62 research outputs found

    Reduced Calcium Signaling Is Associated With Severe Graft-Versus-Host Disease: Results From Preclinical Models and From a Prospective EBMT Study

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    Despite its involvement in various immune functions, including the allogeneic activation of T-lymphocytes, the relevance of calcium (Ca2+) for GVHD pathobiology is largely unknown. To elucidate a potential association between Ca(2+)and GVHD, we analyzed Ca2+-sensing G-protein coupled receptor 6a (GPRC6a) signaling in preclinical GVHD models and conducted a prospective EBMT study on Ca(2+)serum levels prior alloSCT including 363 matched sibling allogeneic peripheral blood stem cell transplantations (alloSCTs). In experimental models, we found decreasedGprc6aexpression during intestinal GVHD. GPRC6a deficient alloSCT recipients had higher clinical and histopathological GVHD scores leading to increased mortality. As possible underlying mechanism, we found increased antigen presentation potential in GPRC6a(-/-)alloSCT recipients demonstrated by higher proliferation rates of T-lymphocytes. In patients with low Ca(2+)serum levels (≤ median 2.2 mmol/l) before alloSCT, we found a higher incidence of acute GVHD grades II-IV (HR = 2.3 Cl = 1.45-3.85p= 0.0006), severe acute GVHD grades III-IV (HR = 3.3 CI = 1.59-7.14,p= 0.002) and extensive chronic GVHD (HR = 2.0 Cl = 1.04-3.85p= 0.04). In conclusion, experimental and clinical data suggest an association of reduced Ca(2+)signaling with increased severity of GVHD. Future areas of interest include the in depth analysis of involved molecular pathways and the investigation of Ca(2+)signaling as a therapeutic target during GVHD

    Multifactorial Activation of NLRP3 Inflammasome: Relevance for a Precision Approach to Atherosclerotic Cardiovascular Risk and Disease

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    Chronic low-grade inflammation, through the specific activation of the NACHT leucine-rich repeat- and PYD-containing (NLRP)3 inflammasome-interleukin (IL)-1\u3b2 pathway, is an important contributor to the development of atherosclerotic cardiovascular disease (ASCVD), being triggered by intracellular cholesterol accumulation within cells. Within this pathological context, this complex pathway is activated by a number of factors, such as unhealthy nutrition, altered gut and oral microbiota, and elevated cholesterol itself. Moreover, evidence from autoinflammatory diseases, like psoriasis and others, which are also associated with higher cardiovascular disease (CVD) risk, suggests that variants of NLRP3 pathway-related genes (like NLRP3 itself, caspase recruitment domain-containing protein (CARD)8, caspase-1 and IL-1\u3b2) may carry gain-of-function mutations leading, in some individuals, to a constitutive pro-inflammatory pattern. Indeed, some reports have recently associated the presence of specific single nucleotide polymorphisms (SNPs) on such genes with greater ASCVD prevalence. Based on these observations, a potential effective strategy in this context may be the identification of carriers of these NLRP3-related SNPs, to generate a genomic score, potentially useful for a better CVD risk prediction, and, possibly, for personalized therapeutic approaches targeted to the NLRP3-IL-1\u3b2 pathway. View Full-Tex

    The Biology of Chronic Graft-versus-Host Disease: A Task Force Report from the National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease

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    Chronic graft-versus-host disease (GVHD) is the leading cause of late, nonrelapse mortality and disability in allogeneic hematopoietic cell transplantation recipients and a major obstacle to improving outcomes. The biology of chronic GVHD remains enigmatic, but understanding the underpinnings of the immunologic mechanisms responsible for the initiation and progression of disease is fundamental to developing effective prevention and treatment strategies. The goals of this task force review are as follows: • Summarize the current state of the science regarding pathogenic mechanisms of chronic GVHD and critical knowledge gaps. • Develop working hypotheses/overriding concepts for chronic GVHD development. • Define the usefulness of current preclinical models to test working hypotheses and ultimately discover and develop new therapeutic strategies. • Identify shortcomings of preclinical models, and define criteria for the creation of additional models to address these limitations. This document is intended as a review of our understanding of chronic GVHD biology and therapies resulting from preclinical studies, and as a platform for developing innovative clinical strategies to prevent and treat chronic GVHD

