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The Spartan Medical Research Journal (SMRJ) is pleased to publish abstracts from the First Annual Research Day hosted by the Michigan State University College of Osteopathic Medicine (MSUCOM), held in Novi, Michigan, on May 15, 2023. Sponsored by MSUCOM, the Statewide Campus System (SCS), and Research, Innovation, and Scholarly Engagement (RISE), this event showcased a total of 139 selected research abstracts following a meticulous blinded review by the MSUCOM Research Day Planning Committee and SMRJ editorial staff. These abstracts were subsequently presented at the MSUCOM First Annual Research Day in 2023, with awards for exceptional oral and poster presentations conferred on May 15, 2023. Of the 139 presentations that were ultimately chosen, 45 authors consented and elected to have their abstracts published in SMRJ. The abstracts from 2023 encompass a wide array of contemporary medical and clinical subjects, incorporating a variety of research designs that cover basic science, clinical research, case reports, medical education, and quality improvement. While abstracts offer concise overview of research projects or presentations, they do not permit a comprehensive evaluation of the scientific rigor employed in the respective works. Although these abstracts offer preliminary results that may necessitate further refinement and validation, they serve a vital function in disseminating novel research concepts and advancements in the discipline of medicine. This knowledge-sharing promotes meaningful dialogue among researchers, clinicians, and educators, thereby making a valuable contribution to the collective body of knowledge in the fields of medical sciences and osteopathic medicine. Andrea Amalfitano, DO, PhD Osteopathic Heritage Foundation Professor of Pediatrics, Microbiology and Molecular Genetics Professor, BioMolecular Science Gateway Editor-in-Chief, Spartan Medical Research Journal (SMRJ) MSU College of Osteopathic Medicine- Statewide Campus System C. Patricia Obando S., PhD Associate Dean and DIO, Graduate Medical Education Associate Professor- MSU College of Osteopathic Medicine- Statewide Campus System Rana Ismail, PhD, MSc, CPHQ Director of Research Editor, Spartan Medical Research Journal (SMRJ) MSU College of Osteopathic Medicine- Statewide Campus System Francis Akenami, BMLS, PhD, MSc, FIMLS Managing Editor Spartan Medical Research Journal (SMRJ) MSU College of Osteopathic Medicine- Statewide Campus System.
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BACKGROUND: Multiple sclerosis (MS) is a chronic, debilitating condition characterized by CNS autoimmunity stemming from a complex etiology involving both environmental and genetic factors. Our current understanding of MS points to dysregulation of the immune system as the pathogenic culprit, however, it remains unknown as to how the many genes associated with increased susceptibility to MS are involved. One such gene linked to MS susceptibility and known to regulate immune function is the self-ligand immune cell receptor SLAMF7. METHODS: We subjected WT and SLAMF7-/- mice to multiple EAE models, compared disease severity, and comprehensively profiled the CNS immune landscape of these mice. We identified all SLAMF7-expressing CNS immune cells and compared the entire CNS immune niche between genotypes. We performed deep phenotyping and in vitro functional studies of B and T cells via spectral cytometry and BioPlex assays. Adoptive transfer studies involving the transfer of WT and SLAMF7-/- B cells into B cell-deficient mice (µMT) were also performed. Finally, B-T cell co-culture studies were performed, and a comparative cell-cell interaction network derived from scRNA-seq data of SLAMF7+ vs. SLAMF7- human CSF immune cells was constructed. RESULTS: We found SLAMF7-/- mice to be more susceptible to EAE compared to WT mice and found SLAMF7 to be expressed on numerous CNS immune cell subsets. Absence of SLAMF7 did not grossly alter the CNS immune landscape, but allowed for altered immune cell subset infiltration during EAE in a model-dependent manner. Global lack of SLAMF7 expression increased myeloid cell activation states along with augmented T cell anti-MOG immunity. B cell profiling studies revealed increased activation states of specific plasma and B cell subsets in SLAMF7-/- mice during EAE, and functional co-culture studies determined that SLAMF7-/- B cells induce exaggerated T cell activation. Adoptive transfer studies revealed that the increased susceptibility of SLAMF7-/- mice to EAE is partly B cell dependent and reconstruction of the human CSF SLAMF7-interactome found B cells to be critical to cell-cell communication between SLAMF7-expressing cells. CONCLUSIONS: Our studies have identified novel roles for SLAMF7 in CNS immune regulation and B cell function, and illuminate underpinnings of the genetic association between SLAMF7 and MS.
