Implications of the Immune Polymorphisms of the Host and the Genetic Variability of SARS-CoV-2 in the Development of COVID-19.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current pandemic affecting almost all countries in the world. SARS-CoV-2 is the agent responsible for coronavirus disease 19 (COVID-19), which has claimed millions of lives around the world. In most patients, SARS-CoV-2 infection does not cause clinical signs. However, some infected people develop symptoms, which include loss of smell or taste, fever, dry cough, headache, severe pneumonia, as well as coagulation disorders. The aim of this work is to report genetic factors of SARS-CoV-2 and host-associated to severe COVID-19, placing special emphasis on the viral entry and molecules of the immune system involved with viral infection. Besides this, we analyze SARS-CoV-2 variants and their structural characteristics related to the binding to polymorphic angiotensin-converting enzyme type 2 (ACE2). Additionally, we also review other polymorphisms as well as some epigenetic factors involved in the immunopathogenesis of COVID-19. These factors and viral variability could explain the increment of infection rate and/or in the development of severe COVID-19.

Major histocompatibility complex class II DR and DQ evolution and variation in wild capuchin monkey species (Cebinae).

The major histocompatibility complex (MHC) is an important gene complex contributing to adaptive immunity. Studies of platyrrhine MHC have focused on identifying experimental models of immune system function in the equivalent Human Leukocyte Antigen (HLA). These genes have thus been explored primarily in captive platyrrhine individuals from research colonies. However, investigations of standing MHC variation and evolution in wild populations are essential to understanding its role in immunity, sociality and ecology. Capuchins are a promising model group exhibiting the greatest habitat diversity, widest diet breadth and arguably the most social complexity among platyrrhines, together likely resulting in varied immunological challenges. We use high-throughput sequencing to characterize polymorphism in four Class II DR and DQ exons for the first time in seven capuchin species. We find evidence for at least three copies for DQ genes and at least five for DRB, with possible additional unrecovered diversity. Our data also reveal common genotypes that are inherited across our most widely sampled population, Cebus imitator in Sector Santa Rosa, Costa Rica. Notably, phylogenetic analyses reveal that platyrrhine DQA sequences form a monophyletic group to the exclusion of all Catarrhini sequences examined. This result is inconsistent with the trans-species hypothesis for MHC evolution across infraorders in Primates and provides further evidence for the independent origin of current MHC genetic diversity in Platyrrhini. Identical allele sharing across cebid species, and more rarely genera, however, does underscore the complexity of MHC gene evolution and the need for more comprehensive assessments of allelic diversity and genome structure.

External Apical Root Resorption and IL-1A, IL-1RN Gene Polymorphisms: A Systematic Review and Meta-Analysis of Prospective Studies

ABSTRACT Objective: To reconnoiter the IL-1A (-889) and IL-1RN (+2018) gene polymorphisms and their association with EARR. Material and Methods: The Science Direct, PubMed and Scopus databases were comprehensively searched by two independent reviewers. In addition, the bibliographies of all relevant publications and textbooks were searched manually. A meta-analysis was performed using data available up to May 9, 2020. Results: A total of 13 and 9 publications were selected for the systematic review and meta-analysis, respectively for both IL-1A and IL-1RN genes. Odds ratio (OR) was used to evaluate the association of the gene polymorphism and the risk of EARR. The risk of EARR was estimated using the overall OR from the published studies. No association was found for IL-1A gene for the risk of EARR. However, the dominant and co-dominant models of IL-1RN gene polymorphism were associated with the risk of EARR. Conclusion: More studies are warranted to determine the relationship between IL-1A and IL-1RN gene polymorphisms and EARR for a clearer understanding of their interactions.

Plasmodium vivax Cysteine-Rich Protective Antigen Polymorphism at Exon-1 Shows Recombination and Signatures of Balancing Selection.

