Resumo: Os objetivos deste trabalho foram a padronização de metodologia alternativa de genotipagem do SNP AY_731081:g.1900T>C do gene CD14 equino por PCR-RFLP, bem como a caracterização em equinos da raça Mangalarga deste e de outros dois polimorfismos, o AY_005808: c.1530A>G do TLR4 e o AX_463789: g.133T>C do Cε, a fim de promover o embasamento necessário para futuras pesquisas visando associação entre marcadores de DNA e características relacionadas à fisiologia do sistema imune na raça. Para tanto, foram utilizados 151 animais Mangalarga, de ambos os sexos e de idades variadas, representativos da população do estado de São Paulo. O método de PCR-RFLP mostrou-se adequado para a genotipagem do SNP AY_731081: g.1900T>C do gene CD14 equino. Entretanto, tal polimorfismo provavelmente não ocorre em equinos Mangalarga, impossibilitando estudos de associação com o marcador na raça. Os parâmetros genético-populacionais obtidos para os polimorfismos AY_005808:c.1530A>G do gene TLR4 e o AX_463789:g.133T>C do gene Cε demonstraram a possibilidade de realização de pesquisas visando a associação entre os marcadores e características relacionadas ao sistema imune na raça Mangalarga / Abstract: The objective of this work was to contribute to the molecular characterization of the equine Mangalarga, aiming at future studies on the association of DNA polymorphisms and traits associated with the immune system physiology of this equine breed. An alternative PCR-RFLP genotyping method was developed for the SNP AY_731081:g.1900T>C, in the gene CD14. Furthermore, this SNP plus the AY_005808: c.1530A>G, in the gene TLR4, and the AX_463789: g.133T>C, in the gene Cε, were used to analyze 151 Mangalarga individuals. The analyzed horses are representative of the São Paulo State population and consisted of male and female animals of several ages. The PCR-RLP method has been demonstrated to be suitable for equine genotyping using the SNP AY_731081: g.1900T>C of the gene CD14. However, this polymorphism is apparently absent in the Mangalarga breed. The population genetic data obtained for the polymorphisms AY_005808:c.1530A>G, of the gene TLR4, and the AX_463789:g.133T>C, in the gene Cε, indicated the feasibility of these SNPs for further studies aiming at the association of molecular markers with traits related to the immune system of the Mangalarga horse breed / Orientador: Marcílio Dias Silveira da Mota / Coorientador: Rogério Abdallah Curi / Banca: Humberto Tonhati / Banca: Lenira El Faro Zadra / Mestre

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Many research articles describes fish oil as a beneficial for health. Mainly for prevention of cardiovascular diseases or that fish oil reduces cholesterol in blood stream. Aim of this study is to investigate effects of fish oil specifically of his parts EPA+DHA, on a modulation of immune system and phenotypic differentiation. For the experiment were chosen monocytes and macrophages obtained from experimental group of animals which was fed a diet consist of the addition of fish oil in an amount 45mg/kg of live weight. Then inflammatory state was induced by LPS. Cells surface receptors were analyze through flow cytometry specifically CD14, CD163, SLA-DR and theirs combinations after they were statistically processed. Results shows proinflammatory effects of fish oil however they were statistically not significant.

OBJECTIVE. To determine if the levels of circulating myeloid-derived suppressor cells increase with progression of prostate cancer (PCa); to determine if such cells could contribute to the relative inefficiency of PCa immunotherapy. MATERIALS AND METHODS. We analyzed peripheral blood mononuclear cells isolated from untreated PCa patients (uPCa; N=18; mean age±SD: 72.1± 6.9 years), tPCa (N = 22; 72.8 ± 9.8 years) and age matched controls (AMC; N = 12; 68.8 ± 7.5 years). We quantified surface marker phenotype, differentiation potential, effects on T cell proliferation and intracellular cytokines. RESULTS. We observed an unexpectedly high percentage of a type of myeloid-derived suppressor cells, CD14+HLA-DR low/- monocytes, in tPCa (30.7±15.0% of CD14+ cells) relative to AMC (4.1+6.5%, P<0.0001) and uPCa (10.6 ± 14.3%, P = 0.0001). The levels of CD14+ HLA-DR low/- cells were significantly correlated with circulating PSA levels and treatment with LHRH-agonist leuprolide in combination with either an antiandrogen or dexamethasone. Monocytes from tPCa inhibited autologous T cell proliferation statistically significantly more effectively than AMC monocytes and were defective in their ability to differentiate into phenotypically mature dendritic cells. Isolated CD14+HLA-DRlow/- cells expressed higher levels of intracellular interleukin-10 and suppressed T cell proliferation more effectively than isolated CD14+HLA-DR+ cells. CONCLUSIONS. This is the first report of CD14+ cells exhibiting reduced expression of HLADRmolecules in PCa patients. These cells suppress immune cell function in vitro and, plausibly, in vivo, a finding that must be factored into the design of immunotherapy protocols for PCa patients.