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Encefalomielite Autoimune Experimental , Esclerose Múltipla , Imunidade Adaptativa , Animais , Autoimunidade , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/patologia , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismoRESUMO
BACKGROUND: In addition to its role in antigen presentation, recent reports establish a new role for endoplasmic reticulum aminopeptidase 1 (ERAP1) in innate immunity; however, the mechanisms underlying these functions are not fully defined. We previously confirmed that loss of ERAP1 functions resulted in exaggerated innate immune responses in a murine in vivo model. Here, we investigated the role of ERAP1 in suppressing inflammasome pathways and their dependence on ER stress responses. RESULTS: Using bone marrow-derived macrophages (BMDMs), we found that loss of ERAP1 in macrophages resulted in exaggerated production of IL-1ß and IL-18 and augmented caspase-1 activity, relative to wild type macrophages. Moreover, an in vivo colitis model utilizing dextran sodium sulfate (DSS) confirmed increased levels of proinflammatory cytokines and chemokines in the colon of DSS treated ERAP1-/- mice as compared to identically stimulated WT mice. Interestingly, stimulated ERAP1-/- BMDMs and CD4+ T cells simultaneously demonstrated exaggerated ER stress, assessed by increased expression of ER stress-associated genes, a state that could be reverted to WT levels with use of the ER stress inhibitor Tauroursodeoxycholic acid (TUDCA). CONCLUSIONS: Together, these results not only suggest that ERAP1 is important for regulating inflammasome dependent innate immune response pathways in vivo, but also propose a mechanism that underlies these changes, that may be associated with increased ER stress due to lack of normal ERAP1 functions.
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Aminopeptidases , Estresse do Retículo Endoplasmático , Inflamassomos , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Imunidade Inata/genética , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genéticaRESUMO
Targeted modulation of the immune system against tumors can achieve responses in otherwise refractory cancers, which has spurred efforts aimed at optimizing such strategies. To this end, we have previously investigated cancer immunotherapy approaches using recombinant adenovirus vectors, as well as via modulation of the self-ligand receptor SLAMF7. Here, we present a gene transfer-based immunotherapy approach using targeted expression of a SLAMF7-Fc fusion construct directly into tumors at high concentrations via a recombinant adenoviral vector (Ad-SF7-Fc). Using multiple murine cancer models, we show that Ad-SF7-Fc can induce tumor control via augmentation of innate immunity; specifically, induction of type I interferons and activation of dendritic cells (DCs) and macrophages. Analogously, we find that modulating SLAMF7 signaling via an adenoviral vector expressing its intracellular adaptor, EAT-2, is also capable of inducing tumor control. Finally, we employ a novel in vivo prediction approach and dataset integration with machine learning to dissect how Ad-SF7-Fc modulates cell-type-specific responses in the tumor microenvironment to achieve tumor control. Thus, our novel combinatorial cancer immunotherapy highlights the benefit of multimodal immune modulation and lays a framework for combination with complementary approaches capable of inducing adaptive immune responses.
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Hundreds of genes have been linked to multiple sclerosis (MS); yet, the underlying mechanisms behind these associations have only been investigated in a fraction of cases. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an endoplasmic reticulum-localized aminopeptidase with important roles in trimming peptides destined for MHC class I and regulation of innate immune responses. As such, genetic polymorphisms in ERAP1 have been linked to multiple autoimmune diseases. In this study, we present, to our knowledge, the first mechanistic studies performed to uncover why polymorphisms in ERAP1 are associated with increased susceptibility to MS. Combining multiple mouse models of CNS autoimmunity with high-dimensional single-cell spectral cytometry, adoptive transfer studies, and integrative analysis of human single-cell RNA-sequencing datasets, we identify an intrinsic defect in B cells as being primarily responsible. Not only are mice lacking ERAP1 more susceptible to CNS autoimmunity, but adoptive transfer of B cells lacking ERAP1 into B cell-deficient mice recapitulates this susceptibility. We found B cells lacking ERAP1 display decreased proliferation in vivo and express higher levels of activation/costimulatory markers. Integrative analysis of single-cell RNA sequencing of B cells from 36 individuals revealed subset-conserved differences in gene expression and pathway activation in individuals harboring the MS-linked K528R ERAP1 single-nucleotide polymorphism. Finally, our studies also led us to create, to our knowledge, the first murine protein-level map of the CNS IL-10+ immune compartment at steady state and during neuroinflammation. These studies identify a role for ERAP1 in the modulation of B cells and highlight this as one reason why polymorphisms in this gene are linked to MS.