Plasmodium vivax Cysteine-Rich Protective Antigen (CyRPA) is a merozoite protein participating in the parasite invasion of human reticulocytes. During natural P. vivax infection, antibody responses against PvCyRPA have been detected. In children, low anti-CyRPA antibody titers correlated with clinical protection, which suggests this protein as a potential vaccine candidate. This work analyzed the genetic and amino acid diversity of pvcyrpa in Mexican and global parasites. Consensus coding sequences of pvcyrpa were obtained from seven isolates. Other sequences were extracted from a repository. Maximum likelihood phylogenetic trees, genetic diversity parameters, linkage disequilibrium (LD), and neutrality tests were analyzed, and the potential amino acid polymorphism participation in B-cell epitopes was investigated. In 22 sequences from Southern Mexico, two synonymous and 21 nonsynonymous mutations defined nine private haplotypes. These parasites had the highest LD-R2 index and the lowest nucleotide diversity compared to isolates from South America or Asia. The nucleotide diversity and Tajima's D values varied across the coding gene. The exon-1 sequence had greater diversity and Rm values than those of exon-2. Exon-1 had significant positive values for Tajima's D, ß-α values, and for the Z (HA: dN > dS) and MK tests. These patterns were similar for parasites of different origin. The polymorphic amino acid residues at PvCyRPA resembled the conformational B-cell peptides reported in PfCyRPA. Diversity at pvcyrpa exon-1 is caused by mutation and recombination. This seems to be maintained by balancing selection, likely due to selective immune pressure, all of which merit further study.

Host genetics contributes to the effectiveness of dendritic cell-based HIV immunotherapy.

Systems biological analysis has recently revealed how innate immune variants as well as gut microbiota impact the individual response to immunization. HIV-infected (HIV+) patients have a worse response rate after standard vaccinations, possibly due to the immune exhaustion, increased gut permeability and microbial translocation. In the last decade, dendritic cells (DC)-based immunotherapy has been proposed as an alternative approach to control HIV plasma viral load, however clinical trials showed a heterogeneity of immunization response. Hypothesizing that host genetics may importantly affects the outcome of immunotherapy in HIV+ patients, genetic polymorphisms' distribution and gene expression modulation were analyzed in a phase I/II clinical trial of DC-based immunotherapy according to immunization response, and quality of vaccine product (DC). Polymorphisms in genes previously associated with progression of HIV infection to AIDS (i.e.: PARD3B, CCL5) contribute to a better response to immunotherapy in HIV+ individuals, possibly through a systemic effect on host immune system, but also directly on vaccine product. Genes expression profile after immunization correlates with different degrees of immune chronic activation/exhaustion of HIV+ patients (i.e. PD1, IL7RA, EOMES), but also with anti-viral response and DC quality (i.e.: APOBEC3G, IL8, PPIA), suggested that an immunocompetent individual would have a better vaccine response. These findings showed once more that host genetics can affect the response to DC-based immunotherapy in HIV+ individuals, contributing to the heterogeneity of response observed in concluded trials; and it can be used as predictor of immunization success.

High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from the Paracoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4 gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4 promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4 polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4 gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.

Development of EST-SSR markers related to disease resistance and their application in genetic diversity and evolution analysis in Gossypium.

Cotton (Gossypium spp) is one of the most economically important crops that provide the world's most widely used natural fiber. Diseases such as Fusarium wilt and particularly Verticillium wilt seriously affect cotton production, and thus breeding for disease resistance is one of the most important goals of cotton breeding programs. Currently, potential exists to improve disease resistance in cultivated cotton. Increasing the understanding of the distribution, structure, and organization of genes or quantitative trait loci for disease resistance will help the breeders improve crop yield even in the event of disease. To facilitate the mapping of disease-resistance quantitative trait loci to achieve disease-resistant molecular breeding in cotton, it is necessary to develop polymorphic molecular markers. The objective of this study was to develop simple sequence repeat markers based on cotton expressed sequence tags for disease resistance. The efficacy of these simple sequence repeat markers, their polymorphisms, and cross-species transferability were evaluated. Their value was further investigated based on genetic diversity and evolution analysis. In this study, the unique sequences used to develop markers were compared with the G. arboretum and G. raimondii genome sequences to investigate their position, homology, and collinearity between G. arboretum and G. raimondii.

High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.

MicroRNAs targeting the immunomodulatory HLA-G gene: a new survey searching for microRNAs with potential to regulate HLA-G.

