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Leukocyte Immunoglobulin (Ig)-like Receptors (LILR) LILRB1 and LILRB2 play a pivotal role in maintaining self-tolerance and modulating the immune response through interaction with classical and non-classical Human Leukocyte Antigen (HLA) molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LIRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next-generation sequencing in a population sample comprising 528 healthy control individuals from São Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes with at least 1% of frequency. For LILRB2, we identified 41 SNVs arranged into 11 haplotypes with frequencies above 1%. We found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have shown that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Taken together, our findings demonstrate that LILRB1 and LILRB2 are highly polymorphic and provide strong evidence supporting the directional selection regime hypothesis.

Background: Combination therapy with lenvatinib plus programmed death-1 (PD-1) immune checkpoint blockades (ICBs) is under investigation in many solid tumors, including thyroid cancer. Lenvatinib is known to reduce angiogenesis and may overturn the immunosuppressive effects of vascular endothelial growth factor in the tumor microenvironment. Previous studies investigating the effects of VEGF receptor inhibition on the immune response were performed in rapidly growing tumor models where immune equilibrium is not established before treatment. We hypothesize that physiologically relevant preclinical models are necessary to define mechanisms of resistance to immune-targeted combination therapies. Methods: We utilized the TPO-CreER/Braf(V600E)(/wt)/Trp53(Δex2–10/Δex2–10) inducible transgenic model of advanced thyroid cancer to investigate lenvatinib treatment in the context of an anti-PD-1 ICB. Following tumor establishment, 3.5 months postinduction, mice were treated with high- (10 mg/kg) or low-dose (2 mg/kg) lenvatinib, anti-PD-1, or combination of lenvatinib with anti-PD-1. Tumor volume and lung metastases were assessed in each group. Immune infiltrate was characterized by flow cytometry and immunohistochemistry, and TCRß sequencing was performed to further investigate the T cell response. Results: Both low- and high-dose lenvatinib reduced tumor volume, while anti-PD-1 had no effect, alone or in combination. Although both low- and high-dose lenvatinib reduced vascular density, low-dose lenvatinib was superior in controlling tumor size. Lung metastases and survival were not improved with therapy despite the effects of lenvatinib on primary tumor size. Low-dose lenvatinib treatment led to a subtle reduction in the dominant Ly6G(+)CD11b(+) myeloid cell population and was associated with increased CD4(+) T cell infiltrate and enrichment in 4–1BB(+) and granzyme B(+) CD4(+) T cells and FoxP3(+) regulatory T cells. Polyclonal T cell expansion was evident in the majority of mice, suggesting that a tumor-specific T cell response was generated. Conclusions: The effects of lenvatinib on the immune response were most pronounced in mice treated with low-dose lenvatinib, suggesting that dose should be considered in clinical application. While the immune-modulating potential of lenvatinib is encouraging, alterations in the immune milieu and T cell activation status were insufficient to sustain durable tumor regression, even with added anti-PD-1. Additional studies are necessary to develop more effective combination approaches in low-mutation burden tumors, such as thyroid cancer.

Background Interleukin (IL)-17A has a dual role in tumor immunity, promotes anti-tumor responses and facilitates angiogenesis by interacting with IL-17 receptor A (IL-17RA). Although IL-17A has been associated with the pathogenesis of papillary thyroid carcinoma (PTC), the nucleotide variability at the IL17A and IL17RA genes is still poorly characterized. Aim To assess the contribution of the IL17A (-197 G >A, rs2275913) and IL17RA (-947 A >G, rs4819554) single nucleotide polymorphisms (SNP) on the development and progression of PTC and on IL-17 plasma levels. Methods We studied 188 PTC patients and 170 healthy controls. SNPs were identified using PCR-amplified DNA and restriction fragment length polymorphism (RFLP) techniques. Plasma levels of IL-17A was evaluated in 83 PTC patients using ELISA. Statistical analyses were performed to evaluate the associations between SNPs and clinicohistopathological features of PTC and IL-17A levels. Results No significant difference was observed regarding the allele and genotype distributions of both SNPs between PTC patients and controls. The IL17A GA was associated with poor biochemical and structural incomplete response to therapy, whereas no influence over the IL-17A expression was observed. The IL17RA AG was significantly associated with small-sized tumors, initial tumor stage at diagnosis and better response to therapy. Conclusions The IL17A SNP may predict an aggressive manifestation of PTC, whereas the IL17RA SNP was associated with a more favorable clinical outcome.