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Doenças Autoimunes , Linfócitos B , Esclerose Múltipla , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Autoimunidade/genética , Sistema Nervoso Central , Camundongos , Antígenos de Histocompatibilidade Menor/genética , Esclerose Múltipla/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
T cell exhaustion represents one of the most pervasive strategies tumors employ to circumvent the immune system. Although repetitive, cognate TCR signaling is recognized as the primary driving force behind this phenomenon, and it remains unknown what other forces drive T cell exhaustion in the tumor microenvironment (TME). In this study, we show that activation of the self-ligand SLAMF7 immune receptor on T cells induced STAT1 and STAT3 phosphorylation, expression of multiple inhibitory receptors, and transcription factors associated with T cell exhaustion. Analysis of The Cancer Genome Atlas revealed that SLAMF7 transcript levels were strongly correlated with various inhibitory receptors and that high SLAMF7 expression was indicative of poor survival in clear cell renal cell carcinoma (ccRCC). Targeted reanalysis of a CyTOF dataset, which profiled the TME in 73 ccRCC patients, revealed cell-type-specific SLAMF7 expression patterns, strong correlations between exhausted T cells and SLAMF7+ tumor-associated macrophages (TAMs), and a unique subset of SLAMF7highCD38high TAMs. These SLAMF7highCD38high TAMs showed the strongest correlations with exhausted T cells and were an independent prognostic factor in ccRCC. Confirmatory ex vivo coculture studies validated that SLAMF7-SLAMF7 interactions between murine TAMs and CD8+ T cells induce expression of multiple inhibitory receptors. Finally, mice lacking SLAMF7 show restricted growth of B16-F10 tumors, and CD8+ T cells from these mice express less PD-1 and TOX and exhibited an impaired ability to progress through the exhaustion developmental trajectory to terminal exhaustion. These findings suggest that SLAMF7 might play an important role in modulating T cell function in the TME.
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Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Melanoma/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Neoplasias Cutâneas/metabolismo , Linfócitos T/imunologia , Animais , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/mortalidade , Células Cultivadas , Reprogramação Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Tolerância Imunológica , Neoplasias Renais/imunologia , Neoplasias Renais/mortalidade , Masculino , Melanoma/imunologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Neoplasias Cutâneas/imunologia , Análise de Sobrevida , Microambiente TumoralRESUMO
The signaling lymphocytic activation molecule (SLAM) family of receptors are expressed on the majority of immune cells. These receptors often serve as self-ligands, and play important roles in cellular communication and adhesion, thus modulating immune responses. SLAM family receptor signaling is differentially regulated in various immune cell types, with responses generally being determined by the presence or absence of two SLAM family adaptor proteins-Ewing's sarcoma-associated transcript 2 (EAT-2) and SLAM-associated adaptor protein (SAP). In addition to serving as direct regulators of the immune system, certain SLAM family members have also been identified as direct targets for specific microbes and viruses. Here, we will discuss the known roles for these receptors in the setting of viral infection, with special emphasis placed on HIV infection. Because HIV causes such complex dysregulation of the immune system, studies of the roles for SLAM family receptors in this context are particularly exciting.
