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In both vertebrates and invertebrates, olfactory perception is mediated by G-protein-coupled receptors. Recent work, in both mouse and Caenorhabditis elegans, sheds light on the role of specific G proteins in olfactory signal transduction, neuronal morphology and axon guidance.
BACKGROUND: The CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF) family of proteins and is transiently expressed on the surface of activated T cells. The binding of CD40L to CD40, which is expressed on the surface of B cells, provides a critical and unique pathway of cellular activation resulting in antibody isotype switching, regulation of apoptosis, and B cell proliferation and differentiation. Naturally occurring mutations of CD40L result in the clinical hyper-IgM syndrome, characterized by an inability to produce immunoglobulins of the IgG, IgA and IgE isotypes. RESULTS: We have determined the crystal structure of a soluble extracellular fragment of human CD40L to 2 A resolution and with an R factor of 21.8%. Although the molecule forms a trimer similar to that found for other members of the TNF family, such as TNF alpha and lymphotoxin-alpha, and exhibits a similar overall fold, there are considerable differences in several loops including those predicted to be involved in CD40 binding. CONCLUSIONS: The structure suggests that most of the hyper-IgM syndrome mutations affect the folding and stability of the molecule rather than the CD40-binding site directly. Despite the fact that the hyper-IgM syndrome mutations are dispersed in the primary sequence, a large fraction of them are clustered in space in the vicinity of a surface loop, close to the predicted CD40-binding site.
Several aminocyclitol-aminoglycoside antibiotics have been studied by tandem mass spectrometry. Glycosidic bond cleavages were the major reactions in the low energy collisionally activated decomposition (CAD) of the protonated antibiotics. Only the glycoside residing on the C6-O of the 2-deoxystreptamine was observed to undergo significant decomposition at the C2-C3 and O-C1 bonds. The comprehension of the CAD of known aminoglycosides aided in the identification of an unknown impurity in tobramycin. The unknown compound was initially detected by reverse phase high-performance liquid chromatography following dinitrofluorobenzene derivatization of the amino groups. The molecular weight of the dinitrobenzene derivative measured by LC mass spectrometry (MS) led to the detection of two isomeric impurities in tobramycin by LC-MS using an amino column. Their CAD spectra were subsequently acquired by LC-MS/MS. One of the two compounds was determined to be a known compound, 6"-O-carbamyltobramycin with the carbamyl group substituted on the glycoside residing on the C6-O of 2-deoxystreptamine. The fragmentation pattern of the other compound was interpreted as that the unknown was also a carbamyltobramycin. The carbamyl group was, however, substituted on 2-deoxystreptamine. It was speculated that the carbamyl group was substituted at the C1 amino group. This compound, to our knowledge, has not been reported before.
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The objective of this study was to determine the effect of screws and keel size on the fixation of an all-metal glenoid component. A prototype stainless-steel glenoid component was designed and implanted in 10 cadaveric scapulae. A testing apparatus capable of producing a loading vector at various angles, magnitudes, and directions was used. The independent variables included six directions and three angles of joint load, and five fixation modalities-three different-sized cross-keels (small, medium, and large), screws, and bone cement. Implant micromotion relative to bone was measured by four displacement transducers at the superior, inferior, anterior, and posterior sites. The components displayed a consistent response to loading of ipsilateral compression and contralateral distraction. Use of progressively larger keels did not significantly improve implant stability. Stability decreased as the angle of load application increased (P <.05). Screw and cement fixation resulted in the most stable fixation (P <.05).
CD8+ T cells control immune responses, and recent studies suggest that this regulation is, in part, specifically directed towards TCR structures expressed by CD4+ cells. To develop a system to study the role of the TCR in regulatory interactions, we isolated clones of CD4+ cells expressing identified TCR V beta chains. These CD4+ clones were used to stimulate and expand autologous CD8+ cells, which kill the inducing CD4+ clone as well as independently isolated autologous CD4+ clones sharing the same TCR V beta as the inducing cell but not CD4+ T cells expressing different V beta TCRs. This V beta-specific cytotoxicity is dependent on the state of activation of the target cells and is not inhibited by an anti-class I monoclonal antibody, W6/32. We envision that V beta-specific CD8+ T cells of this type may regulate immune responses by direct interaction with antigen-activated CD4+ cells.