The HLA-G gene is a non-classical class I MHC, responsible for modulating immune responses by inhibiting Natural Killer and cytotoxic T cells, presenting a crucial role in maternal tolerance to the fetus. In non-pathological conditions, its expression is restricted to certain tissues such as cornea and placenta. The HLA-G 3' untranslated region (3'UTR) has been reported to play an important role in the control of mRNA and protein levels, and polymorphisms in this region may influence mRNA stability and microRNA binding. In this study, we propose an approach to detect and classify microRNAs regarding their ability to bind the target (in this case, HLA-G 3'UTR) and the specificity of such interactions. Then, a panel of microRNAs with potential to modulate HLA-G expression is proposed, in which some microRNAs, such as miR-139-3p, would bind to non-polymorphic sequences of the HLA-G 3'UTR in a stable and specific manner, while others, such as miR-608, binds to polymorphic sequences and therefore the binding might be influenced by the variant actually present. Additionally, both HLA-G 3'UTR polymorphisms and the microRNA microenvironment must be considered when studies correlating HLA-G expression profiles and polymorphisms are being conducted. These new data may provide a remarkable contribution to the understanding of the mechanisms underlying HLA-G post-transcriptional regulation, disclosing the impact of variable and non-variable regions on HLA-G biology and providing a unique microRNA repertoire for future functional studies and therapeutic use.

Association of leishmaniasis with TNF alpha promoter and SLC11A1 gene polymorphisms in patients of two endemic areas in Mexico.

Some Single Nucleotide Polymorphisms (SNPs) of interleukins and other modulatory molecules of the immune response play an important role in susceptibility to infectious diseases, particularly those involving intracellular parasites. In this study, we evaluated allele, genotype and haplotype associations of two SNPs of the TNF-α promoter and seven of the SLC11A1 gene in 79 patients with localized cutaneous leishmaniasis (CL) and 15 with visceral leishmaniasis (VL), compared with 127 and 89 locality paired controls, respectively, from two endemic areas of Chiapas State, Mexico. None of the TNF-α alleles and genotypes was associated either to CL or to VL. Alleles rs2276631-C (P = 0.02; OR [95%CI] = 2.11 [1.16-3.86]) and rs2279015-G (P = 0.005; OR [95%CI] = 2.42 [1.33-4.41]) of SLC11A1, were associated with susceptibility to VL, whereas genotypes rs2276631 C/C (P = 0.003; OR [95%CI] = 2.65 [1.41-5.00]) and rs2279015 G/G (P = 0.018; OR [95%CI] = 2.05 [1.15-3.64]) were significantly increased in CL and VL patients, respectively. Complete haplotypes involved in susceptibility were CGCCGDins with VL and CGCCADins with CL. CGCCA was the minimal susceptibility haplotype for CL and CCG for VL. Our data suggest that SLC11A1 gene polymorphisms might have a relevant role in the pathology of leishmaniasis, directing towards susceptibility outcome of this disease in residents of an endemic area.

Genetic diversity and population structure of genes encoding vaccine candidate antigens of Plasmodium vivax.

BACKGROUND: A major concern in malaria vaccine development is genetic polymorphisms typically observed among Plasmodium isolates in different geographical areas across the world. Highly polymorphic regions have been observed in Plasmodium falciparum and Plasmodium vivax antigenic surface proteins such as Circumsporozoite protein (CSP), Duffy-binding protein (DBP), Merozoite surface protein-1 (MSP-1), Apical membrane antigen-1 (AMA-1) and Thrombospondin related anonymous protein (TRAP). METHODS: Genetic variability was assessed in important polymorphic regions of various vaccine candidate antigens in P. vivax among 106 isolates from the Amazon Region of Loreto, Peru. In addition, genetic diversity determined in Peruvian isolates was compared to population studies from various geographical locations worldwide. RESULTS: The structured diversity found in P. vivax populations did not show a geographic pattern and haplotypes from all gene candidates were distributed worldwide. In addition, evidence of balancing selection was found in polymorphic regions of the trap, dbp and ama-1 genes. CONCLUSIONS: It is important to have a good representation of the haplotypes circulating worldwide when implementing a vaccine, regardless of the geographic region of deployment since selective pressure plays an important role in structuring antigen diversity.

