Polymorphisms at the IL17A and IL17RA Genes are Associated with Prognosis of Papillary Thyroid Carcinoma.

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.

Genetic polymorphism in IL17RA induces susceptibility to Toxoplasma gondii infection in Brazilian pregnant women.

Congenital toxoplasmosis is a parasitic disease caused by Toxoplasma gondii, an obligate intracellular parasite which can cause fetal death/abortion and can induce damage in the brain and eyes of the infected babies. The environmental and genetic factors associated with T. gondii and the maternal immune response, drive part of the pathogenesis of congenital toxoplasmosis. Thus, in this study, we aimed to investigate the allelic and genotypic frequencies of specific single nucleotide polymorphisms (SNPs) in the IL17A and IL17RA genes, as well as the production of IL-17A, IL-33, and CCL2 in pregnant women, from the State of Rio Grande do Norte, Brazil, further relating these along with the clinical parameters, to the toxoplasmosis infection. Through PCR-RFLP techniques, two SNPs implicated in Th17 immune response, IL17A rs2275913 (G> A) and IL17RA rs4819554 (A> G) modulation were evaluated in pregnant women, either infected or not infected by T. gondii. These women were also evaluated in terms of plasma release of CCL2, IL-33, and IL-17A which relate to hypertension, number of abortions, and ethnic pattern. The results showed that the G-allele of the SNP rs2275913 (IL17A) appeared to be protective in this population, while the rs4819554 (IL17RA) SNP G allele was associated with greater susceptibility to T. gondii infection [ρ value = 0.025; OR = 2.815 (1.118-7.089); CI = 95%]. None of the cytokines had any influence on the analyzed parameters (abortion and hypertension). In conclusion, our data suggest an immunogenic evidence of susceptibility to T. gondii infection driven by the rs4819554 (IL17RA) SNP G allele in Brazilian pregnant women. Further studies are needed to reinforce this trial marker in populations from distinct geographical areas as well as to confirm the protective pattern related to the G-allele of the SNP rs2275913 (IL17A) in pregnant women.

Immunohistochemical evaluation of HLA-G and FoxP3+ T regulatory cells in oral cavity and lower lip squamous cell carcinomas.

Human Leukocyte Antigen G (HLA-G) is a molecule involved in the tumor immunosuppression and also in the generation of regulatory T (Treg) cells, thus leading to evasion to the immune system host, and consequently, contributing to tumor progression in several cancers. The aim of this study was to evaluate the immunoexpression of HLA-G by tumor cells and FoxP3+ Treg cells in 25 oral tongue squamous cell carcinomas (SCCs) and 25 lower lip SCCs and analyze their relationship with clinical parameters. HLA-G expression was higher in oral tongue SCCs than in lower lip SCCs. In oral tongue SCCs and lower lip SCCs, no association between HLA-G expression and clinical parameters (tumor size, lymph node status, distant metastasis, and clinical stage) was verified (P>0.05). FoxP3+ Treg cells were detected along the tumor invasive front in all cases of oral tongue and lower lip SCCs. In oral tongue SCC cases, the number of Treg cells tended to be higher in smaller tumors, tumors without regional lymph node metastasis, and tumors in early clinical stages, but the difference was not statistically significant (P>0.05). A significant positive correlation was found between the expression of HLA-G by neoplastic cells and Treg cells in lower lip SCCs (p = 0.008). Our findings suggest the involvement of HLA-G and Treg cells in the modulation of immune responses in oral tongue and lower lip SCCs. This interaction between HLA-G and Treg cells may represent an evasion mechanism in these malignancies.

Immune response and evasion mechanisms in lip carcinogenesis: An immunohistochemical study.