Interactions mediated by TCRs expressed on different T cell subsets may play a role in immunoregulation. To investigate this idea, we studied the regulation of superantigen-induced TCR V beta-restricted responses. We asked whether the in vivo regulation of CD4+ V beta 8+ T cells following SEB injection is controlled by CD8+ T cells. We found that in mice deficient in CD8+ T cells, the down-regulation of CD4+ V beta 8+ T cells below baseline is not observed. Moreover, following SEB administration, CD8+ T cells emerge that preferentially kill subpopulations of activated CD4+ V beta 8+ but not CD4+ V beta 8- T cells in vitro. This TCR V beta-specific cytotoxicity is dependent on beta 2-microglobulin and is inhibited by antisera specific for Qa-1 but not by antibody to MHC class Ia. These data suggest the idea that the specificity of immune regulation may involve CD8+ T cell recognition of TCR V beta determinants and Qa-1 molecules expressed on CD4+ T cells.
Amino acids were identified and quantified in the stomach fluid recovered from animals fasted 8, 24, and 48 h. After in vitro chlorination of stomach fluid, three chloramino acids, N-chloroglycine, either N-chloroleucine or N-chloroisoleucine, and N-chlorophenylalanine, were identified by GC/MS analysis of dansylated methyl ester derivatives. Formation of N-chlorovaline and N-chloroserine was suggested by their HPLC retention times, but their identification could not be confirmed by GC/MS data.
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Cell-mediated cytotoxicity is thought to play an important role in the immunopathogenesis of inflammatory myopathies. We determined whether lymphocytes circulating in patient peripheral blood contain automyocytotoxic precursors. Peripheral blood mononuclear cells, sheep red blood cell rosetting (E+) and nonrosetting (E-) cells, were isolated from patients with polymyositis or dermatomyositis and tested for their ability to kill autologous-cultured myotubes derived from diseased muscle biopsies. Patient-derived as well as normal allogeneic mononuclear cells lysed both polymyositis-dermatomyositis and nonrelated myotubes. However, whereas patient E+ cells were preferentially cytotoxic to autologous myotubes, their E-cells did not discriminate autologous from allogeneic targets. Furthermore, cultures of patient E+ cells triggered by phytohemagglutinin and interleukin-2 maintained myocytotoxic potential. In these cultures, virtually all autologous but only 50% of allogeneic killing was mediated by CD3+ T cells. Moreover, autologous cell-mediated killing was abrogated by anti-CD3 monoclonal antibodies. In conclusion, both myocytotoxic CD3+ T-cell clones specific for autologous myotubes, as well as non-T cells, which are nonspecifically myocytotoxic, are present in the peripheral blood of patients with inflammatory myopathies.
Delayed xenograft rejection (DXR) of pig organs by baboons currently represents the major obstacle to successful xenotransplantation. Although antibodies (Abs) are believed to play a fundamental role in this form of rejection, so far little is known concerning the potential cellular component. Biopsies taken during DXR of human CD55 transgenic pig hearts by non-treated (n = 2), alpha-Gal immunoadsorbed (n = 2), or immunosuppressed (n = 9) baboons were studied. The cellular element was explored by determining not only its phenotype by classical immunohistochemical techniques but also its activity in terms of cytokines, cytolytic enzymes and other mediators using quantitative reverse transcription polymerase chain reaction. All porcine xenografts underwent DXR; within 5 days in non-treated and immunoadsorbed animals but significantly delayed (6 to 29 days) in immunosuppressed animals. Cellular infiltration in non-immunosuppressed grafts consisted predominantly of monocytes/macrophages, CD8 cells and a few CD4 T-cells. The predominant baboon transcripts detectable were the proinflammatory cytokines interleukin1-alpha and tumor necrosis factor-alpha, the lymphokine interferon-gamma and the cytotoxic enzyme granzyme B. However, these cellular components were lacking in the immunosuppressed animals. Despite these differences, strong immunoglobulin M (IgM) and C5b-9 complement deposition was observed in all animals at rejection. Together our findings suggest that although the humoral response plays a predominant role in DXR through IgM Abs and complement activation, there is a clear cellular infiltrate in DXR in this model that is likely to contribute to rejection through a strong pro-inflammatory and cytotoxic environment, necessitating substantial immunosuppression for a prolonged graft survival.