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Cannabis is widely used in the United States, with an estimated prevalence of 9.5%. Certain cannabinoids in Cannabis sativa, Δ9-tetrahydrocannabinol (THC) in particular, possess immune-modulating and anti-inflammatory activity. Depending on the context, the anti-inflammatory activity of cannabinoids may be beneficial (e.g., in treating inflammatory diseases) or detrimental to normal immune defense against pathogens. The potential beneficial effect of cannabinoids on chronic neuroinflammation has gained recent attention. Monocyte migration to the brain has been implicated as a key event in chronic neuroinflammation and in the etiology of central nervous system diseases including viral infection (e.g., human immunodeficiency virus-associated neurocognitive disorder). In the brain, monocytes can contribute to neuroinflammation through interactions with astrocytes, including inducing astrocyte secretion of cytokines and chemokines. In a human coculture system, monocyte-derived interleukin (IL)-1ß due to Toll-like receptor 7 (TLR7) activation has been identified to promote astrocyte production of monocyte chemoattractant protein (MCP)-1 and IL-6. THC treatment of the TLR7-stimulated coculture suppressed monocyte secretion of IL-1ß, resulting in decreased astrocyte production of MCP-1 and IL-6. Furthermore, THC displayed direct inhibition of monocytes, as TLR7-stimulated monocyte monocultures treated with THC also showed suppressed IL-1ß production. The cannabinoid receptor 2 (CB2) agonist, JWH-015, impaired monocyte IL-1ß production similar to that of THC, suggesting that THC acts, in part, through CB2. THC also suppressed key elements of the IL-1ß production pathway, including IL1B mRNA levels and caspase-1 activity. Collectively, this study demonstrates that the anti-inflammatory properties of THC suppress TLR7-induced monocyte secretion of IL-1ß through CB2, which results in decreased astrocyte secretion of MCP-1 and IL-6. SIGNIFICANCE STATEMENT: Because cannabis use is highly prevalent in the United States and has putative anti-inflammatory properties, it is important to investigate the effect of cannabinoids on immune cell function. Furthermore, cannabinoids have garnered particular interest due to their potential beneficial effects on attenuating viral-induced chronic neuroinflammation. This study utilized a primary human coculture system to demonstrate that the major psychotropic cannabinoid in cannabis, Δ9-tetrahydrocannabinol, and a cannabinoid receptor-2 selective agonist suppress specific monocyte-mediated astrocyte inflammatory responses.
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Astrócitos/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/farmacologia , Quimiocina CCL2/metabolismo , Dronabinol/farmacologia , Interleucina-6/metabolismo , Monócitos/efeitos dos fármacos , Receptor 7 Toll-Like/metabolismo , Astrócitos/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Técnicas de Cocultura , Humanos , Indóis/farmacologia , Interleucina-6/genética , Monócitos/metabolismoRESUMO
Toll-like receptor 7 (TLR7)-activation has been implicated as a significant mechanism of neuroinflammation triggered by ssRNA viruses. Infiltration of monocytes into the brain and astrocyte activation occurs during in vivo TLR7-mediated neuroinflammation. The objective here was to determine whether the TLR7 agonist, imiquimod, and interferon-alpha (IFN-α), promote monocyte-mediated astrocyte secretion of pro-inflammatory factors. Using a human primary co-culture system, we demonstrate that monocytes, together with imiquimod and IFN-α, promote astrocyte secretion of MCP-1, IL-6 and IP-10. Furthermore, TLR7-induced monocyte-derived IL-1ß is critical for promoting the astrocyte response. Overall, this study provides a potential mechanism for TLR7-mediated neuroinflammation.
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Astrócitos/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Quimiocina CXCL10/metabolismo , Imiquimode/farmacologia , Interferon-alfa/farmacologia , Interleucina-6/metabolismo , Monócitos/efeitos dos fármacos , Receptor 7 Toll-Like/fisiologia , Astrócitos/fisiologia , Linhagem Celular , Técnicas de Cocultura , Humanos , Inflamação , Interleucina-1beta/farmacologia , Interleucina-1beta/fisiologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Monócitos/fisiologia , Neuroimunomodulação , Proteínas Recombinantes/farmacologia , Receptor 7 Toll-Like/agonistasRESUMO
Autoimmune and autoinflammatory diseases affect millions worldwide. These classes of disease involve abnormal immune activation of both the innate and adaptive immune systems. While both classes of disease represent a spectrum of aberrant immune activation, excessive activation of the innate immune system has been considered causal for the inflammation and tissue damage found in autoinflammatory diseases, while excessive activation of the adaptive immune system has been thought to primarily contribute to end-organ symptoms noted in autoimmune diseases. Interestingly, the endoplasmic reticulum aminopeptidase 1 (ERAP1) protein, well known for its aminopeptidase function as a "molecular ruler", trimming peptides prior to their loading onto MHC-I molecules for antigen presentation in the ER, has also been shown to be genetically associated with both autoinflammatory and autoimmune diseases. Indeed, this multifaceted protein has been found to have many functions that affect both the innate and adaptive immune responses. In this review, we summarize these findings, with an attempt to identify the possible ERAP1 dependent mechanisms responsible for the pathogenesis of multiple, ERAP1 associated diseases.