Non-aggressive basal cell carcinoma (BCC) growth is slow and might be mediated by the immune system. This study analysed the human leukocyte antigen (HLA)-G expression and cytokine profile in non-aggressive BCC subtypes from distinct locations. HLA-G was evaluated via immunohistochemistry and cytokine expression was analysed by a quantitative real-time polymerase chain reaction in 26 primary BCC samples, including nodular BCC (nBCC, n = 16) and superficial BCC (n = 10) from cephalic (ceBCC, n = 12) and non-cephalic (n = 14) locations, and by bioinformatics analysis of public GEO databases. Inflammatory infiltrate was concentrated around the tumour nests. HLA-G-positive inflammatory cells (53.85%) were more abundant than HLA-G-positive tumour cells (21.54%, p

Made available in DSpace on 2021-06-25T10:53:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) KIR2DL4 is an important immune modulator expressed in natural killer cells; HLA-G is its main ligand. We have characterized the KIR2DL4 genetic diversity by considering the promoter, all exons, and all introns in a highly admixed Brazilian population sample and by using massively parallel sequencing. We introduce a molecular method to amplify and to sequence the complete KIR2DL4 gene. To avoid the mapping bias and genotype errors commonly observed in gene families, we have developed and validated a bioinformatic pipeline designed to minimize these errors and applied it to survey the variability of 220 individuals from the State of São Paulo, southeastern Brazil. We have also compared the KIR2DL4 genetic diversity in the Brazilian cohort with the diversity previously reported by the 1000Genomes consortium. KIR2DL4 presents high linkage disequilibrium throughout the gene, with coding sequences associated with specific promoters. There are few but divergent promoter haplotypes. We have also detected many new KIR2DL4 sequences, all bearing nucleotide exchanges in introns and encoding previously described proteins. Exons 3 and 4, which encode the external domains, are the most variable. The ancestry background influences the KIR2DL4 allele frequencies and must be considered for association studies regarding KIR2DL4. Molecular Genetics and Bioinformatics Laboratory - Experimental Research Unit School of Medicine São Paulo State University (UNESP) Genetics Program Institute of Biosciences of Botucatu São Paulo State University (UNESP) Pathology Program School of Medicine São Paulo State University (UNESP) School of Agronomical Sciences São Paulo State University (UNESP) Department of Medicine Ribeirão Preto Medical School University of São Paulo (USP) Departamento de Química Faculdade de Filosofia Ciências E Letras de Ribeirão Preto Universidade de São Paulo Molecular Genetics and Bioinformatics Laboratory - Experimental Research Unit School of Medicine São Paulo State University (UNESP) Genetics Program Institute of Biosciences of Botucatu São Paulo State University (UNESP) Pathology Program School of Medicine São Paulo State University (UNESP) School of Agronomical Sciences São Paulo State University (UNESP) FAPESP: 2017/05042-4 FAPESP: 2017/19223-0

The present study assessed the effect of systemic lupus erythematosus (SLE) activity, a chronic and inflammatory autoimmune disease, on the sinusoidal uptake transporter OATP1B1 using atorvastatin (ATV) as a probe drug. Fifteen healthy subjects, 13 patients with controlled SLE (SLEDAI 0–4), and 12 patients with uncontrolled SLE (SLEDAI from 6 to 15), all women, were investigated. Apparent total clearance of midazolam (MDZ), a marker of CYP3A4 activity, did not vary among the three investigated groups. The controlled and uncontrolled SLE groups showed higher plasma concentrations of MCP‐1 and TNF‐α, while the uncontrolled SLE group also showed higher plasma concentrations of IL‐10. The uncontrolled SLE group showed higher area under the curve (AUC) for ATV (60.47 (43.76–83.56) vs. 30.56 (22.69–41.15) ng⋅hour/mL) and its inactive metabolite ATV‐lactone (98.74 (74.31–131.20) vs. 49.21 (34.89–69.42) ng⋅hour/mL), and lower apparent total clearance (330.7 (239.30–457.00) vs. 654.5 (486.00–881.4) L/hour) and apparent volume of distribution (2,609 (1,607–4,234) vs. 7,159 (4,904–10,450) L), when compared to the healthy subjects group (geometric mean and 95% confidence interval). The pharmacokinetics of ATV and its metabolites did not differ between the healthy subject group and the patients with controlled SLE group. In conclusion, uncontrolled SLE increased the systemic exposure to both ATV and ATV‐lactone, inferring inhibition of OATP1B1 activity, once in vivo CYP3A4 activity assessed by oral clearance of MDZ was unaltered. The inflammatory state, not the disease itself, was responsible for the changes described in the uncontrolled SLE group as a consequence of inhibition of OATP1B1, because systemic exposure to ATV and its metabolites were not altered in patients with controlled SLE.

Aim: Human Leukocyte Antigen-G (HLA-G) is a non-classical class I molecule that is involved in maternal–fetal immunotolerance. In cancer, this molecule contributes to the tumor escape. The aim of t...