No description available.

Human milk contains ~ 25 µg/mL of soluble cluster of differentiation 14 (sCD14) protein, a pattern recognition receptor (PRR) that triggers the innate immune system to respond to bacterial lipopolysaccharide (LPS). To date, the role of CD14 in the digestive tract of breast fed infants has not been well characterized and is the subject of this thesis. To investigate the biodistribution of proteins such as CD14 in vivo, a novel method for 14C radiolabeling of proteins to high specific radioactivity was developed using in vacuo methylation. Bovine serum albumin (BSA) and casein were used as test proteins to determine the following: 1) The efficacy of the in vacuo radiolabeling procedure; 2) The extent of incorporation of the 14C-label into the organs of oro-gastric gavaged 10 day old Sprague Dawley rats. [14C]BSA, [14C]casein and [14C]CD14 were prepared with specific radioactivities of 10 400, 10 800 and 163 000 dpm/µg, respectively. After feeding 6.25 µg of 14C-labeled proteins, quantifiable levels of 14C were found in the stomach, jejunum, duodenum, ileum, large intestine, intestinal luminal flushes, blood, liver, spleen and kidneys of rats. The accumulation of radiolabel in the organs of [14C]CD14 fed rats was temporally and spatially distinct from [14C]BSA and [14C]casein. Most notably, the label persisted in the stomach 480 min post-gavage. To design a neonate animal model for biodistribution, the segmental and total gastrointestinal transit times (GItt) were measured in two litters of 10 and 15 day old Sprague Dawley rat pups using barium sulfate. Ten day old rat pups that remained with and without the dam had a total gastrointestinal transit time of 13.8 ± 0.9 hr and 9.3 ± 0.7 hr, respectively. This decrease (p<0.05) in total gastrointestinal transit time in the absence of the dam was age dependent, as it was not observed (p>0.05) in the 15 day old rat pup litter. The immunological impact of an exogenous sCD14 source was examined in human peripheral blood mononuclear cells (PBMC). Pre-treatment of CD14+ monocytes with sCD14 had a protective effect, one of reducing the production of proinflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) when challenged with LPS. 14C was absorbed by neonate rats upon ingestion of [14C]CD14 and exposure to relatively high concentrations of rCD14 led to a reduction in inflammation. This may be beneficial to initial gut colonization in breast-fed newborns. / Alexander Graham Bell NSERC CGS M scholarship. Japan Society for the Promotion of Sciences, Summer in Japan Fellowship. Funded by the Canadian Institutes of Health Research, Institute of Nutrition Metabolism and Diabetes Grant #82816 “Fate and function of breast milk and recombinant human CD14 at mammary and newborn gastrointestinal mucosal epithelia”.

