The presence of donor-specific HLA antibodies before or after transplantation may have different implications based on the antibody strength. Yet, current approaches do not provide information regarding the true antibody strength as defined by antigen-antibody dissociation rate. To assess currently available methods, we compared between neat mean fluorescence intensity (MFI) values, C1q MFI values, ethylenediaminetetraacetic acid (EDTA)-treated samples, as well as titration studies and peak MFI values of over 7000 Luminex-based single-antigen HLA antibody data points. Our results indicate that neat MFI values do not always accurately depict antibody strength. We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with C1q or titration studies. In this study of patients presenting with multiple antibody specificities, a prozone effect was observed in 71% of the cohort (usually not affecting all antibody specificities within a single serum sample, though). Similar to titration studies, the C1q assay was able to address the issue of potential inhibition; however, its limitation is its low sensitivity and inability to detect the presence of weak antibodies. Titration studies are the only method among the approaches used in this study to provide information suggesting antigen-antibody dissociation rates and are, therefore, likely to provide better indication of true antibody strength.Abbreviations: AMR, antibody-mediated rejection; DSA, donor-specific antibody; EDTA, ethylenediaminetetraacetic acid; MFI, mean fluorescence intensity; SAB, single antigen beads; SPA, solid phase assays
Identifying the formation of de novo HLA-directed antibodies following heart transplantation may predict allograft outcome. This, in turn, may serve as a tool for individualization of immunosuppression protocols in heart transplant recipients.
Immunosuppression withdrawal from calcineurin inhibitors is only possible in ~20% of liver transplant recipients. However, mTOR inhibitors (sirolimus) appear to be more immunoregulatory and might promote a tolerant state for withdrawal. Our purpose was to determine if systemic (blood, marrow, allograft) signatures of immunoregulation are promoted by conversion from tacrolimus to sirolimus. We therefore performed the following serial assays before and after sirolimus conversion in liver transplant recipients to test for enhanced markers of immunoregulation: 1) Flow cytometry immunophenotyping of PBMC and bone marrow aspirates for regulatory T cells (Tregs: CD4+CD25+++FOXP3+) and regulatory dendritic cells (DCregs: ILT3+/4+); 2) liver biopsy immunohistochemical staining (FOXP3:CD3, CD4:CD8 ratios) and immunophenotyping of biopsy-derived Tregs after growth in culture; 3) effects of pre-vs. post-conversion sera on Treg generation in mixed lymphocyte reactions; 4) peripheral blood non-specific CD4 responses (Cylex® ImmuKnow); 5) peripheral blood gene transcripts and proteomic profiles. We successfully converted 20 non-immune, non-viremic recipients (age 57.2±8; 3.5±2.1 years post-LT) from tacrolimus to sirolimus for renal dysfunction. Our results demonstrated significant increases in Tregs in PBMC and marrow and DCregs in PBMC (p<0.01) following conversion. In biopsy immunohistochemistry, FOXP3:CD3 and CD4:CD8 ratios were significantly higher after conversion and a number of biopsy cultures developed new or higher FOXP3+ cell growth. Non-specific CD4 responses (Cylex® ImmuKnow) did not change. Both pre- and post-conversion sera inhibited mixed lymphocyte reactions, although only tacrolimus sera suppressed Treg generation. Finally, 289 novel genes and 22 proteins, several important in immunoregulatory pathways, were expressed after conversion. Conclusions Tacrolimus to sirolimus conversion increases systemic Tregs, DCregs and immunoregulatory proteogenomic signatures in liver transplant recipients and may therefore facilitate immunosuppression minimization or withdrawal.
Neurotoxicology considers that chemicals perturb neurological functions by interfering with the structure or function of neural pathways, circuits and systems. Using in vitro methods for neurotoxicity studies should include evaluation of specific targets for the functionalism of the nervous system and general cellular targets. In this review we present the neuronal characteristics of primary cultures of cortical neurons and of cerebellar granule cells and their use in neurotoxicity studies. Primary cultures of cortical neurons are constituted by around 40% of GABAergic neurons, whereas primary cultures of cerebellar granule cells are mainly constituted by glutamatergic neurons. Both cultures express functional GABAA and ionotropic glutamate receptors. We present neurotoxicity studies performed in these cell cultures, where specific neural targets related to GABA and glutamate neurotransmission are evaluated. The effects of convulsant polychlorocycloalkane pesticides on the GABAA, glycine and NMDA receptors points to the GABAA receptor as the neural target that accounts for their in vivo acute toxicity, whereas NMDA disturbance might be relevant for long-term toxicity. Several compounds from a list of reference compounds, whose severe human poisoning result in convulsions, inhibited the GABAA receptor. We also present cell proteomic studies showing that the neurotoxic contaminant methylmercury affect mitochondrial proteins. We conclude that the in vitro assays that have been developed can be useful for their inclusion in an in vitro test battery to predict human toxicity.
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