Reconsidering the association between the major histocompatibility complex and bipolar disorder.

Bipolar disorder (BD) is a cyclical and chronic affective disorder, globally recognized as an important public health problem and characterized by mood changes with recurring phases such as mania and depression. It is considered a complex disease, depending on the interaction of genetic and environmental triggers (stressors factors), but with a poorly known pathogenesis. Recent studies have implicated immune factors in the pathogenesis of BD and more particularly associated with different human major histocompatibility complex (MHC) regions. A major consortium study have recently linked BD to hundreds of variations with stronger associations in the MHC region, such as the rs3130297 SNP, located in the NOTCH4 gene, with an additional overlapping association with schizophrenia. This short review focuses on studies that investigated the association between bipolar disorder and the MHC, and the involvement of the immune system in the pathogenesis of the disease, in order to provide further information for additional diagnostic and therapeutic strategies. Fully understanding the etiology and pathophysiology of BD is extremely important to define new approaches for intervention and prevention, maybe through the modulation of the immune system.

Identificación de polimorfismos en RXam2, un gen candidato de resistencia a la bacteriosis vascular de yuca / Identification of polymorphisms in RXam2 a cassava bacterial blight resistance gene candidate

La yuca se constituye en la base de la alimentación para más de mil millones de personas en el mundo. Una de las principales enfermedades de la yuca y que podría comprometer la seguridad alimentaria es la bacteriosis vascular ocasionada por la bacteria Xanthomonas axonopodis pv. manihotis (Xam). El gen candidato de resistencia RXam2 codifica para una proteína con dominios NBS (Nucleotide Binding Site) y LRR (Leucine Rich Repeats) y colocaliza con un QTL (Quantitative Trait Loci) que explica el 61.6% de la resistencia a la cepa CIO151 de Xam. En este trabajo se secuenció parcialmente el gen RXam2 en tres variedades de yuca: MCOL2246, TMS60444 y SG107-35 con el objetivo de tener una visión preliminar del grado de polimorfismos tipo SNP (Single Nucleotide Polymorphism) que se presenta en este gen. La región secuenciada incluye 507 pb de la región promotora y 1309 pb de la secuencia codificante. Se logró identificar 5 y 31 SNPs al interior de las variedades MCOL2246 y TMS60444, respectivamente. Al mismo tiempo el número de SNPs entre las variedades fue de 44, 34 y 23 para MCOL2246-TMS60444, TMS60444-SG107-35 y MCOL2246-SG107-35, respectivamente. El mayor número de SNPs estuvieron localizados en la región -500 a -300 pb que corresponden a un fragmento de la región promotora del gen, aunque también se identificó un importante número de polimorfismos en la región codificante. Este estudio permitirá identificar el número de polimorfismos en este gen en un mayor grupo de variedades de yuca con el fin de asociar estos polimorfismos con el fenotipo de resistencia/susceptibilidad.

Cassava is a staple food for more than a billion people. One of the major diseases of cassava which could compromise food security is known as cassava bacterial blight, caused by the bacterium Xanthomonas axonopodis pv. manihotis (Xam). The RXam2 resistance gene candidate encodes a protein with Nucleotide Binding Site and Leucine Rich Repeats domains, and colocalizes with a QTL (Quantitative Trait Loci) that accounts for 61.6% of the resistance to the Xam strain CIO151. In this work we sequenced 1816 bp corresponding to a partial sequence of this gene in three cassava varieties with the aim of having a preliminary overview of the degree of Single Nucleotide Polymorphisms (SNPs). The sequenced regions included 507 bp of the promotor and 1309 bp of the coding sequence. It was possible to identify five and 31 intravarietal SNPs in MCOL2246 and TMS60444, respectively. In addition, the number of SNPs between varieties was 44, 34 and 23 for MCOL2246-TMS60444, TMS60444-SG107-35 and MCOL2246-SG107-35, respectively. The largest number of SNPs was located in the promoter region -500 to -300 bp. This study may help to develop alternatives to identify SNPs in a more diverse group of cassava varieties, and possibly associate them with resistance/susceptible phenotypes.