The in silico and in vitro binding of a peptide covering a part of the autoimmune regulator (AIRE) SAND domain with the SIRT1 protein provides a powerful model system for studying the mechanism of the dominant SAND G228W mutation, which is the causative of APS-1 autoimmune syndrome. It is known that the mutant G228W AIRE protein accumulates more within the nucleus of cells than its wild-type counterpart does. This accumulation is not physiological and is associated with loss of AIRE function. However, the precise molecular mechanism that leads to AIRE accumulation is not yet known. AIRE works as a tetramer and interacts with partner proteins to form the "AIRE complex" that pushes RNA Pol II stalling in the chromatin of medullary thymic epithelial cells. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates Lys residues of the SAND domain. Once AIRE is deacetylated, the binding with SIRT1 is undone, allowing the complex to proceed downstream. Here, we integrate molecular modeling, docking, dynamics, and surface plasmon resonance approaches to compare the structure and energetics of binding/release between AIRE G228 (wild-type) or W228 (mutant) peptides to SIRT1. We find that the proximity of G228W mutation to a K aminoacid residue in the SAND domain promotes a longer-lasting AIRE-SIRT1 interaction. The lasting interaction might cause a delay in the AIRE SAND domain to be released from the SIRT1 catalytic site, which might cause accumulation of the defective AIRE mutant protein in the nuclei of cells. SignificanceThis report reveals the mechanism of the pathogenic and dominant G228W mutation in the AIRE SAND domain. The G228W mutation is found in APS-1 syndrome patients, and it is critical to understand the molecular basis of loss of self-representation, a challenging aspect for immunology. Through modeling, molecular dynamics, and protein binding kinetics, we found that the G228W mutation leads to a stronger physical interaction between the AIRE SAND domain and the SIRT1 protein when compared to the equivalent wild-type segment. The short-term consequence of this stronger interaction is that the release of the AIRE-SIRT1 binding is slower. This might explain the reason that cells carrying the G228W mutation accumulate AIRE protein in their nuclei. This finding reveals with precision the AIRE-SIRT1 binding and the molecular mechanism of the human AIRE G228W mutation.

Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown thatAIREknockout (KO) mice have a small and disorganized thymic medulla; however, whetherAireinfluences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed anin vitromodel system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. The effect ofAireon mTEC-mTEC adhesion was evaluated by comparingAIREwild type (AIREWT)versus Aire KO (AIRE-/-)mTECs. Considering the 3D spheroid model evaluated, we reported that the absence ofAIREdisorganizes the 3D structure of mTEC spheroids, promotes a differential regulation of mTEC classical surface markers, and modulates genes encoding adhesion and other molecules.

Background: Chronic Chagas disease (CChD), one of the infectious parasitic diseases with the greatest social and economic impact upon a large part of the American continent, has distinct clinical manifestations in humans (cardiac, digestive, or mixed clinical forms). The mechanisms underlying the development of the most common and ominous clinical form, the chronic Chagas cardiomyopathy (CCC) have not been completely elucidated, despite the fact that a high intensity of parasite persistence in the myocardium is deemed responsible for an untoward evolution of the disease. The present study aimed to assess the parasite load CCC and its relation to left ventricular ejection fraction (LVEF), a definite prognostic marker in patients with CCC.Methods: Patients with CCC were clinically evaluated using 12-lead-electrocardiogram, echocardiogram, chest X-ray. Peripheral blood sampling (5 ml of venous blood in guanidine/EDTA) was collected from each patient for subsequent DNA extraction and the quantification of the parasite load using real-time PCR.Results: One-hundred and eighty-one patients with CCC were evaluated. A total of 140 (77.3%) had preserved left ventricular ejection fraction (of ≥40%), and 41 individuals had LV dysfunction (LVEF of Conclusion: The blood parasite load is highly variable and seems to be directly related to the reduction of LVEF, an important prognostic factor in CCC patients.

The high-risk oncogenic human papillomavirus (HPV) has developed mechanisms for evasion of the immune system, favoring the persistence of the infection. The chronic inflammation further contributes to the progression of tissue injury to cervical cancer. The programmed cell death protein (PD-1) after contacting with its ligands (PD-L1 and PD-L2) exerts an inhibitory effect on the cellular immune response, maintaining the balance between activation, tolerance, and immune cell-dependent lesion. We evaluated 295 patients exhibiting or not HPV infection, stratified according to the location (injured and adjacent non-injured areas) and severity of the lesion (benign, pre-malignant lesions). Additionally, we investigated the role of the promoter region PDCD1 -606G>A polymorphism (rs36084323) on the studied variables. PD-1 and PDCD1 expression were evaluated by immunohistochemistry and qPCR, respectively, and the PDCD1 polymorphism was evaluated by nucleotide sequencing. Irrespective of the severity of the lesion, PD-1 levels were increased compared to adjacent uninjured areas. Additionally, in cervical intraepithelial neoplasia (CIN) I, the presence of HPV was associated with increased (P = 0.0649), whereas in CIN III was associated with decreased (P = 0.0148) PD-1 levels, compared to the uninjured area in absence of HPV infection. The PDCD1 -606A allele was rare in our population (8.7%) and was not associated with the risk for development of HPV infection, cytological and histological features, and aneuploidy. In contrast, irrespective of the severity of the lesion, patients exhibiting the mutant PDCD1 -606A allele at single or double doses exhibited increased protein and gene expression when compared to the PDCD1 -606GG wild type genotype. Besides, the presence of HPV was associated with the decrease in PDCD1 expression and PD-1 levels in carriers of the -606 A allele presenting severe lesions, suggesting that other mediators induced during the HPV infection progression may play an additional role. This study showed that increased PD-1 levels are influenced by the -606G>A nucleotide variation, particularly in low-grade lesions, in which the A allele favors increased PDCD1 expression, contributing to HPV immune system evasion, and in the high-grade lesion, by decreasing tissue PD-1 levels.