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Aminopeptidases/genética , Aminopeptidases/metabolismo , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Autoimunidade , Inflamação/etiologia , Inflamação/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Imunidade Adaptativa , Aminopeptidases/química , Animais , Apresentação de Antígeno/imunologia , Suscetibilidade a Doenças , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunidade Inata , Antígenos de Histocompatibilidade Menor/químicaRESUMO
Current advances in combined antiretroviral therapy have rendered HIV infection a chronic, manageable disease; however, the problem of persistent immune activation still remains despite treatment. The immune cell receptor SLAMF7 has been shown to be upregulated in diseases characterized by chronic immune activation. In this study, we studied the function of the SLAMF7 receptor in immune cells of HIV patients and the impacts of SLAMF7 signaling on peripheral immune activation. We observed increased frequencies of SLAMF7+ PBMCs in HIV+ individuals in a clinical phenotype-dependent manner, with discordant and long-term nonprogressor patients showing elevated SLAMF7 levels, and elite controllers showing levels comparable to healthy controls. We also noted that SLAMF7 was sensitive to IFN-⺠stimulation, a factor elevated during HIV infection. Further studies revealed SLAMF7 to be a potent inhibitor of the monocyte-derived proinflammatory chemokine CXCL10 (IP-10) and other CXCR3 ligands, except in a subset of HIV+ patients termed SLAMF7 silent (SF7S). Studies utilizing small molecule inhibitors revealed that the mechanism of CXCL10 inhibition is independent of known SLAMF7 binding partners. Furthermore, we determined that SLAMF7 activation on monocytes is able to decrease their susceptibility to HIV-1 infection in vitro via downregulation of CCR5 and upregulation of the CCL3L1 chemokine. Finally, we discovered that neutrophils do not express SLAMF7, are CXCL10+ at baseline, are able to secrete CXCL10 in response to IFN-⺠and LPS, and are nonresponsive to SLAMF7 signaling. These findings implicate the SLAMF7 receptor as an important regulator of IFN-âº-driven innate immune responses during HIV infection.
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Infecções por HIV/imunologia , HIV-1/fisiologia , Interferon-alfa/metabolismo , Neutrófilos/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Células Cultivadas , Quimiocina CCL3/metabolismo , Quimiocina CXCL10/metabolismo , Progressão da Doença , Suscetibilidade a Doenças , Humanos , Fenótipo , Receptores CCR5/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Monocyte infection by HIV-1 is an important component of chronic HIV pathogenesis. Following infection by HIV-1, monocytes are able to cross the blood brain barrier and set up a viral reservoir in the central nervous system. Additionally, in the setting of chronic HIV-1 infection, monocytes can become activated either directly through HIV-1 infection or indirectly via HIV-1-mediated systemic immune activation. Currently, there are few studies looking at HIV-1 infection of primary human monocytes in vitro. Furthermore, detection of successful HIV-1 infection of monocytes can be laborious requiring an ELISA for p24 or assessing levels of HIV-1 mRNA or DNA. This protocol utilizes an HIV-1 strain expressing GFP to allow for easy quantification of HIV-1 infection by fluorescence-assisted cell sorting (FACS). By determining HIV-1 infection by FACS one can take advantage of its multiparametric nature allowing for the use of less cells and the ability to assess the expression of other markers on HIV-1+ and HIV-1- cells in the same experiment. Additionally, this protocol could be modified to study HIV-1 infection of other cells including CD4+ T cells.
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Ankylosing spondylitis (AS) is a prototypical sero-negative autoimmune disease that affects millions worldwide. Single nucleotide polymorphisms in the Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) gene have been linked to AS via GWAS studies, however, the exact mechanism as to how ERAP1 contributes to pathogenesis of AS is not understood. We undertook µCT imaging and histologic analysis to evaluate bone morphology of the axial skeletons of ERAP1-/- mice and discovered the hallmark skeletal features of AS in these mice, including spinal ankylosis, osteoporosis, and spinal inflammation. We also confirmed the presence of spontaneous intestinal dysbiosis and increased susceptibility to Dextran Sodium Sulfate (DSS)-induced colitis in ERAP1-/- mice, however the transfer of healthy microbiota from wild type mice via cross-fostering experiments did not resolve the skeletal phenotypes of ERAP1-/- mice. Immunological analysis demonstrated that while ERAP1-/- mice had normal numbers of peripheral Foxp3+ Tregs, they had reduced numbers of both "Tr1-like" regulatory T cells and tolerogenic dendritic cells, which are important for Tr1 cell differentiation. Together, our data suggests that ERAP1-/- mice may serve as a useful animal model for studying pathogenesis of intestinal, skeletal, and immunological manifestations of Ankylosing Spondylitis.