Interaction of human complement factor H variants Tyr402 and His402 with leptospira spp

Leptospirosis isazoonosis caused by pathogenic bacteria from the genus Leptospira.Thediseaserepresents a seriouspublic health problem inunderdeveloped tropical countries.Leptospire sinfecthosts throughsmallabrasions in the skin or mucousmembranesand they rapidly disseminateto target organs.Thecapacityof some pathogenic leptospira lstrains toacquire thenegative complementregulators factorH(FH)andC4bbindingpro teincorrelateswith their abilityto survivein humans e rum.Inthis study weasses sed the functional consequences oft heagemaculardegeneration-associatedpolymorphismFHHis402orFHTyr402onFH Leptospira interactions.Inbinding assaysusing sub-satura tingamounts ofFH, theFHTyr402 variant interacted with all the strainstested more stronglythanthe FHHis402variant.Athigher concentrations, differences tendedto disappear. We then compared co factor activities displayed by FH. His402and FH Tyr402 bound tothe surface ofL.interrogans. Bothvariantsex ibit similar activity as cofactors for FactorI mediated cleavage of C3b,thusindicating that they do not differin their capacity toregulate the complement cascade.

Variantes polimórficas Ala513Pro y Gly972Arg del gen IRS-1 no se asocian a la diabetes mellitus tipo 2 en un grupo de la población cubana / The Ala513Pro and Gly972ARg polymorphous variants of IRS-1 gen are not associated with type diabetes mellitus in a group of the Cuban population

Introducción: la diabetes mellitus tipo 2 es una enfermedad heterogénea y multifactorial, que está determinada por factores genéticos y no genéticos. El sustrato 1 del receptor de la insulina (IRS-1) cumple una función fundamental en la transmisión de la señal insulínica, por tanto sus variantes génicas constituyen blancos importantes en el estudio de la susceptibilidad genética a esta enfermedad en las diferentes poblaciones. Objetivo: explorar el papel de las variantes polimórficas Gly972Arg y Ala513Pro del gen IRS-1 en la susceptibilidad genética de la diabetes mellitus tipo 2 en un grupo de la población cubana.Métodos: se determinó la frecuencia de los polimorfismos Gly972Arg y Ala513Pro del IRS-1 en 499 ciudadanos cubanos, con un índice de masa corporal entre 22-30, con edades comprendidas entre los 40 y 70 años: de ellos 272 (54,5 por ciento) diabéticos y 227 (45,5 por ciento) no diabéticos.Resultados: la frecuencia del alelo Pro513 fue baja (1,2 por ciento) y similar para ambos grupos (1,1 por ciento vs. 1,3 por ciento para el grupo de diabéticos y el grupo control, respectivamente). La frecuencia del polimorfismo Gly972Arg fue de 16,2 por ciento, superior a la reportada para la mayoría de las poblaciones estudiadas. No se encontraron diferencias significativas en la frecuencia del alelo Arg972 entre el grupo de diabéticos y el grupo control (15,4 por ciento vs. 17,3 por ciento), ni cambios en los niveles de glucemia e insulinemia asociados a la presencia del alelo polimórfico Arg972.Conclusiones: en este grupo de sujetos de la población cubana, las variantes polimórficas Ala513Pro y Gly972Arg del gen IRS-1 no participan en la etiología de la diabetes mellitus tipo 2(AU)

Introduction: the type 2 diabetes mellitus is a heterogeneous and multifactor disease determined by genetic and no-genetic factors. The substrate 1 of insulin receptor (IRS-1) has a fundamental function in transmission of insulin signal, thus its genic variants are significant targets in study of genetic susceptibility to this disease in different populations. Objective: to explore the role of the Gly972Arg and Ala513PRo of IRS-1 gen polymorphous variants in the genetic susceptibility of type 2 diabetes mellitus in Cuban population. Methods: the frequency of above mentioned polymorphous variants in 499 Cuban citizens with a 22-30 body mass index aged between 40 to 70 including 272 diabetics (54,5 por ciento) and 227 non-diabetic (45,5 por ciento). Results: frequency of Pro513 allele was low (1,2 por ciento) and similar en both groups (1,1 por ciento versus 1,3 por ciento for diabetic group and the control one, respectively). Frequency of Gly972Arg polymorphism was of 16,2 por ciento, higher than that reported for most of study populations. There were not significant differences in frequency of Arg972 allele between the diabetic group and the control one (15,4 por ciento versus 17,3 por ciento). Also, there were not changes in glycemia and insulinemia levels associated to presence of polymorphous allele. Conclusions: in present group of Cuban population the above mentioned polymorphous variants are not involved in etiology of type 2 diabetes mellitus(AU)