Background Little information is available regarding the expression of the immunomodulatory Human Leukocyte Antigen (HLA)-G and -E molecules in pancreatic disorders. Aim To analyze HLA-G and -E expression in specimens of alcoholic chronic pancreatitis (ACP), idiopathic chronic pancreatitis (ICP), type 1 (T1D) and type 2 diabetes (T2D) and in histologically normal pancreas (HNP). Methods HLA-G and -E expression (ACP = 30, ICP = 10, T1D = 10, T2D = 30 and HNP = 20) was evaluated by immunohistochemistry in three different areas (acini, islets and inflammatory infiltrate). Results Acini and islets from HNP specimens exhibited higher HLA-G and -E expression compared to corresponding areas from all other patient groups. In inflammatory infiltrate, HLA-G and -E expression was observed only among the pancreatic disorders. We observed higher HLA-G and -E expression in acini from T2D compared to ACP, as well as higher HLA-G expression compared to ICP. Conclusion The decreased expression of HLA-G and -E in islets and acini together with the expression of these molecules in the inflammatory infiltrating cells were shared features among chronic inflammatory and autoimmune pancreatic disorders evaluated in this study, possibly reflecting tissue damage.

The Autoimmune Regulator (Aire) protein coordinates the negative selection of developing thymocytes by inducing the expression of hundreds of tissue‐specific antigens within the thymic medulla, which is also a primary site of the expression of the immune checkpoint HLA‐G molecule. Considering the immunomodulatory properties of Aire and HLA‐G, and considering that the role of the constitutive thymus expression of HLA‐G has not been elucidated, we studied the effect of AIRE cDNA transfection on HLA‐G expression in 4D6 thymic cells and in the HLA‐G‐positive JEG‐3 choriocarcinoma cells. Aire promoted the transactivation of HLA‐G gene by increasing the overall transcription, inducing the transcription of at least G1 and G2/G4 isoforms, and incrementing the occurrence and distribution of intracellular HLA‐G protein solely in 4D6 thymic cells. Luciferase‐based assays and chromatin immunoprecipitation experiments performed in 4D6 cells revealed that Aire targeted at least two regions within the 5′‐untranslated regulatory region (5′‐URR) extending 1·4 kb from the first ATG initiation codon. The interaction occurs independently of three putative Aire‐binding sites. These results indicate that the Aire‐induced upregulation of HLA‐G in thymic cells is likely to act through the interaction of Aire with specific HLA‐G 5′‐URR DNA‐binding factors. Such a multimeric transcriptional complex might operate in the thymus during the process of promiscuous gene expression.

BACKGROUND Patients with hepatitis C virus (HCV) and hepatocellular carcinoma (HCC) may or not develop iron overload (IO), which is associated with worst prognosis, because can cause serious damage to organs. HFE gene controls the iron uptake from gut, particularly in patients with hereditary hemochromatosis (HH). AIM To identify associations between HFE coding region in patients exhibiting hereditary hemochromatosis and in diseases associated with acquired IO. METHODS We sequenced exons 2 to 5 and boundary introns of HFE gene, evaluating all polymorphic sites in patients presenting hereditary (hemochromatosis) or acquired iron overload HCV and HCC) and in healthy controls, using Sanger sequencing. We also determined the ensemble of extended haplotype in healthy control individuals, including several major histocompatibility complex loci, using sequence specific probes. Haplotype reconstruction was performed using the Arlequin and Phase softwares, and linkage disequilibrium (LD) between histocompatibility loci and HFE gene was performed using the Haploview software. RESULTS The HFE*003 allele was overrepresented (f = 71%) and HFE*001 allele was underrepresented (f = 14%) in HH patients compared to all groups. A strong linkage disequilibrium was observed among the H63D-G, IVS2(+4)-C and C282Y-G gene variants, particularly in HH; however, the mutation IVS2(+4)T>C was not directly associated with HH susceptibility. The HFE*001/HFE*002 genotype conferred susceptibility to HCC in HCV patients exhibiting IO (P = 0.02, OR = 14.14). Although HFE is telomeric to other histocompatibility genes, the H63D-G/IVS2(+4)-C (P ≤ 0.00001/P ≤ 0.0057) combination was in LD with HLA-B*44 allele group in healthy controls. No LD was observed between HFE alleles and other major histocompatibility loci. CONCLUSION A differential HFE association was observed for HH and for diseases associated with acquired IO (HCV, HCC). Since HFE is very distant from other histocompatibility loci, only weak associations were observed with these alleles.