    Genetic variation of tight junction structures in intestinal inflammation

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    The epithelial barrier facing the external environment in the gastrointestinal (GI) tract is comprised of several components, including the tight junction (TJ) structures observed for the first time in 1963. TJ structures, which are multiprotein complexes composed of transmembrane proteins and a diverse spectrum of intracellular components, create a primary barrier to diffusion of ions, solutes, and water and they concomitantly prevent permeation of pro-inflammatory factors, such as pathogens, toxins, and antigens. Recent studies suggested that disturbance of epithelial integrity is associated with intestinal inflammatory conditions, such as inflammatory bowel disease (IBD), graft-versus-host disease (GVHD), celiac disease, but also diabetes. The general aim of this thesis was to identify novel genetic variants related to the development of intestinal inflammation with a specific focus on the TJ structures, yielding implications for epithelial integrity and paracellular permeability. Using a case-control study approach (Swedish cases and controls) in paper I, potential associations were investigated between IBD and three selected genetic markers in the genetic region of CLDN1, CLDN2, and CLDN4 (one marker per gene). The strongest association was observed between Crohn’s disease (CD) and the single nucleotide polymorphism (SNP) marker in the genetic region of CLDN2. The same SNP markers were further investigated using a family-based approach in non-Swedish families, but none of the identified associations from the Swedish case-control approach were confirmed. MORC4 which is located in the same genetic region as CLDN2 was also included in the investigation. A significant association was observed between a nonsynonymous SNP in MORC4 and CD in the Swedish case-control cohort. Similarities between IBD and GVHD include intestinal barrier defects and genetic contributions. GVHD is considered to be multifactorial, where the human leucocyte antigen (HLA) acts as a cornerstone; however, non-HLA genes have been identified in association with the outcome after stem cell transplantation (SCT). By using a casecontrol approach the relationship between non-HLA polymorphisms and emergence of GVHD as well as overall mortality after SCT was analyzed in paper II. The markers of MORC4, CD14, TLR4, and NOD2 were found to associate with the outcome (overall mortality) after SCT. The SNP marker of CD14 was the only analyzed marker that associated with acute GVHD. In paper III, the associations between IBD and several TJ genes that encode proteins reported to interact with each other were analyzed in a Swedish population. The strongest associations were observed between IBD and SNP markers in the membraneassociated guanylate kinase inverted genes MAGI2 and MAGI3. The MAGI3 SNP was also associated with ileal MAGI3 expression level in the non-inflamed non-IBD subgroup. Furthermore, no overlap between the expression levels of PTEN in inflamed colonic mucosa from patients with CD and those in the non-inflamed mucosa was detected, suggesting that PTEN is an inflammatory marker in CD. In paper IV, the genetic associations between microscopic colitis (MC) and TJ genes were analyzed in a Swedish population. The strongest association was identified between a SNP marker in PTEN and MC and also the sub-phenotype collagenous colitis (CC). Furthermore, significant associations were observed between genetic variations of MAGI1 and MC and also between a SNP marker in F11R and CC. Moreover, decreased expression levels of PTEN and MAGI1 were primarily associated to CC and the MC subtype lymphocytic colitis (LC), respectively, in comparison with controls. In conclusion, genes encoding proteins involved in the regulation of the intestinal epithelial integrity, including those contributing to TJ structures, may predispose individuals to intestinal inflammation, such as IBD and MC. Furthermore, MORC4 may be a predisposing factor for CD and one-year mortality after SCT for hematological malignancies

    Computational analysis of innate and adaptive immune responses

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    Both innate and adaptive immune processes rely on the activation of differentiated haematopoietic stem cell lineages to affect an appropriate response to pathogens. This thesis employs a largely network biology focused approach to better understand the specificity of immune cell responses in two distinct cases of pathogenic challenge. In the context of adaptive immunity, I studied the transcriptional responses of T cells during Graft-versus-Host Disease (GvHD). GvHD represents one of the major complications to arise following allogeneic hematopoietic stem cell transplantation and yet why only particular organs are damaged as a result of this pathology is still unclear. To investigate whether key GvHD transcriptional signatures seen in effector CD8+ T cells compared to naïve T cells are triggered in target organs or the secondary lymphoid organs, a module-based association test was developed to combine the output of gene clustering algorithms with that of differential expression analysis. This methodology significantly aided the identification of skin specific effector T cell transcriptional programs believed to drive murine GvHD pathogenesis at this site. Turning to the innate immune response, I investigated the transcriptional profiles of resting and activated macrophages in the setting of Tuberculosis (TB), the second leading cause of death from infectious disease worldwide. Regression-based analyses and clustering of macrophage expression data provided insight into the variations in gene expression profiles of naïve macrophages compared to those infected with Mycobacterium tuberculosis (MTB) or a vaccine strain of mycobacteria (BCG). The availability of genotype data as part of the macrophage dataset facilitated an expression quantitative trait loci (eQTL) study which highlighted a novel association between the cytoskeleton gene BCAR1 and TB risk, together with a previously undescribed trans-eQTL module specific to MTB infected macrophages. Potential genetic variants impacting expression of the aforementioned GvHD specific T cell transcriptional signatures were additionally investigated using external trans-eQTL datasets