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Aminopeptidases/genética , Predisposição Genética para Doença/genética , Antígenos de Histocompatibilidade Menor/genética , Polimorfismo de Nucleotídeo Único/genética , Espondilite Anquilosante/genética , Espondilite Anquilosante/imunologia , Linfócitos T Reguladores/imunologia , Aminopeptidases/imunologia , Animais , Colite/genética , Colite/imunologia , Disbiose/genética , Disbiose/imunologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Inflamação/genética , Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/imunologia , Fenótipo , Polimorfismo de Nucleotídeo Único/imunologiaRESUMO
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in phenotypically diverse dominant and recessive human diseases. The charging of tRNAPHE with phenylalanine is performed by a tetrameric enzyme that contains two alpha (FARSA) and two beta (FARSB) subunits. To date, mutations in the genes encoding these subunits (FARSA and FARSB) have not been implicated in any human disease. Here, we describe a patient with a severe, lethal, multisystem, developmental phenotype who was compound heterozygous for FARSB variants: p.Thr256Met and p.His496Lysfs*14. Expression studies using fibroblasts isolated from the proband revealed a severe depletion of both FARSB and FARSA protein levels. These data indicate that the FARSB variants destabilize total phenylalanyl-tRNA synthetase levels, thus causing a loss-of-function effect. Importantly, our patient shows strong phenotypic overlap with patients that have recessive diseases associated with other ARS loci; these observations strongly support the pathogenicity of the identified FARSB variants and are consistent with the essential function of phenylalanyl-tRNA synthetase in human cells. In sum, our clinical, genetic, and functional analyses revealed the first FARSB variants associated with a human disease phenotype and expand the locus heterogeneity of ARS-related human disease.
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Aminoacil-tRNA Sintetases/genética , Predisposição Genética para Doença , Mutação com Perda de Função/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/deficiência , Regulação da Expressão Gênica , Humanos , Masculino , Fenótipo , Fenilalanina-tRNA Ligase/genéticaRESUMO
Tight junctions (TJs) involve close apposition of transmembrane proteins between cells. Although TJ proteins have been studied in detail, the role of lipids is largely unknown. We addressed the role of very long-chain (VLC ≥26) ceramides in TJs using diabetes-induced loss of the blood-retinal barrier as a model. VLC fatty acids that incorporate into VLC ceramides are produced by elongase elongation of very long-chain fatty acids protein 4 (ELOVL4). ELOVL4 is significantly reduced in the diabetic retina. Overexpression of ELOVL4 significantly decreased basal permeability, inhibited vascular endothelial growth factor (VEGF)- and interleukin-1ß-induced permeability, and prevented VEGF-induced decrease in occludin expression and border staining of TJ proteins ZO-1 and claudin-5. Intravitreal delivery of AAV2-hELOVL4 reduced diabetes-induced increase in vascular permeability. Ultrastructure and lipidomic analysis revealed that ω-linked acyl-VLC ceramides colocalize with TJ complexes. Overall, normalization of retinal ELOVL4 expression could prevent blood-retinal barrier dysregulation in diabetic retinopathy through an increase in VLC ceramides and stabilization of TJs.