Delayed xenograft rejection (DXR) remains a major obstacle in discordant xenotransplantation. As strategies of complement inhibition and xenogeneic natural antibody (Ab) removal have been shown to give prolonged xenograft survival, we endeavored to determine whether combining these two strategies would lead to an additive effect in terms of graft survival. The study was initiated with two groups, A and B, where group A received normal kidneys and group B received hCD55 transgenic kidneys. Both groups underwent pre-transplant (day-1) total immunoglobulin (Ig) immunoadsorption (IA) and received an immunosuppression of cyclophosphamide, cyclosporine A, mycophenolate mofetil and corticosteroids. Two subsequent groups (C and D) receiving hCD55 transgenic pig kidneys were then performed with an 'optimized' immunosuppression (Cyclophosphamide starting 1 day earlier) but only group D recipients were immunoadsorbed. Biopsies taken during the post-transplantation period were analyzed for Ab deposition, compliment activation and cellular infiltration. No hyperacute rejection was observed. In the initial immunoadsorbed groups A and B, all baboons underwent DXR, which started surprisingly early (day 5 in most cases. In the subsequent two groups, the immunoadsorbed group D baboons also underwent DXR, again as early as day 5. In contrast, group C baboons did not show any signs of DXR on their day 6 biopsy or at their time of death. Analysis of graft biopsies from the kidneys undergoing rejection or with stable function showed strong deposition of anti-Gal IgM in all cases whereas strong complement C5b-9 deposits were only observed in biopsies at rejection. Cellular infiltration consisted mostly of monocytes/macrophages, was more pronounced in biopsies taken at rejection and was associated with a pro-inflammatory environment involving interleukins 1alpha, 6 and 8. Our findings suggest non-specific Ig (anti-Gal and non-Gal Ig of all isotypes) IA or even incomplete IA in immunosuppressed baboon recipients of transgenic pig kidneys is detrimental to graft survival by being associated with an Ab and compliment driven rejection. We speculate that the IA were insufficient in terms of Ig depletion or frequency inducing an Ab rebound or that this total Ig depletion also removed components facilitating graft survival.
Besides virological and physiological concerns, the success of xenotransplantation (Xt) is still dependent on the prevention of delayed xenograft rejection (DXR). Although multifactorial, DXR is mainly due to xenonatural antibody (Ab) recognizing their xenogenic antigen (Ag) followed by complement activation. Despite the use of intensive treatments capable of inhibiting the humoral response, DXR can still not be avoided and always occurs within weeks following transplantation. Moreover, these latter treatments currently used in Xt could not be used clinically in humans because of their high risk of over-immunosuppressing the patients. Mitoxantrone (Mx) is a drug well known for its antiproliferative properties and is used clinically in oncology and in the treatment of relapsing multiple sclerosis. In models of arthritis in rats, it has been shown to be 10 to 20 times more powerful than cyclophosphamide (CyP) at blocking both inflammatory and B-cell responses. Because of its B-cell inhibitory capacity and considering the implication of the humoral response in xenograft rejection, we have compared Mx with CyP for its ability to block in vivo anti-pig immunization induced via subcutaneous injections of pig red blood cells into baboons. Neither drug was able to inhibit the anti-pig responses following the first and second immunizations, emphasizing the particularity of preformed Ab responses. However, the rise in Ab in the Mx treated animals was significantly delayed as compared with the non-treated as well as the CyP treated animals and was mainly because of a profound depletion of circulating B-cells. Mx displays an interesting antihumoral effect that we now intend to test in a pig kidney to baboon Xt model, with anticipated administration of the drug allowing an early B-cell depletion.
This study assessed basic learning and attention abilities in Wistar-Kyoto hyperactive (WKHA) rats using appetitive conditioning preparations. Two measures of conditioned responding to a visual stimulus, orienting behavior (rearing on the hind legs), and food cup behavior (placing the head inside the recessed food cup) were measured. In Experiment 1, simple conditioning, but not extinction, was impaired in WKHA rats compared with Wistar rats. In Experiment 2, nonreinforced presentations of the visual cue preceded the conditioning sessions. WKHA rats displayed less orienting behavior than Wistar rats but comparable levels of food cup behavior. These data suggest that WKHA rats exhibit specific abnormalities in attentional processing as well as in learning stimulus-reward relationships.