    Characterization of human gamma delta T cells in allogeneic hematopoietic stem cell transplantation

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    Over the last five decades, allogeneic hematopoietic stem cell transplantation (HSCT) has evolved rapidly, continuing to offer a cure for several hematological diseases. Nevertheless, associated life-threatening complications remain an obstacle against exploiting its full therapeutic benefit. Among these complications, infection, relapse, and graft-versus-host disease (GVHD) represent not only the most common but also the most serious ones. Though commonly regarded as distinct clinical events, their underlying pathophysiology is firmly related from an immunological perspective. T lymphocytes are key players in HSCT complications and their proper reconstitution following allogeneic HSCT is central for beneficial clinical outcome. The last two decades have witnessed a growing interest in a subset of T cells known as gamma delta (γδ) T cells. The immunological capabilities of these unconventional cells have been intensively explored. However, more efforts aimed at unraveling the immunobiological features of different γδ subsets are warranted to effectively exploit their full immunotherapeutic potential. In the present work, I tried to tackle several immune-related aspects that directly influence allogeneic HSCT outcome with a special focus on γδ T cells. In paper I, the main objective was to address the impact of different GVHD prophylaxis regimens on de novo generation of T and B lymphocytes. Using PCR methods, T cell receptor recombination excision circle (TREC), kappa deleting recombination excision circle (KREC), and telomere length (TL) were quantified in the peripheral blood (PB) of transplanted patients at several time intervals. Although there was no significant difference between the two GVHD prophylaxis groups, we identified other transplant related factors that were associated with reduced TREC and/or KREC levels after HSCT. Furthermore, we showed that high levels of these excision circles correlated with favorable outcome post HSCT. In paper II-IV, more attention was paid to explore the role of γδ T cells in donor grafts. Using multicolor flow cytometry together with other molecular and functional assays, we found a significant association between graft frequencies of CD8+γδ T cells and acute GVHD (aGVHD) grade II-III in Paper II. Additionally, we showed that higher frequencies of CD27+ γδ T cells in the stem cell grafts were correlated with both less relapse and CMV incidences. The results from paper II highlighting a potential role of CD8+γδ T cells in donor grafts raised our interest to further investigate this subset to elucidate their immunological characteristics. In paper III we thoroughly analysed γδ T cells in BM grafts using multicolor flow cytometry and TCR repertoire analysis using next generation sequencing (NGS). We showed that grafts from CMV+ donors contained higher proportions of CD8+γδ T that preferentially expressed Vγ9- and differentiated towards terminal effector memory phenotype. Additionally, analysis of TCRγ chain revealed a clonally focused repertoire in CMV+ donor grafts. We also showed that CD8+γδ T cells differentially respond to TCR stimuli suggesting adaptive-like phenotype In paper IV, we sought to address whether allogeneic HSCT outcome is influenced by γδ TCR repertoire composition in donor grafts. Immunosequencing of TCRγ chain by NGS revealed a more public repertoire and increased presence of long sequence clonotypes in graft given to non-relapsed patients. Further analysis of the amino acid sequences identified 12 public and 4 private sequences that were exclusively found in high frequencies in grafts given to nonrelapsed patients. Finally, in paper V we aimed to optimize a protocol for efficient in-vitro expansion of Vγ9Vδ2 T cells from umbilical cord blood (UCB). Phenotypical and functional characterization of expanded cells was comparable to PB and suggests that UCB can be a reliable source for Vγ9Vδ2 T cell expansion