OBJECTIVES: Programmed death ligand-1 (PD-L1) and human leukocyte antigen-G (HLA-G) are considered immune checkpoint molecules that inhibit T-cell effectiveness, contributing to tumor immune escape. This study investigated PD-L1, HLA-G, CD8, and granzyme B (GrB) expression at different stages of lip carcinogenesis. DESIGN AND RESULTS: Forty cases of lip squamous cell carcinoma (LSCC), 55 actinic cheilitis (AC), and 10 healthy lip mucosa (HLM) were submitted to immunohistochemistry. Semiquantitative (PD-L1, HLA-G), and quantitative (CD8, GrB) analysis were performed. PD-L1 and HLA-G expression in neoplastic cells/keratinocytes and stroma/connective tissue was significantly higher in LSCC and AC, compared to HLM (p<0.05). PD-L1 was not associated with clinicopathological features of the lesions. HLA-G expression by malignant cells was significantly higher in LSCCs with distant metastasis (p = 0.041).CD8+ and GrB+ cell numbers progressively increased from HLMs to LSCC, with AC exhibiting intermediate numbers (p<0.01). Most LSCCs showed coexistence of PD-L1+ and CD8+ cells (72.5%). PD-L1 was directly correlated to CD8+ and GrB+ lymphocytic infiltration in LSCCs (p<0.05). Low cytotoxic immune response was associated with lymph node metastasis in LSCC (p<0.05). CONCLUSIONS: PD-L1 and HLA-G-mediated immune evasion mechanisms are likely to occur from early pre-malignant to advanced malignant stages of lip carcinogenesis, which might provide a rationale for therapeutic blockade of these pathways. PD-L1 expression in LSCCs was correlated with the cytotoxic markers, suggesting that PD-L1 may appear as an escape mechanism in response to an active antitumor response.

Immunohistochemical evaluation of HLA-G and FoxP3+ T regulatory cells in oral cavity and lower lip squamous cell carcinomas

Abstract: Human Leukocyte Antigen G (HLA-G) is a molecule involved in the tumor immunosuppression and also in the generation of regulatory T (Treg) cells, thus leading to evasion to the immune system host, and consequently, contributing to tumor progression in several cancers. The aim of this study was to evaluate the immunoexpression of HLA-G by tumor cells and FoxP3+ Treg cells in 25 oral tongue squamous cell carcinomas (SCCs) and 25 lower lip SCCs and analyze their relationship with clinical parameters. HLA-G expression was higher in oral tongue SCCs than in lower lip SCCs. In oral tongue SCCs and lower lip SCCs, no association between HLA-G expression and clinical parameters (tumor size, lymph node status, distant metastasis, and clinical stage) was verified (P>0.05). FoxP3+ Treg cells were detected along the tumor invasive front in all cases of oral tongue and lower lip SCCs. In oral tongue SCC cases, the number of Treg cells tended to be higher in smaller tumors, tumors without regional lymph node metastasis, and tumors in early clinical stages, but the difference was not statistically significant (P>0.05). A significant positive correlation was found between the expression of HLA-G by neoplastic cells and Treg cells in lower lip SCCs (p = 0.008). Our findings suggest the involvement of HLA-G and Treg cells in the modulation of immune responses in oral tongue and lower lip SCCs. This interaction between HLA-G and Treg cells may represent an evasion mechanism in these malignancies.

Early SIV and HIV infection promotes the LILRB2/MHC-I inhibitory axis in cDCs.

Classical dendritic cells (cDCs) play a pivotal role in the early events that tip the immune response toward persistence or viral control. In vitro studies indicate that HIV infection induces the dysregulation of cDCs through binding of the LILRB2 inhibitory receptor to its MHC-I ligands and the strength of this interaction was proposed to drive disease progression. However, the dynamics of the LILRB2/MHC-I inhibitory axis in cDCs during early immune responses against HIV are yet unknown. Here, we show that early HIV-1 infection induces a strong and simultaneous increase of LILRB2 and MHC-I expression on the surface of blood cDCs. We further characterized the early dynamics of LILRB2 and MHC-I expression by showing that SIVmac251 infection of macaques promotes coordinated up-regulation of LILRB2 and MHC-I on cDCs and monocytes/macrophages, from blood and lymph nodes. Orientation towards the LILRB2/MHC-I inhibitory axis starts from the first days of infection and is transiently induced in the entire cDC population in acute phase. Analysis of the factors involved indicates that HIV-1 replication, TLR7/8 triggering, and treatment by IL-10 or type I IFNs increase LILRB2 expression. Finally, enhancement of the LILRB2/MHC-I inhibitory axis is specific to HIV-1 and SIVmac251 infections, as expression of LILRB2 on cDCs decreased in naturally controlled chikungunya virus infection of macaques. Altogether, our data reveal a unique up-regulation of LILRB2 and its MHC-I ligands on cDCs in the early phase of SIV/HIV infection, which may account for immune dysregulation at a critical stage of the anti-viral response.