Análise de polimorfismos em genes relacionados à virulência e resistência de Streptococcus suis em uma população suína / Analysis of genetic polymorphisms related to virulence and resistance in a population ofStreptococcus suis

Background: Streptococcus suis is a bacterium that causes several diseases in swines and can infect humans; in addition, it represents an important risk factor for professionals from this area. In cases of outbreak, despite some animals may remain as asymptomatic disease carriers, mortality can vary between 4 and 14%. Virulence of this bacterium may be associated with different serotypes exist, as well as with cellular proteins present in the capsule, or by mutations in the genomes of different serotypes, or even between samples of the same serotype. The strains of serotype 2 are considered to be the most virulent, although they can have moderate virulence, or even being less virulent. The objective of this work is to analyze genetic variability in S. suis samples. Materials, Methods & Results: Forty S. suis samples provided by the Laboratório Microvet (Minas Gerais, Brazil) and isolated between 1995 and 2003 on a farm located in Ponte Nova, MG, have been used. The lyophilized samples were reactivated in BHI, for 24 h, at 37°C; after that, they were cultivated and isolated in Todd Hewitt nutrient medium plus agar-agar and 10% defibrinated sheep blood and kept at 37°C, for 18 h. Serotyping of samples was conducted by Microvet and the data was compiled for comparison with the results. Genomic DNA was extracted from 3x106 cells, using DNAzol kit (Invitrogen®), under the protocol proposed by the manufacturer, followed by PCR individually for each primer pair - dpr and aroA, using 200 µM dNTP, 3.4 mM MgCl2, 2 U of Taq DNA polymerase (Invitrogen®), 1.6x amplification buffer and 0.20 µM of the primers in a final volume of 25 µL. The annealing temperature was 59°C, for aroA gene, and 60°C, for dpr gene. The amplified fragments of genes were digested with 0.2 µL of the restriction enzymes Alu I, AflIII, EcoRI and MPS I, at 37°C, for 2 h. All products were analyzed by separation on 3% agarose gel. Eight in the forty samples analyzed showed polymorphisms in the dpr gene for cleavage site of Alu I enzyme, thus generating samples with two and three DNA fragments. Analysis with the other endonucleases does not show differences; besides, no cleavage has occurred in the fragment. All samples analyzed for the aroA gene were observed to be identical for the cleavage sites of the used enzymes. During the analysis of serotypes, 39 (97.5%) samples were identified as type 2 and one (2.5%) as type 3. Discussion: The dpr gene showed different patterns between the assessed S. suis colonies, what indicates the existence of polymorphisms in the restriction sites. These data partially corroborate those already reported in the literature, where the percentage of amino acid polymorphisms was 17.9% in the dpr gene. In this study, changes identified in the dpr gene are not conclusive; still, modifications in nucleotide bases of certain genes may be related to expressions of differential proteins, associated with pathogenicity and virulence of pathogens. Even with the use of antibiotics and hygienic-sanitary control measures, records of difficult-to-control outbreaks are constant in the swine populations from which the samples were isolated. The sample of serotype 3 was collected from healthy animal, whereas the serotype 2 samples were collected from sick animals. These factors reinforce the idea that alterations in genomes of bacteria may be establishing new mechanisms to escape, hence favoring the survival of pathogens.(AU)

Análise de polimorfismos em genes relacionados à virulência e resistência de Streptococcus suis em uma população suína / Analysis of genetic polymorphisms related to virulence and resistance in a population ofStreptococcus suis