    Aspectos imunogenéticos da imunotolerância na gestação e transplantes

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    O estudo da imunologia reprodutiva data da década de 1950. Seus estudos emergiram das observações pioneiras de Peter Medawar sobre a existência de antígenos de tecido, mais especificamente, em como antígenos expressos em enxertos de pele eram reconhecidos e rejeitados quando transplantados em indivíduos geneticamente distintos. Na gestação, a aceitação do feto é um evento único, e, demonstra como o sistema imunológico materno se adapta e tolera as células fetais semi-alogênicas sem rejeitá-las, mesmo considerando que o feto herda e expressa metade dos antígenos paternos e metade maternos. Diante disso, sugere-se que muitas desordens gestacionais, tais como, a pré-eclâmpsia e abortamentos, sejam causadas por desequilíbrios na tolerância materna. Tal tolerância é alcançada, entre outros mecanismos, através da elevada expressão das moléculas do MHC de classe I não clássico (HLA-E, -F e -G) e ausência de expressão de MHC de classe I clássico (exceto a baixa expressão de HLA-C) e de moléculas de MHC-II, as quais conferem baixa antigenicidade às células fetais. No contexto dos transplantes (histocompatibilidade), a tolerância imunológica é estabelecida de maneira mais complexa, uma vez que deve se considerar dois genomas distintos. A rejeição do transplante é um fenômeno "criado pelo homem", uma vez que o transplante de órgãos não é evidenciado no mundo natural. Embora a rejeição seja altamente dependente da incompatibilidade entre os diferente alelos de HLA entre os pares de doador e receptor, a rejeição aguda ou crônica pode ocorrer mesmo em pares compatíveis, sugerindo a influência de outras moléculas não clássicas do MHC-I e moléculas não-HLA no desfecho dos transplantes. Apesar de serem fenômenos biológicos distintos, as semelhanças entre a gravidez e o transplante não podem ser negligenciadas, especificamente, em relação ao papel imunorregulatório das moléculas do MHC-I não clássico e moléculas não HLA. Na presente tese, apresentaremos o papel das variantes genéticas em genes não clássicos do MHC-I (HLA-E, HLA-G, MIC-A) e receptores cognatos (NKG2C e NKG2D) no contexto da gravidez saudável ou patológica. Para complementar esta tese, avaliamos como os eixos imunológicos NKG2C/HLA-E e NKGD2/MIC-A influenciam na manutenção e sobrevivência do transplante. No total, três artigos publicados em periódicos internacionais e, adicionalmente, três artigos em fase de preparação para publicação compõem está tese. Os resultados desses artigos (publicados) podem ser resumidos como seguem: 1. Michita et al. (2016) A tug-of-war between tolerance and rejection - New evidence for 3'UTR HLAG haplotypes influence in recurrent pregnancy loss (Hum Immunol. 77:892-897). Neste estudo observamos que as variantes genéticas +3010C/G, +3142G/C e +3187A/G localizados na região 3’UTR do HLA-G influenciam na suscetibilidade para o abortamento de repetição numa população brasileira. Além disso, haplótipos observados nessa região foram caracterizados na população estudada, e, a associação do haplótipo UTR-1 com o risco de abortamentos foi observada. 2. Michita et al. (2018) A Valine Mismatch at Position 129 of MICA Is an Independent Predictor of Cytomegalovirus Infection and Acute Kidney Rejection in Simultaneous Pancreas–Kidney Transplantation Recipients (Int J Mol Sci. 4;19). Nesse estudo avaliamos o impacto da variante MICA Val129Met no desfecho do primeiro ano após o transplante simultâneo de rim e pâncreas. Brevemente, observamos que incompatibilidade no MHC (HLAA, -B, -DR) não influenciaram no desfecho (infecção pelo citomegalovírus, função do órgão e rejeição aguda). Porém, um acentuado efeito no desfecho foi influenciado pela incompatibilidade do alelo MICA 129Val (doador → receptor). Estas observações foram observadas pela primeira vez no contexto de transplantes de órgão sólidos e destacam a importância de outros genes não-HLA no desfecho dos transplantes. 3. Michita et al. (2018) Genetic Variants in Preeclampsia: Lessons From Studies in Latin-American populations (Front Physiol. 