Caracterización clínica de los pacientes con síncope / Clinical profile of patients with syncope

Resumen Introducción: Se hizo una caracterización clínica de los pacientes con síncope en un Hospital Universitario en Bogotá. Objetivo: Describir las características clínicas de los pacientes con síncope que consultaron al Hospital Militar Central en Bogotá en el período 2012-2015, y analizar la contribución de las ayudas diagnósticas y de las escalas EGSYS y OESIL para orientar el diagnóstico etiológico. Métodos: Se realizó un estudio observacional, descriptivo, de pacientes mayores de 18 años que ingresaron a urgencias del Hospital Militar Central por síncope; se analizaron características clínicas, estudios solicitados y puntajes de las escalas EGSYS y OESIL. Resultados: Se evaluaron 705 historias clínicas, de las cuales 116 fueron excluidas por datos faltantes; la edad promedio fue 58 años y el 46,52% eran mujeres. El 41,6% tenía hipertensión arterial y el 21% enfermedad cardiaca previa. Según el diagnóstico etiológico, 75% fueron clasificados como síncope no cardiaco, 23% como síncope cardiaco y en 2% no se identificó la etiología. El examen más solicitado fue el electrocardiograma (79%), seguido por troponina (63%) y TAC cerebral (58%). Al aplicar las escalas, 60% de los pacientes tuvo un puntaje menor a 3 (EGSYS) y 2 puntos (OESIL), lo que sugería que eran de etiología no cardiogénica/bajo riesgo de mortalidad respectivamente. Conclusiones: La etiología del síncope en la mayoría de los casos fue no cardiaca. El electrocardiograma debe ser solicitado a todos los pacientes con síncope. El uso rutinario de las escalas de riesgo puede contribuir a disminuir la solicitud de estudios no indicados, optimizar el uso de recursos y reducir los días de hospitalización.

Abstract Introduction: A clinical profile was constructed on patients with syncope in a Bogota University Hospital. Objective: To describe the clinical characteristics of patients with syncope that were seen in the Hospital Militar Central in Bogota in the period 2012-2015, as well as to analyse the contribution of diagnostic aids and the Evaluation of Guidelines in Syncope Study (EGSYS) and the Lazio epidemiological syncope Observation (OESIL) scores in order to determine the aetiological diagnosis. Methods: A descriptive observational study was performed on patients over 18 years admitted to the Emergency Department of the Hospital Militar Central due to syncope. An analysis was carried out on the clinical characteristics, examinations requested, and the scores on the EGSYS and OESI L scales. Results: A total of 705 clinical histories were evaluated, of which 116 were excluded due to lack of data. The mean age was 58 years, and 46.52% were women. Arterial hypertension was observed in 41.6%, and 21% had a previous heart disease. According to the aetiological diagnosis, 75% were classified as non-cardiac syncope, 23% as cardiac syncope, and 2% of unknown origin. The most requested examination was the electrocardiogram (79%), followed by troponin (63%), and a computed tomography brain scan (58%). On applying the scales, 60% of the patients had a score of less than 3 (EGSYS) and 2 points (OESIL), which suggested that they were of non-cardiogenic origin/low mortality risk, respectively. Conclusions: The origin of syncope in the majority of cases was non-cardiac. An electrocardiogram must be requested on all patients with syncope. The routine use of risk scales can contribute to reducing the number of non-indicated examinations, as well as optimise the use of resources and reduce hospital stay.

Haplotypes of the HLA-G 3' Untranslated Region Respond to Endogenous Factors of HLA-G+ and HLA-G- Cell Lines Differentially.

The immune checkpoint HLA-G prevents maternal rejection of the fetus and contributes in cancer invasion and acceptance of allografts. The 5' and 3' regulatory regions of the HLA-G gene are polymorphic and balancing selection probably maintains this variability. It is proposed that nucleotide variations may affect the level of HLA-G expression. To investigate this issue we aimed to analyze how haplotypes of the 3' untranslated region (3'UTR) with highest worldwide frequencies, namely UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-18 and UTR-7, impact the expression of a luciferase reporter gene in vitro. Experiments performed with the HLA-G positive cell lines JEG-3 (choricarcinoma) and FON (melanoma), and with the HLA-G negative cell lines M8 (melanoma) and U251MG (glioblastoma) showed that the HLA-G 3'UTR polymorphism influences the response to endogenous cellular factors and may vary according to the cell type. UTR-5 and UTR-7 impact the activity of luciferase the most whereas UTR-2, UTR-3, UTR-4, and UTR-18 have intermediate impact, and UTR-1 has the lowest impact. These results corroborate the previous associations between amounts of plasma sHLA-G levels and 3'UTR haplotypes in healthy individuals and reinforce that 3'UTR typing may be a predictor of the genetic predisposition of an individual to express different levels of HLA-G.