HLA-A is the second most polymorphic locus of the human leucocyte antigen (HLA) complex encoding a key molecule for antigen presentation and NK cell modulation. Many studies have evaluated HLA-A variability in worldwide populations, focusing mainly on exons, but the regulatory segments have been poorly characterized. HLA-A variability is particularly high in the segment encoding the peptide-binding groove (exons 2 and 3), which is related to the antigen presentation function and the balancing selection in these segments. Here we evaluate the genetic diversity of the HLA-A gene considering a continuous segment encompassing the extended promoter (1.5 kb upstream of the first translated ATG), all exons and introns, and the entire 3' untranslated region, by using massively parallel sequencing. To achieve this goal, we used a freely available bioinformatics workflow that optimizes read mapping for HLA genes and defines complete sequences using either the phase among variable sites directly observed in sequencing data and probabilistic models. The HLA-A variability detected in a highly admixed population sample from Brazil shows that the HLA-A regulatory segments present few, but divergent sequences. The regulatory segments are in close association with the coding alleles. Both exons and introns are highly variable. Moreover, patterns of molecular diversity suggest that the promoter, in addition to the coding region, might be under the same selective pressure, but a different scenario arises when it comes to exon 4 and the 3'UTR segment.

Made available in DSpace on 2021-06-25T10:58:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação Oswaldo Cruz Introduction: Non-classical class I human leukocyte antigens (HLA) molecules are known to modulate the function of cytotoxic cells (NK and T CD8+) during viral infection by interacting with inhibitory/activating receptors. However, little is known about the HLA-E/-F genetic variability on arbovirus infections. Methods: We evaluated by massive parallel sequencing the full HLA-E/-F genetic diversity among patients infected during the arbovirus (ZIKV, DENV, and CHIKV) outbreak leading to a broad range of neurological complications in the Brazilian State of Pernambuco. In parallel, healthy blood donors from the same area were also studied. Plink and R software were used for genetic association study. To limit the false-positive results and enhance the reliability of the results, we adopted P-values

Hepatitis C is considered a major public health problem caused by the hepatitis C virus (HCV). Viral infections are known to induce production of IL1β through the signaling pathway of inflammasomes. Emerging evidences suggest that Inflammasome genes may influence the immune response against HCV as the host genetic background may contribute to the balance between acute and chronic inflammation. We investigated in 151 patients with chronic hepatitis C and 206 healthy blood donors’ individuals (HD). Polymorphisms in the IL1B and IL18 genes were genotyped by PCR-RFLP, while NLRP3, CARD8, CTSB and AIM2 by RT- PCR. Serum assay of IL-1β cytokine was performed by ELISA. 84 patients presented mild fibrosis (NLRP3-rs10754558 C/C genotype correlated with higher IL-1β levels compared to the G/G genotype. Similar pattern was observed in patients with hepatitis C, mean circulating IL-1β levels were 21,96 ± 4.5 and 10,62 ± 3.3pg/mL among the C/C and G/G genotypes, respectively. This pattern holds even after stratification of the patients into mild fibrosis and advanced fibrosis, demonstrating that the NLRP3-rs10754558 or another polymorphism in linkage disequilibrium with it possibly has an influence on the processing of pro-IL-1β. Notably, higher levels of IL-1β (Mann–Whitney test, pCARD8-rs2009373 and IL1B-rs16944 are less prone to hepatitis C development (padj = 0.039). Similarly, herozygote carriers for CTSB-rs1692816 and AIM2-rs1103577 (padj = 0.008) or for IL18-rs187238 and NLRP3-rs10754558 (padj = 0.005), have less chances to the development of hepatitis C. However, between subgroups of CARD8-rs2009373 and heterozygous for IL18-rs187238 (padj = 0.028), have mild form of fibrosis.

Severe neurological complications following arbovirus infections have been a major concern in seasonal outbreaks, as reported in the Northeast region of Brazil, where the same mosquito transmitted Zika (ZIKV), Dengue (DENV), and Chikungunya (CHIKV) viruses. In this study, we evaluated the levels of 36 soluble markers, including cytokines, chemokines, growth factors, and soluble HLA-G (Luminex and ELISA) in: i) serum and cerebrospinal fluid (CSF), during the acute phase and two years after the infection (recovery phase, only serum), ii) the relationship among all soluble molecules in serum and CSF, and iii) serum of infected patients without neurological complications, during the acute infection. Ten markers (sHLA-G, IL-10, IL-22, IL-8, MIP-1α, MIP-1β, MCP-1, HGF, VEGF, and IL-1RA) exhibited differential levels between the acute and recovery phases, with pronounced increases in MIP-1α (PPP= 0.0001), and VEGF (P

Since HLA-G is an immune checkpoint molecule and since Crohn’s disease (CD) and ulcerative colitis (UC) exhibit deregulated immune-mediated mechanisms, we aimed to evaluate intestinal HLA-G expression and soluble HLA-G (sHLA-G) levels in CD/UC patients stratified according to the CD phenotype/localization and UC extension. HLA-G tissue expression was assessed by immunohistochemistry in biopsies collected from 151 patients (90 CD, 61 UC) and in surgical resection specimens (28 CD, 12 UC). Surgical material from 24 healthy controls was also assessed. Plasma sHLA-G levels (97 CD, 81 UC, and 120 controls) were evaluated using ELISA. HLA-G expression was similarly observed in the intestinal epithelial cells of control and CD/UC specimens. However, in biopsies, the plasma cells/lymphocytes infiltrating the lamina propria in CD/UC presented (1) increased HLA-G expression compared to controls (P