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Barreira Hematorretiniana/metabolismo , Permeabilidade Capilar/genética , Ceramidas/metabolismo , Células Endoteliais/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Vasos Retinianos/metabolismo , Junções Íntimas/metabolismo , Animais , Bovinos , Claudina-5/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/etiologia , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Células Endoteliais/ultraestrutura , Humanos , Interleucina-1beta/metabolismo , Camundongos , Ocludina/metabolismo , Retina/metabolismo , Vasos Retinianos/ultraestrutura , Junções Íntimas/ultraestrutura , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
OBJECTIVE: Chronic immune activation and elevated numbers of circulating activated monocytes (CD16) are implicated in HIV-associated neuroinflammation. The objective was to compare the level of circulating CD16 monocytes and IFN-γ-inducible protein 10 (IP-10) between HIV-infected cannabis users (HIV+MJ+) and noncannabis users (HIV+MJ-) and determine whether in-vitro Δ-Tetrahydrocannabinol (THC), a constituent of cannabis, affected CD16 expression as well as IP-10 production by monocytes. DESIGN: The levels of circulating CD16 monocytes and IP-10 from HIV+MJ- and HIV+MJ+ donors were examined. In-vitro experimentation using THC was performed on primary leukocytes isolated from HIV-MJ-, HIV+MJ- and HIV+MJ+ donors to determine if THC has an impact on CD16 monocyte and IP-10 levels. METHODS: Flow cytometry was used to measure the number of blood CD16 monocytes and plasma IP-10 from HIV+MJ- and HIV+MJ+ donors. Peripheral blood mononuclear cells were isolated from HIV-MJ- and HIV+ (MJ- and MJ+) donors for in-vitro THC and IFNα treatment, and CD16 monocytes and supernatant IP-10 were quantified. RESULTS: HIV+MJ+ donors possessed a lower level of circulating CD16 monocytes and plasma IP-10, compared with HIV+MJ- donors. Further, monocytes from HIV+MJ+ donors were unable to induce CD16 expression when treated with in-vitro IFNα, whereas HIV-MJ- and HIV+MJ- donors displayed pronounced CD16 induction, suggesting anti-inflammatory effects by cannabis. Lastly, in-vitro THC treatment impaired CD16 monocyte transition to CD16 and monocyte-derived IP-10. CONCLUSION: Components of cannabis, including THC, may decelerate peripheral monocyte processes that are implicated in HIV-associated neuroinflammation.
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Cannabis/efeitos adversos , Quimiocina CXCL10/sangue , Infecções por HIV/complicações , Infecções por HIV/patologia , Monócitos/imunologia , Receptores de IgG/análise , Transtornos Relacionados ao Uso de Substâncias/complicações , Adulto , Idoso , Dronabinol/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI/análise , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Monócitos/química , Monócitos/efeitos dos fármacosRESUMO
Purpose of review Lysosomal storage disorders (LSDs) are a class of genetic disorders that are a testing ground for the invention of novel therapeutics including enzyme replacement therapy (ERT), substrate reduction therapy (SRT), gene therapy, and hematopoietic stem cell transplant (HSCT). This review summarizes recently approved drugs, then examines the successful clinical trials in gene therapy and HSCT. Recent findings The FDA has recently approved a second SRT by reversing an earlier FDA decision, suggesting a favorable regulatory landscape going forward. Adeno-associated virus therapies, adenovirus therapies, and HSCT have overcome limitations of earlier clinical and preclinical trials, suggesting that gene therapy may be a reality for LSDs in the near future. At the same time, the first EU-approved gene therapy drug, Glybera, has been discontinued, and other ex vivo-based therapies although approved for clinical use have failed to be widely adapted and are no longer economically viable. Summary There are now 11 ERTs and two SRTs approved for LSDs in the USA. Gene therapy approaches and HSCT have also demonstrated promising clinical trial results suggesting that these therapies are on the frontier. Challenges that remain include navigating immune responses, developing drugs capable of crossing the blood-brain barrier (BBB), developing therapies that can reverse end-organ damage, and achieving these goals in a safe, ethical, and financially sustainable manner. The amount of active development and a track record of iterative progress suggest that treatments for LSDs will continue to be a field of innovation, problem solving, and success.
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Specific variants of endoplasmic reticulum-associated aminopeptidase 1 (ERAP1) identified by genome-wide association study modify the risk for developing ankylosing spondylitis. We previously confirmed that disease-associated ERAP1 variants have altered enzymatic abilities that can impact upon the production of pro-inflammatory cytokines from cells expressing the same ERAP1 variants. To determine if these ERAP1 variants also impacted immune responses in vivo, we generated two strains of transgenic mice expressing human ERAP1 genes containing non-synonymous single-nucleotide polymorphisms associated with an increased (ERAP1-High) or decreased (ERAP1-Low) risk for developing autoimmune disease. After vaccination with foreign antigens, ERAP1-High mice generated unique populations of antigen-specific T-cell clones. The expression of ERAP1-High also reduced MHC-I expression on the surface of multiple cell types, demonstrating a global impact on the MHC-I peptidome. ERAP1 variants also affected the innate immune system, because NK cells from murine ERAP1 (mERAP1) knockout mice and ERAP1-High/mERAP1-/- mice had decreased surface expression of the activating receptor NKG2D on their NK and T cells, and NK cells derived from mERAP1-/- mice or ERAP1-Low mice demonstrated more active NK cell killing than NK cells derived from wild-type or ERAP1-High mice. Finally, these studies were conducted in female mice, as all male ERAP1-High mice died in utero or shortly after birth, making ERAP1-High one of the only dominant lethal autosomal genes known in mammals. Together, these results present the first direct evidence that human disease-associated ERAP1 variants can greatly alter survival, as well as antigen presentation, T-cell repertoire and NK cell responses in vivo.