Costos de bolsillo de pacientes con diagnóstico de tuberculosis en Colombia / Out-of-pocket costs of patients diagnosed with tuberculosis in Colombia

Introducción. La tuberculosis (TB) es una enfermedad infecto-contagiosa producida por micobacterias; según datos de la Organización Mundial de la Salud, un tercio de la población mundial está infectada. Para combatirla se ha empleado la estrategia DOTS (Directly Observed Therapy Short Course), efectiva para el diagnóstico, tratamiento y monitoreo de la tuberculosis. Objetivo. Estimar costos de bolsillo que asumen los pacientes con tuberculosis, que reciben tratamiento bajo la estrategia DOTS. Diseño. Estudio observacional descriptivo, prospectivo. Lugar. Tres ciudades de Colombia (Medellín, Montería y Quibdó). Participantes. Pacientes con diagnóstico de TB. Intervenciones. Se utilizó un instrumento de recolección que incluía variables relacionadas con los costos de bolsillo directos e indirectos. El análisis se hizo en el programa SPSS versión 17,0 y STATA 11; a las variables cuantitativas se les estimó media y desviación estándar, mientras que a las cualitativas proporciones. Resultados. Participaron 91 pacientes que se encontraban en tratamiento bajo la estrategia DOTS. El promedio de edad fue 39,3±20 años; la mayoría vivía con sus familiares. Los ingresos mensuales de los pacientes tuvieron una media de 422 863 COP (1€ = 3 149 COP) y los gastos directos más altos generados por el tratamiento fueron los destinados al desplazamiento y ayudas diagnósticas, con una media de 8 181 y 7 630 COP, respectivamente. Conclusiones. Los costos asumidos por los pacientes bajo la estrategia DOTS fueron altos, incluso cuando el tratamiento se entrega gratuitamente. La modificación de la estrategia para evitar costos en los pacientes podría impactar disminuyendo el abandono del tratamiento por los mismos.

Introduction: Tuberculosis (TB) is a contagious disease caused by mycobacteria. According to the World Health Organization, one third of the world's population is infected. The directly observed therapy short course (DOTS) strategy has been used effective for the diagnosis, treatment and monitoring of tuberculosis. Objective: To estimate the out-of-pocket costs of TB patients who receive treatment under the DOTS strategy. Design: Descriptive prospective and observational study. Setting: Three cities in Colombia (Medellin, Monteria and Quibdo). Participants: Patients diagnosed with TB. Interventions: An instrument was used that included variables related to direct and indirect out-of-pocket costs. The analysis was done using the SPSS version 17.0 and STATA 11; mean and standard deviation were estimated for quantitative variables, and proportions for qualitative variables. Results: The DOTS strategy was applied in 91 patients. The average age was 39.3 ± 20 years; most patients lived with their families. The monthly income of the patients was 422 863 COP (1€ = 3 149 COP) in average and the higher direct costs generated by the treatment were those for traveling and diagnostic aids, with an average cost of 8 181 and 7 630 COP respectively. Conclusions: The costs assumed by patients under the DOTS strategy were high, even when treatment was provided free of charge. The modification of the strategy to avoid costs in patients could decrease treatment dropout.

Expression of HLA-E molecules in the placental tissue of women infected with HIV-1 and uninfected women.

INTRODUCTION: Expression of HLA-E molecule in the placental extravillous trophoblast is associated with immune system cell inhibition, resulting in immune tolerance to fetus during pregnancy. HIV-1 can infect trophoblast cells and modify the expression of HLA-E, which may inhibit the cytotoxic activity of the immune system. AIM: The aim of this study was to evaluate HLA-E expression in third trimester placental tissue of women infected with HIV-1 and uninfected women. METHODS: We performed an immunohistochemistry assay to evaluate HLA-E staining in the placental tissue of 99 HIV-1 infected and 85 uninfected women. A pathologist analyzed and classified the HLA-E expression in the placental cells. RESULTS: Irrespective of the HIV status, HLA-E staining was observed in the extravillous trophoblast cells, endothelial cells and Hofbauer cells, but not in the syncytiotrophoblast. HLA-E staining showed no significant difference between the placental tissue of women infected with HIV-1 and uninfected women (P = 0.76). Considering HIV-1 infected women, HLA-E staining was not influenced by HIV-1 viral load (P = 0.48), CD4+ T-cell count (P = 0.10) and antiretroviral therapy used during pregnancy (P = 0.54). DISCUSSION: Despite the presence of HIV-1 infection, the expression of HLA-E molecules in the placental tissue was not modified when the infection was under antiretroviral therapy control.