The central nucleus (CN) of the amygdala and basal forebrain cholinergic projections to the posterior parietal cortex (PPC) are involved in regulating changes in attentional processing of conditioned stimuli. In a previous study, lesions of the CN produced a deficit in conditioned orienting behavior (rearing on the hind legs) when a visual stimulus was paired with food. Unconditioned orienting (rearing to nonreinforced presentations of the stimulus) and conditioned food cup behavior were unaffected. The present study examined the contribution of the PPC to attentional orienting behavior. Damage to the PPC did not affect orienting behavior but produced deficits in food cup behavior. These findings help define the specific contributions of the PPC to attentional processing and associative learning.
The known T-cell receptors (TCRs) involved in the recognition of antigen and major histocompatibility complex (MHC) molecules are glycoproteins comprised of polymorphic disulphide-linked alpha- and beta-chains. The genes encoding these chains are homologous to immunoglobulin genes and consist of V (variable), J (joining) and C (constant) regions that rearrange during development. TCRs are expressed relatively late in thymocyte development and only in association with an invariant molecular complex of proteins termed T3. Immature thymocytes do not express the TCR-T3 complex but do express messenger RNA encoding a third rearranging T-cell receptor-like gene, termed T gamma. Here we report a clone of normal immature T4-T8- human thymocytes, designated CII, which does not express mature mRNA for T alpha or T beta genes, but does express high levels of T gamma mRNA. This clone also expresses high levels of surface T3, and antibodies to T3 induce immunologically relevant functions in CII cells. Immunoprecipitation of CII surface-labelled proteins with anti-T3 co-precipitates a T3 molecular complex together with two additional and novel peptides of relative molecular mass (Mr), 44,000 (44K) and 62,000 (62K).
Mature T cells segregate phenotypically into one of two classes: those that express the surface glycoprotein CD4, and those that express the glycoprotein CD8. The CD4 molecule is expressed primarily on helper T cells whereas CD8 is found on cytotoxic and suppressor cells. A more stringent association exists, however, between these T-cell subsets and the major histocompatibility complex (MHC) gene products recognized by their T-cell receptors (TCRs). CD8+ lymphocytes interact with targets expressing class I MHC gene products, whereas CD4+ cells interact with class II MHC-bearing targets. To explain this association, it has been proposed that these 'accessory' molecules bind to monomorphic regions of the MHC proteins on the target cell, CD4 to class II and CD8 to class I products. This binding could hold the T cell and its target together, thus improving the probability of the formation of the trimolecular antigen: MHC: TCR complex. Because the TCR on CD4+ cells binds antigen in association with class II MHC, it has been difficult to design experiments to detect the association of CD4 with a class II molecule. To address this issue, we devised a xenogeneic system in which human CD4 complementary DNA was transfected into the murine CD4-, CD8- T-cell hybridoma 3DT-52.5.8, the TCR of which recognizes the murine class I molecule H-2Dd. The murine H-2Dd-bearing target cell line, P815, was cotransfected with human class II HLA-DR alpha, beta and invariant chain cDNAs. Co-culture of the parental T-cell and P815 lines, or of one parental and one transfected line resulted in a low baseline response. In contrast, a substantial increase in response was observed when CD4+ 3DT-52.5.8 cells were co-cultured with HLA-DR+ P815 cells. This result strongly indicates that CD4:HLA-DR binding occurs in this system and that this interaction augments T-cell activation.
In the vascular system, endothelium-derived relaxing factor (EDRF) is the name of the local hormone released from endothelial cells in response to vasodilators such as acetylcholine, bradykinin and histamine. It diffuses into underlying smooth muscle where it causes relaxation by activating guanylate cyclase, so producing a rise in cyclic GMP levels. It has been known for many years that in the central nervous system (CNS) the excitatory neurotransmitter glutamate can elicit large increases in cGMP levels, particularly in the cerebellum where the turnover rate of cGMP is low. Recent evidence indicates that cell-cell interactions are involved in this response. We report here that by acting on NMDA (N-methyl-D-aspartate) receptors on cerebellar cells, glutamate induces the release of a diffusible messenger with strikingly similar properties to EDRF. This messenger is released in a Ca2+-dependent manner and its activity accounts for the cGMP responses that take place following NMDA receptor activation. In the CNS, EDRF may link activation of postsynaptic NMDA receptors to functional modifications in neighbouring presynaptic terminals and glial cells.