Made available in DSpace on 2021-06-25T10:58:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Human pigmentation is a complex trait, probably involving more than 100 genes. Predicting phenotypes using SNPs present in those genes is important for forensic purpose. For this, the HIrisPlex tool was developed for eye and hair color prediction, with both models achieving high accuracy among Europeans. Its evaluation in admixed populations is important, since they present a higher frequency of intermediate phenotypes, and HIrisPlex has demonstrated limitations in such predictions; therefore, the performance of this tool may be impaired in such populations. Here, we evaluate the set of 24 markers from the HIrisPlex system in 328 individuals from Ribeirão Preto (SP) region, predicting eye and hair color and comparing the predictions with their real phenotypes. We used the HaloPlex Target Enrichment System and MiSeq Personal Sequencer platform for massively parallel sequencing. The prediction of eye and hair color was accomplished by the HIrisPlex online tool, using the default prediction settings. Ancestry was estimated using the SNPforID 34-plex to observe if and how an individual’s ancestry background would affect predictions in this admixed sample. Our sample presented major European ancestry (70.5%), followed by African (21.1%) and Native American/East Asian (8.4%). HIrisPlex presented an overall sensitivity of 0.691 for hair color prediction, with sensitivities ranging from 0.547 to 0.782. The lowest sensitivity was observed for individuals with black hair, who present a reduced European contribution (48.4%). For eye color prediction, the overall sensitivity was 0.741, with sensitivities higher than 0.85 for blue and brown eyes, although it failed in predicting intermediate eye color. Such struggle in predicting this phenotype category is in accordance with what has been seen in previous studies involving HIrisPlex. Individuals with brown eye color are more admixed, with European ancestry decreasing to 62.6%; notwithstanding that, sensitivity for brown eyes was almost 100%. Overall sensitivity increases to 0.791 when a 0.7 threshold is set, though 12.5% of the individuals become undefined. When combining eye and hair prediction, hit rates between 51.3 and 68.9% were achieved. Despite the difficulties with intermediate phenotypes, we have shown that HIrisPlex results can be very helpful when interpreted with caution. Departamento de Química Laboratório de Pesquisas Forenses e Genômicas Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo, Av. Bandeirantes, 3900 Departamento de Genética Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Divisão de Imunologia Clínica Departamento de Clínica Médica Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Departamento de Patologia Faculdade de Medicina de Botucatu Unesp - Universidade Estadual Paulista Departamento de Patologia Faculdade de Medicina de Botucatu Unesp - Universidade Estadual Paulista

Made available in DSpace on 2021-06-25T11:03:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-01 Institut de Recherche pour le Développement Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: Little attention has been devoted to the role of the immunoregulatory HLA-E/-F/−G genes in malaria. We evaluated the entire HLA-E/-F/−G variability in Beninese children highly exposed to Plasmodium falciparum (P.f.) malaria. Methods: 154 unrelated children were followed-up for six months and evaluated for the presence and number of malaria episodes. HLA-E/-F/−G genes were genotyped using massively parallel sequencing. Anti P.f. antibodies were evaluated using ELISA. Results: Children carrying the G allele at HLA-F (−1499,rs183540921) showed increased P.f. asymptomatic/symptomatic ratio, suggesting that these children experienced more asymptomatic P.f. episodes than symptomatic one. Children carrying HLA-G-UTR-03 haplotype exhibited increased risk for symptomatic P.f. episodes and showed lower IgG2 response against P.f. GLURP-R2 when compared to the non-carriers. No associations were observed for the HLA-E gene. Conclusion: HLA-F associations may be related to the differential expression profiles of the encoded immunomodulatory molecules, and the regulatory sites at the HLA-G 3'UTR may be associated to posttranscriptional regulation of HLA-G and to host humoral response against P.f. Post-graduate Program in Basic and Applied Immunology Ribeirão Preto Medical School University of São Paulo, Avenida Bandeirantes, 3900, Monte Alegre Immunogenetic Laboratory Immunology Department Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Av. Moraes rego, s/n, Campus da UFPE, Cidade Universitária Centre d'Etude et de Recherche sur le Paludisme Associé à la Grossesse et à l'Enfance (CERPAGE) faculté des Sciences de la Santé Cotonou Université de Paris, UMR 261 MERIT, IRD Département de Zoologie faculté des Sciences et Techniques Université d'Abomey-Calavi Intertryp IRD Cirad University of Montpellier, Avenue Agropolis São Paulo State University (UNESP) School of Medicine Molecular Genetics and Bioinformatics Laboratory, Av. Prof. Dr. Walter Maurício Correa, s/n Centre Médical Saint Joseph Sô Ava CEA DRF-Institut François Jacob Service de Recherches en Hémato-Immunologie, Hopital Saint-Louis Université de Paris CEA U976 HIPI Unit (Human Immunology Physiopathology Immunotherapy) Institut de Recherche Saint-Louis Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo, AV Bandeirantes, 3900 Laboratoire de Biologie et Physiologie Cellulaire Université d'Abomey-Calavi São Paulo State University (UNESP) Department of Pathology School of Medicine São Paulo State University (UNESP) School of Medicine Molecular Genetics and Bioinformatics Laboratory, Av. Prof. Dr. Walter Maurício Correa, s/n São Paulo State University (UNESP) Department of Pathology School of Medicine CAPES: #304931/2014-1 CAPES: #466036/2013-5 CAPES: 302060/2019-07 CAPES: 406594/2013-9 CNPq: PROCAD#88881-068436/2014-1