Background: This bioinformatics study aimed to reveal potential cross-talk genes, related pathways, and transcription factors between periimplantitis and rheumatoid arthritis (RA). Methods: The datasets GSE33774 (seven periimplantitis and eight control samples) and GSE106090 (six periimplantitis and six control samples) were included from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO). A differential expression analysis (p < 0.05 and |logFC (fold change)| ≥ 1) and a functional enrichment analysis (p < 0.05) were performed. Based on this, a protein–protein interaction (PPI) network was constructed by Cytoscape. RA-related genes were extracted from DisGeNET database, and an overlap between periimplantitis-related genes and these RA-related genes was examined to identify potential cross-talk genes. Gene expression was merged between two datasets, and feature selection was performed by Recursive Feature Elimination (RFE) algorithm. For the feature selection cross-talk genes, support vector machine (SVM) models were constructed. The expression of these feature genes was determined from GSE93272 for RA. Finally, a network including cross-talk genes, related pathways, and transcription factors was constructed. Results: Periimplantitis datasets included 138 common differentially expressed genes (DEGs) including 101 up- and 37 downregulated DEGs. The PPI interwork of periimplantitis comprised 1,818 nodes and 2,517 edges. The RFE method selected six features, i.e., MERTK, CD14, MAPT, CCR1, C3AR1, and FCGR2B, which had the highest prediction. Out of these feature genes, CD14 and FCGR2B were most highly expressed in periimplantitis and RA. The final activated pathway–gene network contained 181 nodes and 360 edges. Nuclear factor (NF) kappa B signaling pathway and osteoclast differentiation were identified as potentially relevant pathways. Conclusions: This current study revealed FCGR2B and CD14 as the most relevant potential cross-talk genes between RA and periimplantitis, which suggests a similarity between RA and periimplantitis and can serve as a theoretical basis for future research.

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Les progresseurs lents du VIH-1 sont de rares sujets asymptomatiques pendant plusieurs années sans thérapie antirétrovirale. Parmi ces sujets à progression lente vers le SIDA, il est possible qu’un sous-groupe perde le contrôle de leur infection après plusieurs années de contrôle. Notre laboratoire a analysé l’expression différentielle de différentes protéines et voies moléculaires associées à la perte de contrôle de l’infection: l’interleukine-32 (IL-32) est une cytokine pro-inflammatoire dont le niveau des isoformes alpha et delta a significativement diminué chez les progresseurs lents lors de la perte de contrôle. Par ailleurs, des études antérieures ont attribué, de façon intrigante, à l’IL-32 aussi bien des propriétés anti-VIH-1 que des propriétés immunosuppressives induisant un environnement propice à la réplication du VIH-1. Ce projet de maitrise s’est penché sur l’implication de l’IL-32 dans la progression de l’infection à VIH-1 avec un accent particulier sur les progresseurs lents. Nous avons principalement mesuré les niveaux d’IL-32 des sujets séropositifs comparativement aux sujets VIH négatif et estimé les fonctions de cette cytokine à travers des études longitudinales et de corrélation. Nous avons observé que l’IL-32 total demeure plus élevé chez les séropositifs comparativement aux sujets VIH négatif. Également, l’infection par le VIH-1 entraine une augmentation du niveau d’IL-32 total. De plus, après une année de thérapie antirétrovirale, les taux plasmatiques d’IL-32 total demeurent significativement plus élevés que ceux des sujets VIH négatif. Comme attendu, le taux d’IL-32 total augmente lors de la perte de contrôle de l’infection chez les progresseurs lents. Une forte concentration plasmatique d’IL-32 total coïncide avec: 1) une augmentation du taux plasmatique de sCD14 et de la cytokine pro-inflammatoire IL-6, 2) une baisse du compte cellulaire CD4 et une augmentation de la charge virale. Un taux plasmatique élevé de CCL5 pourrait prédire une faible concentration d’IL-32 total. L’isoforme alpha de l’IL-32 est plus élevée dans le plasma des sujets VIH négatif tandis que l’IL-32 gamma semble induire un environnement pro-inflammatoire et immunosuppressif. Il ressort à l’issue de ces observations que l’augmentation de l’IL-32 total est associée à la progression de l’infection à VIH-1 et pourrait constituer un biomarqueur permettant d’apprécier le pronostic de cette infection. / HIV-1 slow progressors constitute a rare population of subjects who remain asymptomatic for many years without antiretroviral therapy. Among this population, some individuals will lose control of their infection after several years of immunological control. Our laboratory has analyzed the differential expression profile of various proteins and molecular pathways associated with the loss of control of HIV infection. The pro-inflammatory cytokine interleukin-32 alpha and delta isoforms significantly decreased in slow progressors as they were losing control of their infection. Furthermore, previous studies have attributed to IL-32 both antiviral property against HIV-1 and immunosuppressive properties that can induce an environment conducive to HIV-1 replication. This project addresses the role of IL-32 in HIV-1 disease progression with a particular emphasis on slow progressors. We compared the levels of IL-32 in HIV-1 positive versus HIV-1 negative subjects and evaluated the role of this cytokine using longitudinal studies. We observed that levels of IL-32 remains higher in HIV-positive compared to HIV-negative subjects. Also, HIV-1 infection leads to increased level of IL-32. In addition, after one year of antiretroviral therapy, IL-32 plasma levels remain significantly higher than those of HIV-negative subjects. As expected, the levels of IL-32 increased as slow progressors lost control of their infection. A high plasma concentration of IL-32 predicts: 1) an increase in plasma levels of sCD14 as well as pro-inflammatory cytokine IL-6, 2) a decrease in CD4 cell count and an increase in viral load. High plasma CCL5 predicted a low concentration of IL-32. The alpha isoform of IL-32 is elevated in the plasma of HIV negative subjects while IL-32 gamma appears to induce a pro-inflammatory and immunosuppressive environment. We conclude that increased IL-32 levels are associated with progression of HIV-1 disease and could be used as a biomarker for assessing HIV-1 prognosis.

