Structure-based selection of human metabolite binding P4 pocket of DRB1*15:01 and DRB1*15:03, with implications for multiple sclerosis.

Binding of small molecules in the human leukocyte antigen (HLA) peptide-binding groove may result in conformational changes of bound peptide and an altered immune response, but previous studies have not considered a potential role for endogenous metabolites. We performed virtual screening of the complete Human Metabolite Database (HMDB) for docking to the multiple sclerosis (MS) susceptible DRB1*15:01 allele and compared the results to the closely related yet non-susceptible DRB1*15:03 allele; and assessed the potential impact on binding of human myelin basic peptide (MBP). We observed higher energy scores for metabolite binding to DRB1*15:01 than DRB1*15:03. Structural comparison of docked metabolites with DRB1*15:01 and DRB1*15:03 complexed with MBP revealed that PhenylalanineMBP92 allows binding of metabolites in the P4 pocket of DRB1*15:01 but ValineMBP89 abrogates metabolite binding in the P1 pocket. We observed differences in the energy scores for binding of metabolites in the P4 pockets of DRB1*15:01 vs. DRB1*15:03 suggesting stronger binding to DRB1*15:01. Our study confirmed that specific, disease-associated human metabolites bind effectively with the most polymorphic P4 pocket of DRB1*15:01, the primary MS susceptible allele in most populations. Our results suggest that endogenous human metabolites bound in specific pockets of HLA may be immunomodulatory and implicated in autoimmune disease

Expression of Regulatory Platelet MicroRNAs in Patients with Sickle Cell Disease

Background: Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile. Methods and Findings: We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD = 19, controls = 10) and a validation cohort (SCD = 7, controls = 7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1. Conclusions: We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets. © 2013 Jain et al

Deciphering the mechanisms underlying the role of interleukin-10 in cognitive function