Made available in DSpace on 2021-06-25T12:38:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Hepatitis C virus usually produces chronic infection and liver damage. Considering that: i) the human leukocyte antigen-E (HLA-E) molecule may modulate the immune response, and ii) little is known about the role of HLA-E gene variability on chronic hepatitis C, we studied the impact of HLA-E polymorphisms on the magnitude of HLA-E liver expression and severity of hepatitis C. HLA-E variability was evaluated in terms of: i) single nucleotide polymorphism (SNP) alleles and genotypes along the gene (beginning of the promoter region, coding region and 30UTR), and ii) ensemble of SNPs that defines the coding region alleles, considered individually or as genotypes. The comparisons of the HLA-E variation sites between patients and controls revealed no significant results. The HLA-E + 424 T > C (rs1059510), +756 G > A (rs1264457) and + 3777 G > A (rs1059655) variation sites and the HLA-E*01:01:01:01 and HLAE*01:03:02:01 alleles, considered at single or double doses, were associated with the magnitude of HLA-E liver expression in Kupfer cell, steatosis, inflammatory activity and liver fibrosis. Although these associations were lost after corrections for multiple comparisons, these variable sites may propitiate biological clues for the understanding of the mechanisms associated with hepatitis C severity. (C) 2021 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. Univ Sao Paulo, Ribeirao Preto Med Sch, Div Gastroenterol, BR-14048900 Ribeirao Preto, SP, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, BR-14048900 Ribeirao Preto, SP, Brazil Univ Toronto Mississauga, Dept Anthropol, Mississauga, ON, Canada Hosp Israelita Albert Einstein, Liver Transplant Dept, BR-05652900 Sao Paulo, SP, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Pathol Dept, BR-14048900 Ribeirao Preto, SP, Brazil Sao Paulo State Univ, Sch Med, Dept Pathol, BR-18618687 Botucatu, SP, Brazil Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, BR-14048900 Ribeirao Preto, SP, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Immunol Div, BR-14048900 Ribeirao Preto, SP, Brazil Sao Paulo State Univ, Sch Med, Dept Pathol, BR-18618687 Botucatu, SP, Brazil CNPq: 302060/2019-7 CAPES: 001 FAPESP: 2015/26556-0

HLA-G is a promiscuous immune checkpoint molecule. The HLA-G gene presents substantial nucleotide variability in its regulatory regions. However, it encodes a limited number of proteins compared to classical HLA class I genes. We characterized the HLA-G genetic variability in 4640 individuals from 88 different population samples across the globe by using a state-of-the-art method to characterize polymorphisms and haplotypes from high-coverage next-generation sequencing data. We also provide insights regarding the HLA-G genetic diversity and a resource for future studies evaluating HLA-G polymorphisms in different populations and association studies. Despite the great haplotype variability, we demonstrated that: (1) most of the HLA-G polymorphisms are in introns and regulatory sequences, and these are the sites with evidence of balancing selection, (2) linkage disequilibrium is high throughout the gene, extending up to HLA-A, (3) there are few proteins frequently observed in worldwide populations, with lack of variation in residues associated with major HLA-G biological properties (dimer formation, interaction with leukocyte receptors). These observations corroborate the role of HLA-G as an immune checkpoint molecule rather than as an antigen-presenting molecule. Understanding HLA-G variability across populations is relevant for disease association and functional studies., We acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil, #302060/2019-7), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/Brazil, Finance code: 001), INCTC/FAPESP (Grant #465539/2014-9) for financial support, and FAPESP/Brazil (#Grant 2017/19223-0).

Background Antigens from the Rh and Kell systems are recognized as the most immunogenic in clinical practice. This study evaluated the possible molecular mechanisms involved in the interaction of antigenic peptides with the DRB1 molecules, which help to explain the high frequency of anti-K and association of D + C antibodies in transfusion and incompatible pregnancy. Study design and methods We included 201 patients with antibodies against antigens from the Rh and Kell systems and compare them with 174,015 controls. HLA-DRB1 genotyping and in silico analysis were performed. The NetMHCIIpan software was used to identify RhD-, RhCE-, and KEL-derived anchor peptides that bind to DRB1 molecules. Results HLA-DRB1*15 is associated with an increased risk of D, C, E, and K alloimmunization, while the HLA-DRB1*01 and *12 alleles are overrepresented in patients with anti-C and anti-D, respectively. In silico analysis showed that three polymorphic points (60I, 68S, and 103S) common to C and D antigens can be presented by several DRB1 molecules, including DRB1*15:01. The DRB1*09:01 molecule, although not showing statistical significance, was able to interact strongly with almost all five anchor peptides from the sequence containing the polymorphic determinants of E antigen, except 217-WMFWPSVNS-225. Conclusion The DRB1*15 molecule has specific physicochemical characteristics in residues 11P and 13R in the P4 pocket that can favor the response to various antigenic peptides. Anti-K alloimmunization is unrestricted for interaction with specific DRB1 molecules, which suggests that almost all individuals in our population have DRB1 molecules capable of binding to KEL-derived anchor peptides and produce anti-K when stimulated.

