     

Studies on the resistance to tolerance induction against human IgG in DDD mice. III. Development of the resistance with age and cellular events.

Three-week-old DDD mice were easily rendered tolerant to human IgG while 12-week-old mice were tolerized only partially. Mechanisms of the development of the resistance with age were investigated. It was shown by the cell transfer experiments that spleen T cells, purified on a Tetron wool column, from older mice were responsible for the resistance, which was not associated with the loss of suppressor cells with age. To elucidate the possibility of whether tolerogen-sensitive spleen T cells differentiate into resistant ones, cell transfer experiments were carried out in which thymectomized, lethally irradiated mice were reconstituted with spleen cells from 3-week-old mice and then treated with the tolerogen on various days afterward. The results indicated that tolerance was inducible in these hosts to the same degree, irrespective of the timing of the tolerogen injection, while age-matched intact mice gradually acquired the resistance. Then the possibility of whether age of thymus affected tolerance inducibility of the hosts or not was examined. The tolerogen was injected into irradiated, bone-marrow-reconstituted mice which bore either 4- or 7-week-old thymus. It was shown that helper T cells newly generated under younger thymus acquired higher susceptibility to the tolerogen. There was no difference in tolerance inducibility irrespective as to whether bone marrow cells were prepared from younger or older mice. From these observations it was suggested that the resistance to tolerance induction in DDD mice is acquired through the appearance of resistant T cells which are generated from T-cell precursors in bone marrow under the influence of a radioresistant thymic constitution and predominantly located in the spleen.