Dissertação de mestrado em Ciências da SaúdeA função cognitiva refere-se aos processos mentais internos críticos para as atividades quotidianas, como a memória. A imunovigilância do cérebro é crucial para a cognição. A ausência de células T e níveis aumentados de citocinas pró-inflamatórias têm sido associados a comprometimentos cognitivos. No entanto, o papel das citocinas anti-inflamatórias, como a interleucina-10 (IL-10), tem sido pouco estudado. Aqui, o efeito da ausência de IL-10 na função cognitiva foi investigado em murganhos fêmeas BALB/c jovens-adultas IL-10 knockout (KO) e irmãs de tipo selvagem (WT). A ausência de IL-10 prejudicou a memória de referência espacial dependente do hipocampo no Barnes-maze test. Curiosamemente, neste teste, os murganhos IL-10 KO mostraram uma redução nas estratégias dependentes do hipocampo principalmente durante as fases de metestro e diestro do ciclo estríco. Não foram observados problemas locomotores nos murganhos IL-10 KO, no entanto, a deficiência de IL-10 comprometeu a exploração no open-field test. Embora a ausência de IL-10 tivesse aumentado os níveis basais de corticosterona e de expressão genética de marcadores pró-inflamatórios no cólon, estes parâmetros não se correlacionaram com o desempenho comportamental. Usando as variáveis comportamentais analisadas, o genótipo (WT vs IL-10 KO) foi classificado por support vector machine models com uma precisão de até 89,3%. Adicionalmente, a ausência de IL-10 diminui o número de neurónios e volume do hipocampo dorsal, mas não do ventral. Além disso, no hipocampo, a deficiênciade IL-10 modulou negativamente a dinâmica das espinhas dendríticas e diminuiu a arborização dendrítica dos neurónios granulares do giro dentado dorsal e ventral e piramidais do cornu ammonis-1 e -3, que são conhecidos por suportar a aprendizagem e memória. Ademais, análises por citometria de fluxo mostraram que a ausência de IL-10 influenciou o perfil leucocitário no sangue pelo aumento do número total de neutrófilos e da sua percentagem dentro dos leucócitos e diminuição da percentagem dentro dos leucócitos de eosinófilos, células natural-killer, células B e células T. Além disso, no sangue, a deficiência de IL-10 aumentou a percentagem de células T CD4+ efetoras de memória, que foram previamente associadas a uma pior função cognitiva em idosos saudáveis. A ausência de IL-10 também aumentou o número total de leucócitos nos nódulos linfáticos cervicais profundos, sugerindo um aumento do recrutamento de células para o sistema linfático do cérebro. Por fim, através de um tratamento antibiótico, um protocolo para a depleção do microbioma intestinal de murganhos IL-10 KO foi otimizado. Após 3 dias de tratamento, os antibióticos reduziram os níveis de expressão genética de16s nas fezessem proliferação fúngica, proporcionando uma etapa inicial para explorar o papel do microbioma intestinal na função cognitiva de murganhos IL-10 KO. No geral, estes resultados não só suportaram que a ausência de IL-10 impactou as habilidades cognitivas, mas também destacaram potenciais mecanismos subjacentes à ação dessa citocina anti-inflamatória, que podem ser contribuintes importantes para o desenvolvimento de novas terapias para comprometimentos cognitivas baseadas em IL-10.Cognitive function refers to internal mental processes critical for daily life activities, such as memory. Brain immune surveillance has proven to be crucial for cognitive function. T cell absence and increased levels of pro-inflammatory cytokines have been associated with impaired cognition. However, the role of anti-inflammatory cytokines, such as interleukin-10 (IL-10), has been poorly studied. Here, the effect of IL-10 absence in cognitive function was investigated in young-adult female BALB/c IL-10 knockout (KO) and wild-type (WT) littermate mice. IL-10 absence impaired the hippocampal-dependent spatial reference memory in the Barnes-maze test. Interestingly, in this test, IL-10 KO mice showed a reduction in hippocampal-dependent strategies mainly during the metestrus and diestrus phases of the estrous cycle. No locomotor disabilities were observed in IL-10 KO mice however IL-10 deficiency impaired exploration in the open-field test. Although IL-10 absence has led to higher basal levels of corticosterone and increased gene expression levels of pro-inflammatory markers in the colon, these parameters did not correlate with the behavioral performance. Using the behavioral variables analyzed, the genotype (WT vsIL-10 KO) was classified by support vector machine models with an accuracy of up to 89.3%. Moreover, IL-10 absence led to a decreased number of neurons and volumetric atrophy of the dorsal, but not of the ventral hippocampus. Additionally, in the hippocampus, IL-10 deficiency negatively modulated the dendritic spine dynamics and decreased the dendritic arborization of dorsal and ventral dentate gyrus granule neurons and cornu ammonis-1 and -3 pyramidal neurons, which are known to support learning and memory. Furthermore, flow cytometry analysis showed that IL-10 absence impacted the leukocyte profile in the blood by increasing the total number of neutrophils and its percentage within leukocytes and decreasing the percentage of eosinophils, natural-killer cells, B cells, and T cells within leukocytes. Also, in the blood, IL-10 deficiency increased the percentage of effector memory CD4+ T cells, which were previously associated with a worst cognitive function of healthy aged individuals. IL-10 absence also increased the total number of leukocytes in the deep cervical lymph nodes, suggesting an increased cell recruitment to the lymphatic system of the brain. Lastly, through an antibiotic treatment, a protocol for gut microbiome depletion of IL-10 KO mice was optimized. After 3 days of treatment, antibiotics reduced the gene expression levels of 16s in the feces without fungal overgrowth, providing an initial step to explore the role of the gut microbiome in the cognitive function of IL-10 KO mice. Overall, these results not only supported that IL-10 absence impacted cognitive abilities but also highlighted the potential mechanisms underlying the action of this anti-inflammatory cytokine, which may be important contributors to the development of new IL-10-based therapies for cognitive impairments.E, por fim, ao Programa Operacional Regional do Norte de Portugal – NORTE 2020 no âmbito dos projetos NORTE-01-0145-FEDER-000013 e NORTE-01-0145-FEDER-000023; à Plataforma de Microscopia Científica do ICVS, membro da infraestrutura nacional da Plataforma Portuguesa de Bioimagem – PPBI no contexto do projeto PPBI-POCI-01-0145-FEDER-022122, ambos ao abrigo do Acordo de Parceria – Portugal 2020, através do Fundo Europeu de Desenvolvimento Regional; e à Fundação para a Ciência e Tecnologia mediante fundos nacionais vinculados aos projetos UIDB/50026/2020 e UIDP/50026/2020 pelo suporte financeiro

Definitions, Criteria and Global Classification of Mast Cell Disorders with Special Reference to Mast Cell Activation Syndromes: A Consensus Proposal

Activation of tissue mast cells (MCs) and their abnormal growth and accumulation in various organs are typically found in primary MC disorders also referred to as mastocytosis. However, increasing numbers of patients are now being informed that their clinical findings are due to MC activation (MCA) that is neither associated with mastocytosis nor with a defined allergic or inflammatory reaction. In other patients with MCA, MCs appear to be clonal cells, but criteria for diagnosing mastocytosis are not met. A working conference was organized in 2010 with the aim to define criteria for diagnosing MCA and related disorders, and to propose a global unifying classification of all MC disorders and pathologic MC reactions. This classification includes three types of `MCA syndromes' (MCASs), namely primary MCAS, secondary MCAS and idiopathic MCAS. MCA is now defined by robust and generally applicable criteria, including (1) typical clinical symptoms, (2) a substantial transient increase in serum total tryptase level or an increase in other MC-derived mediators, such as histamine or prostaglandin D 2, or their urinary metabolites, and (3) a response of clinical symptoms to agents that attenuate the production or activities of MC mediators. These criteria should assist in the identification and diagnosis of patients with MCAS, and in avoiding misdiagnoses or overinterpretation of clinical symptoms in daily practice. Moreover, the MCAS concept should stimulate research in order to identify and exploit new molecular mechanisms and therapeutic targets. Copyright (C) 2011 S. Karger AG, Base