KIR2DL4 is an important immune modulator expressed in Natural Killer cells, being HLA-G its main ligand. We characterizeKIR2DL4gene diversity considering the promoter, all exons, and all introns, in a highly admixed Brazilian population sample using massively parallel sequencing. We also introduce a molecular method to amplify and sequence the completeKIR2DL4gene. To avoid mapping bias and genotype errors commonly observed in gene families, we have developed a bioinformatic pipeline designed to minimize mapping, genotyping, and haplotyping errors. We have applied this method to survey the variability of 220 samples from the State of São Paulo, southeastern Brazil. We have also compared theKIR2DL4genetic diversity in Brazilian samples with the previously reported by the 1000Genomes consortium.KIR2DL4presents high linkage disequilibrium throughout the gene, with coding sequences associated with specific promoters. There were few, but divergent, promoter haplotypes. We have also detected many newKIR2DL4sequences, all with nucleotide exchanges in introns and encoding previously described proteins. Exons 3 and 4, which encode the external domains, were the most variable ones. The ancestry background influencesKIR2DL4allele frequencies and must be considered for association studies regardingKIR2DL4.

Objective To assess the cognitive function, prevalence, and risk factors associated with cognitive decline and dementia in people above 65 years of age in Santa Cruz Island, Galápagos, Ecuador. Methods This is a cross-sectional observational study that was carried out in adults over 65 years of age in Santa Cruz Island, Galápagos, Ecuador. The mini-mental state examination (MMSE) and ascertain dementia eight-item informant questionnaire (AD8)-validated Ecuador Spanish versions were used to assess cognition. Results There were a total of 80 participants, 55 (67%) women and 25 (31.2%) men. The majority of participants were Mestizos (85.3%), with the remainder classified as White (4.8%), Afro-Ecuadorians (2.4%), or Indigenous (3.6%). The prevalence of cognitive impairment is 30.0%-43.7%. The MMSE results showed that older age and lack of education are risk factors for cognitive decline (p < 0.01). There was high correlation between MMSE and AD8 scores. The AD8 showed that older age, widowhood, and living in Santa Rosa were risk factors for cognitive decline (p < 0.01). According to the AD8, the group with the highest education (six years or more) had the lowest risk of cognitive decline and dementia (p < 0.01). Conclusions The main risk factors for cognitive decline and dementia in individuals above 65 years old in Santa Cruz Island, Galápagos, Ecuador are increased age, lack of education, and widowhood. The prevalence of cognitive impairment is similar to previous studies in Ecuador.

Human pigmentation is a complex trait, probably involving more than 100 genes. Predicting phenotypes using SNPs present in those genes is important for forensic purpose. For this, the HIrisPlex tool was developed for eye and hair color prediction, with both models achieving high accuracy among Europeans. Its evaluation in admixed populations is important, since they present a higher frequency of intermediate phenotypes, and HIrisPlex has demonstrated limitations in such predictions; therefore, the performance of this tool may be impaired in such populations. Here, we evaluate the set of 24 markers from the HIrisPlex system in 328 individuals from Ribeirão Preto (SP) region, predicting eye and hair color and comparing the predictions with their real phenotypes. We used the HaloPlex Target Enrichment System and MiSeq Personal Sequencer platform for massively parallel sequencing. The prediction of eye and hair color was accomplished by the HIrisPlex online tool, using the default prediction settings. Ancestry was estimated using the SNPforID 34-plex to observe if and how an individual's ancestry background would affect predictions in this admixed sample. Our sample presented major European ancestry (70.5%), followed by African (21.1%) and Native American/East Asian (8.4%). HIrisPlex presented an overall sensitivity of 0.691 for hair color prediction, with sensitivities ranging from 0.547 to 0.782. The lowest sensitivity was observed for individuals with black hair, who present a reduced European contribution (48.4%). For eye color prediction, the overall sensitivity was 0.741, with sensitivities higher than 0.85 for blue and brown eyes, although it failed in predicting intermediate eye color. Such struggle in predicting this phenotype category is in accordance with what has been seen in previous studies involving HIrisPlex. Individuals with brown eye color are more admixed, with European ancestry decreasing to 62.6%; notwithstanding that, sensitivity for brown eyes was almost 100%. Overall sensitivity increases to 0.791 when a 0.7 threshold is set, though 12.5% of the individuals become undefined. When combining eye and hair prediction, hit rates between 51.3 and 68.9% were achieved. Despite the difficulties with intermediate phenotypes, we have shown that HIrisPlex results can be very helpful when interpreted with caution.