A doença de Behçet tem sua fisiopatologia caracterizada por hiperatividade neutrofílica, particularmente em relação à quimiotaxia, e períodos de atividade da doença podem ser desencadeados por exposição a estreptococos. Uma vez que células do sistema imune inato são ativadas pelo ácido lipoteicoico (LTA) de bactérias gram-positivas via receptor toll-like (TLR) 2 e CD14, cujas expressões são reguladas pelos fatores estimulantes de colônias de granulócitos (G-CSF) e granulócitos-macrófagos (GM-CSF), o objetivo principal deste estudo foi determinar se há hiperexpressão de TLR2 em neutrófilos de DB ativa e se a quimiotaxia de polimorfonucleares (PMN) neutrófilos na DB poderia ser hiperestimulada pelo LTA. Além do TLR2, foram medidas as expressões de TLR4, CD14, CD114 (receptor de G-CSF) e CD116 (receptor de GM-CSF) nos neutrófilos e nos monócitos de pacientes com doença de Behçet (DB), as concentrações séricas de CD14 solúvel (CD14s) e as respostas quimiotáxicas dos PMNs de DB sob diferentes estímulos. A expressão dos receptores foi medida pela citometria de fluxo, as concentrações séricas por ELISA e as respostas quimiotáxicas foram avaliadas em câmara de Boyden. Nos PMNs, os receptores foram igualmente expressos nos dois grupos e, estimulados com LTA, suas respostas quimiotáxicas também foram similares. Somente à incubação com plasma os PMNs de DB desenvolveram hiperquimiotaxia em relação aos PMNs controles. A expressão do TLR2 foi maior em monócitos de DB em relação aos controles, e a concentração de CD14s sérica, de origem monocitária, foi maior nos pacientes com DB ativa. Em conjunto, os resultados demonstram que PMNs de DB, isoladamente, não reagem exacerbadamente ao LTA, e suas respostas migratórias são estritamente dependentes de fatores estimulantes solúveis. / Expressions of toll-like receptor (TLR) 2, TLR4, CD14, CD114 and CD116 were assessed on polymorphonuclear (PMN) neutrophils and monocytes of patients with Behçets disease (BD). PMN chemotactic responses under different stimulations were also measured. The objective was to determine if BD PMN chemotaxis may be overstimulated by lipoteichoic acid (LTA) from gram-positive bacteria. Receptor expressions were measured by flow cytometry and PMN chemotaxis was assessed in a Boyden chamber. Only TLR2 expression was higher on monocytes of the BD group than in control group. On PMNs, however, TLR2 expression was similar in both groups and, when stimulated with LTA, BD PMN cells showed chemotactic responses similar to the controls. These cells only exhibited increased chemotaxis when incubated with plasma. In conclusion, isolated BD PMN did not overreact to LTA, and its hyperchemotaxis is strictly dependent on soluble stimulating factors