Background: Streptococcus suis is a bacterium that causes several diseases in swines and can infect humans; in addition, it represents an important risk factor for professionals from this area. In cases of outbreak, despite some animals may remain as asymptomatic disease carriers, mortality can vary between 4 and 14%. Virulence of this bacterium may be associated with different serotypes exist, as well as with cellular proteins present in the capsule, or by mutations in the genomes of different serotypes, or even between samples of the same serotype. The strains of serotype 2 are considered to be the most virulent, although they can have moderate virulence, or even being less virulent. The objective of this work is to analyze genetic variability in S. suis samples. Materials, Methods & Results: Forty S. suis samples provided by the Laboratório Microvet (Minas Gerais, Brazil) and isolated between 1995 and 2003 on a farm located in Ponte Nova, MG, have been used. The lyophilized samples were reactivated in BHI, for 24 h, at 37°C; after that, they were cultivated and isolated in Todd Hewitt nutrient medium plus agar-agar and 10% defibrinated sheep blood and kept at 37°C, for 18 h. Serotyping of samples was conducted by Microvet and the data was compiled for comparison with the results. Genomic DNA was extracted from 3x106 cells, using DNAzol kit (Invitrogen®), under the protocol proposed by the manufacturer, followed by PCR individually for each primer pair - dpr and aroA, using 200 µM dNTP, 3.4 mM MgCl2, 2 U of Taq DNA polymerase (Invitrogen®), 1.6x amplification buffer and 0.20 µM of the primers in a final volume of 25 µL. The annealing temperature was 59°C, for aroA gene, and 60°C, for dpr gene. The amplified fragments of genes were digested with 0.2 µL of the restriction enzymes Alu I, AflIII, EcoRI and MPS I, at 37°C, for 2 h. All products were analyzed by separation on 3% agarose gel. Eight in the forty samples analyzed showed polymorphisms in the dpr gene for cleavage site of Alu I enzyme, thus generating samples with two and three DNA fragments. Analysis with the other endonucleases does not show differences; besides, no cleavage has occurred in the fragment. All samples analyzed for the aroA gene were observed to be identical for the cleavage sites of the used enzymes. During the analysis of serotypes, 39 (97.5%) samples were identified as type 2 and one (2.5%) as type 3. Discussion: The dpr gene showed different patterns between the assessed S. suis colonies, what indicates the existence of polymorphisms in the restriction sites. These data partially corroborate those already reported in the literature, where the percentage of amino acid polymorphisms was 17.9% in the dpr gene. In this study, changes identified in the dpr gene are not conclusive; still, modifications in nucleotide bases of certain genes may be related to expressions of differential proteins, associated with pathogenicity and virulence of pathogens. Even with the use of antibiotics and hygienic-sanitary control measures, records of difficult-to-control outbreaks are constant in the swine populations from which the samples were isolated. The sample of serotype 3 was collected from healthy animal, whereas the serotype 2 samples were collected from sick animals. These factors reinforce the idea that alterations in genomes of bacteria may be establishing new mechanisms to escape, hence favoring the survival of pathogens.

Marcadores de inflamação na morbidade da malária por Plasmodium vivax: contribuição de micropartículas, ácidos nucléicos circulantes e polimorfismos genéticos do hospedeiro vertebrado

Os estudos sobre os mecanismos patogênicos que levam à malária grave foram durante muitos anos restritos basicamente ao Plasmodium falciparum. Entretanto, o número de casos de P. vivax vem aumentando em todo o mundo, incluindo aqueles de malária grave. Evidências recentes sugerem que a infecção pelo P. vivax induz uma resposta imune inflamatória mais intensa que aquela induzida pelo P. falciparum. Diante disso, os objetivos deste trabalho foram: (i) investigar mediadores plasmáticos relacionados à inflamação que poderiam ser utilizados como marcadores de morbidade da malária por P. vivax; (ii) identificar indivíduos geneticamente susceptíveis e/ou resistentes à malária clínica. Na busca de biomarcadores, a hipótese investigada foi a de que as micropartículas (MPs) – vesículas resultantes da ativação e/ou apoptose celular – e os ácidos nucléicos circulantes (CNAs) estariam associados à infecção pelo P. vivax