Assuntos
Aminopeptidases/genética , Citotoxicidade Imunológica/genética , Células Matadoras Naturais/fisiologia , Antígenos de Histocompatibilidade Menor/genética , Espondilite Anquilosante/genética , Linfócitos T/fisiologia , Imunidade Adaptativa/genética , Animais , Apresentação de Antígeno , Células Clonais , Feminino , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunidade Inata/genética , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos T/genética , Risco , Transgenes/genéticaRESUMO
Recombinant adenovirus serotype 5 (Ad5) vectors are promising vaccine candidates due to their intrinsic immunogenicity and potent transgene expression; however, widespread preexisting Ad5 immunity has been considered a developmental impediment to the use of traditional, or conventional, E1 and E3 gene-deleted Ad5 (Ad5[E1-]) vaccines. Even in the presence of anti-Ad5 immunity, recent murine and human studies have confirmed E2b gene-deleted Ad5 (Ad5[E1-,E2b-]) vaccines to be highly efficacious inducers of transgene-specific memory responses and significantly less toxic options than Ad5[E1-] vaccines. While these findings have been substantially confirmed, the molecular mechanisms underlying the different reactions to these vaccine platforms are unknown. Using cultures of human peripheral blood mononuclear cells (hPBMCs) derived from multiple human donors, we found that Ad5[E1-,E2b-] vaccines trigger higher levels of hPBMC proinflammatory cytokine secretion than Ad5[E1-] vaccines. Interestingly, these responses were generated regardless of the donors' preexisting anti-Ad5 humoral and cell-mediated immune response status. In vitro hPBMC infection with the Ad5[E1-,E2b-] vaccine also provoked greater Th1-dominant gene responses yet smaller amounts of Ad-derived gene expression than Ad5[E1-] vaccines. These results suggest that Ad5[E1-,E2b-] vaccines, in contrast to Ad5[E1-] vaccines, do not promote activities that suppress innate immune signaling, thereby allowing for improved vaccine efficacy and a superior safety profile independently of previous Ad5 immunity.
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Vacinas contra Adenovirus/imunologia , Adenovírus Humanos/imunologia , Portadores de Fármacos , Expressão Gênica , Vetores Genéticos , Vacinas contra Adenovirus/genética , Adenovírus Humanos/genética , Células Cultivadas , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/imunologia , Sorogrupo , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
The CD2-like receptor activating cytotoxic cell (CRACC) receptor is a member of the SLAM family of receptors that are found on several types of immune cells. We previously demonstrated that increasing the abundance of the adaptor protein EAT-2 during vaccination enhanced innate and adaptive immune responses to vaccine antigens. Engagement of the CRACC receptor in the presence of the EAT-2 adaptor generally results in immune cell activation, while activating CRACC signaling in cells that lack EAT-2 adaptor inhibits their effector and regulatory functions. As EAT-2 is the only SAP adaptor that interacts with the CRACC receptor, we hypothesized that technologies that specifically modulate CRACC signaling during vaccination may also improve antigen specific adaptive immune responses. To test this hypothesis, we constructed a CRACC-targeting Fc fusion protein and included it in vaccination attempts. Indeed, mice co-vaccinated with the CRACC-Fc fusion protein and an adenovirus vaccine expressing the HIV-Gag protein had improved Gag-specific T cell responses, as compared to control mice. These responses are characterized by increased numbers of Gag-specific tetramer+ CD8+ T cells and increases in production of IFNγ, TNFα, and IL2, by Gag-specific CD8+ T cells. Moreover, our results revealed that use of the CRACC-Fc fusion protein enhances vaccine-elicited innate immune responses, as characterized by increased dendritic cells (DCs) maturation and IFNγ production from NK cells. This study highlights the importance of CRACC signaling during the induction of an immune response generally, and during vaccinations specifically, and also lends insight into the mechanisms underlying our prior results noting EAT-2-dependent improvements in vaccine efficacy.