4;9:1771). Nesse estudo foi realizado um extensa revisão da literatura no que se refere aos estudos genéticos – variantes genéticas – realizados em populações latinoamericanas investigando a patogênese da pré-eclâmpsia. Este artigo de revisão representa uma importante fonte de dados para estudos futuros devido a sua abrangência e profundidade. Além disso, o artigo destaca os principais grupos engajados na caracterização das bases genéticas da pré-eclâmpsia. Os resultados da presente tese, destacam que apesar das diferenças entre os transplantes e a gestação, diversos mecanismos imunorregulatórios podem se sobrepor nestes dois fenômenos biológicos. Principalmente, no que se refere a investigação das moléculas não clássicas do MHC de classe-I.The field of reproductive immunology dates from the 1950s. It emerged from the pioneering observations of Peter Medawar on tissue antigens, more specifically, in how skin grafts were recognized and rejected when transplanted to a genetically different individual. In pregnancy, the acceptance of the fetus is a unique event, and shows how the maternal immune system should shape to tolerate and do not reject the semi-allogeneic fetal cells, even considering that the fetus inherited half of antigens from each parental - mother and father. Not surprisingly, many pregnancy disorders such as preeclampsia and miscarriages are thought to originate from a breakdown of tolerance. Such tolerance is achieved, amongst other mechanisms, by a high expression of non-classical MHC class I molecules such as HLA-E, -F and –G, and the lack of expression of classical MHC-I (or low expression of HLA-C) and MHC-II molecules. Both such mechanisms allow poor antigenicity to fetal cells. In the context of allotransplantation, immunological tolerance is achieved in a more complex way (histocompatibility), as the attention must be directed to antigens derived from more than one genome. Graft rejection is a “man-made” phenomenon since tissue transplantation is no evidenced in the natural world. Although rejection is highly dependent on HLA mismatches between donor and recipient pairs, even fully matched patients can undergo acute or chronic rejection, suggesting the existence of other non-classical MHC-I and non-HLA molecules driving transplantation outcome. Despite being different biological phenomena, the similarities between pregnancy and transplantation cannot be neglected, is essential to consider the overlapped functions towards a tolerogenic phenotype conferred by non-classical MHC-I and non-MHC molecules. In the current thesis, we will present the role of functional genetic variants in non-classical MHC-I (HLA-E, HLA-G, MIC-A) and cognate receptors (NKG2A and NKG2D) in the context of a healthy or a pathological pregnancy. In order to complement this thesis, we evaluated how the immunological axis NKG2C/HLA-E and NKGD2/MIC-A are involved in the maintenance of organ transplantation. In total, three articles published in international journals and, additionally, three articles in preparation compose the current thesis. The results of these (published) articles can be summarized as follows: 1. Michita et al. (2016) A tug-of-war between tolerance and rejection - New evidence for 3'UTR HLAG haplotypes influence recurrent pregnancy loss (Hum Immunol 77: 892- 897). In this study, we observed that the genetic variants +3010C/G, +3142G/C and +3187A/G located in the 3'UTR region of HLA-G influence the susceptibility to recurrent pregnancy loss in a Brazilian population. In addition, haplotypes observed in this region were characterized in the population, and an association of the UTR-1 haplotype with the risk of miscarriages was observed. 2. Michita et al. (2018) Valine Mismatch at Position 129 of MICA Is an Independent Predictor of Cytomegalovirus Infection and Acute Kidney Rejection in Simultaneous Pancreas-Kidney Transplantation Recipients (Int J Mol Sci. 4;19). In this study, we evaluated the impact of the MICA Val129Met variant on the first year outcome after simultaneous transplantation of the kidney and pancreas. Briefly, we found that MHC mismatch (HLA-A, -B, -DR) did not influence the outcome (cytomegalovirus infection, organ function, and acute rejection). However, a pronounced effect on the outcome was influenced by the incompatibility of the MICA 129Val allele (donor → recipient). These observations were first observed in the context of solid organ transplants and highlight the importance of other non-HLA genes in the outcome of transplants. 3. Michita et al. (2018) Genetic Variants in Preeclampsia: Lessons From Studies in Latin American Populations (Front Physiol. 4: 9: 1771). In this study, an extensive review of the literature was carried out regarding genetic studies - genetic variants - carried out in Latin American populations investigating the pathogenesis of preeclampsia. This review article represents an essential source of data for future studies because of its breadth and depth. In addition, the article highlights the main groups engaged in characterizing the genetic basis of preeclampsia in Latin America. The results of this thesis point out that despite the differences between transplants and gestation, several immunoregulatory mechanisms can overlap in these two biological phenomena. Mainly for the investigation of non-classical MHC class I molecules
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