Em pacientes bem caracterizados clinicamente foi possível demonstrar que as MPs, de diferentes origens, estavam significativamente aumentadas na malária por P. vivax, sendo as MPs de origem plaquetária associadas à sintomatologia da doença. Os níveis de CNAs plasmáticos estavam aumentados nos pacientes infectados pelo P. vivax sendo estes níveis associados ao espectro clínico da doença. Um achado interessante no presente trabalho foi que os níveis de CNAs também correlacionaram- se com a trombocitopenia. Um resultado de grande importância foi que os níveis de MPs e CNAs no plasma retornaram aos valores basais após o tratamento antimalárico específico, o que sugere que ambos os fatores podem ser bons marcadores de morbidade por P. vivax.Na segunda parte do estudo, buscou- se a associação entre os polimorfismos genéticos de citocinas (MIF, TNF-α, IL-10 e TGF-β) e de glicoproteínas plaquetárias (GPIa/IIa e GPIIb/IIIa) com as complicações clínicas e hematológicas da infecção pelo P. vivax. Os resultados obtidos evidenciaram associações entre os polimorfismos dos genes GPIa/IIa, TNF-α, MIF e IL-10 e alterações clínicas e hematológicas na malária causada pelo P. vivax. Em resumo, os dados apresentados reforçam a influência de múltiplos genes do hospedeiro vertebrado na malária por P. vivax. Espera-se que os resultados observados no presente trabalho possam contribuir no esclarecimento dos mecanismos envolvidos na patogenia do P. vivax

Marcadores de inflamação na morbidade da malária por Plasmodium vivax: contribuição de micropartículas, ácidos nucléicos circulantes e polimorfismos genéticos do hospedeiro vertebrado / Markers of inflammation in the morbidity of Plasmodium vivax malaria: contribution of microparticles circulating nucleic acids and genetic polymorphisms of the vertebrate host

Os estudos sobre os mecanismos patogênicos que levam à malária grave foram durante muitos anos restritos basicamente ao Plasmodium falciparum. Entretanto, o número de casos de P. vivax vem aumentando em todo o mundo, incluindo aqueles de malária grave. Evidências recentes sugerem que a infecção pelo P. vivax induz uma resposta imune inflamatória mais intensa que aquela induzida pelo P. falciparum. Diante disso, os objetivos deste trabalho foram: (i) investigar mediadores plasmáticos relacionados à inflamação que poderiam ser utilizados como marcadores de morbidade da malária por P. vivax; (ii) identificar indivíduos geneticamente susceptíveis e/ou resistentes à malária clínica. Na busca de biomarcadores, a hipótese investigada foi a de que as micropartículas (MPs) – vesículas resultantes da ativação e/ou apoptose celular – e os ácidos nucléicos circulantes (CNAs) estariam associados à infecção pelo P. vivax. Em pacientes bem caracterizados clinicamente foi possível demonstrar que as MPs, de diferentes origens, estavam significativamente aumentadas na malária por P. vivax, sendo as MPs de origem plaquetária associadas à sintomatologia da doença. Os níveis de CNAs plasmáticos estavam aumentados nos pacientes infectados pelo P. vivax sendo estes níveis associados ao espectro clínico da doença. Um achado interessante no presente trabalho foi que os níveis de CNAs também correlacionaram- se com a trombocitopenia. Um resultado de grande importância foi que os níveis de MPs e CNAs no plasma retornaram aos valores basais após o tratamento antimalárico específico, o que sugere que ambos os fatores podem ser bons marcadores de morbidade por P. vivax.Na segunda parte do estudo, buscou- se a associação entre os polimorfismos genéticos de citocinas (MIF, TNF-α, IL-10 e TGF-β) e de glicoproteínas plaquetárias (GPIa/IIa e GPIIb/IIIa) com as complicações clínicas e hematológicas da infecção pelo P. vivax. Os resultados obtidos evidenciaram associações entre os polimorfismos dos genes GPIa/IIa, TNF-α, MIF e IL-10 e alterações clínicas e hematológicas na malária causada pelo P. vivax. Em resumo, os dados apresentados reforçam a influência de múltiplos genes do hospedeiro vertebrado na malária por P. vivax. Espera-se que os resultados observados no presente trabalho possam contribuir no esclarecimento dos mecanismos envolvidos na patogenia do P. vivax.