DNA damage increase in peripheral neutrophils from patients with rheumatoid arthritis is associated with the disease activity and the presence of shared epitope.

OBJECTIVES: Neutrophils play a major role in rheumatoid arthritis (RA) pathogenesis. We aimed to evaluate if neutrophil DNA damage in RA patients is associated with the disease activity, autoantibodies status, carriage of the RA shared epitope (SE) and treatment. METHODS: DNA damage was assessed by alkaline comet assay in peripheral blood (77 patients and 55 healthy controls) and in 10 RA synovial fluid neutrophils. Evaluation of the respiratory burst of 30 patients with RA and 30 healthy controls was done. RESULTS: Compared to controls, RA patients exhibited increased neutrophil DNA damage. RA synovial fluid cells DNA damage was increased when compared to OA synovial fluids cells. In addition, our study shows that anti-TNF-α therapy reduces the frequency of DNA damage. Patients with simple or double dose of shared epitope presented a higher frequency of DNA damage compared to patients without the allele. Positive correlation was found between neutrophil DNA damage and DAS-28 and ROS production. CONCLUSIONS: Our results suggest that an increase of respiratory burst of neutrophils reflects the higher levels of DNA damage in neutrophils and a positive correlation between DNA damage and disease activity shows the importance of oxidative stress in the pathogenesis of RA.

HLA-G 3' untranslated region polymorphic sites associated with increased HLA-G production are more frequent in patients exhibiting differentiated thyroid tumours.

BACKGROUND: HLA-G is a nonclassical class I histocompatibility molecule implicated on the immune escape mechanism of tumour cells. We evaluated the genetic diversity of HLA-G 3' untranslated region (3'UTR) and associated polymorphic sites with clinical presentation and with the magnitude of HLA-G thyroid expression. PATIENTS AND METHODS: Polymorphic sites at 3'UTR (14bpINS/DEL, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142C/G, +3187A/G, +3196C/G) were characterized by sequencing analyses in blood samples of 72 patients exhibiting papillary thyroid carcinoma (PTC), 22 follicular thyroid carcinomas (FTC), 19 follicular adenomas (FA), 21 colloid goitres and 156 healthy controls. RESULTS: Compared to goitre and/or controls, patients with PTC exhibited higher frequency of 14bpDEL (P = 0·030), +3010G (P = 0·034), +3010CG (P = 0·044), +3142CG (P = 0·040), +3035C (P = 0·050) and +3187GG (P = 0·032). Patients with FTC presented higher frequency of 14bpINS/DEL (P = 0·020). The UTR-5 haplotype was underrepresented in PTC (P = 0·050). The +3003TT was more frequent in patients with PTC older than 45 years (P = 0·030). Male patients had a higher frequency of +3196GG (P = 0·040). Tumour multicentricity was associated with UTR-2 (P = 0·030). The following associations were observed in PTC and FTC combined: i) tumour size <2 cm with 14bpINS/INS (P = 0·030); ii) multicentricity with +3035CC (P = 0·030) and +3196GG (P = 0·030); iii) decreased thyroid HLA-G expression with +3196C and +3196CC; and iv) moderate HLA-G thyroid staining with UTR-2. CONCLUSIONS: HLA-G 3'UTR polymorphisms associated with a greater magnitude of HLA-G production were associated with differentiated thyroid tumours and with variables implicated in poor prognosis. These findings corroborate the unfavourable role of HLA-G in thyroid cancer.

HLA-G 3'-untranslated region polymorphisms are associated with HTLV-1 infection, proviral load and HTLV-associated myelopathy/tropical spastic paraparesis development.

Most human T-lymphotropic virus type 1 (HTLV-1)-infected patients remain asymptomatic throughout life. The factors associated with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development have not been fully elucidated; immunological and genetic factors may be involved. The association of 14 bp INS/DEL HLA-G polymorphism with HTLV-1 infection susceptibility has been reported previously. Here, other polymorphic sites at the HLA-G 3'-UTR (14-bp D/I, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142C/G, +3187A/G and +3196C/G) were evaluated in 37 HTLV-1-infected individuals exhibiting HAM/TSP, 45 HTLV-1 asymptomatic carriers (HAC) and 153 uninfected individuals, followed up at University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil. It was observed that: (i) 14bpDI genotype is a risk factor for HTLV-1 infection, while the 14bpDD and +3142CC genotypes were associated with protection against infection; (ii) the +3142C allele and the +3003CT and +3142CC genotypes were associated with susceptibility, while 14bpII and +3003TT genotypes were associated with protection against HAM/TSP development; and (iii) the 14bpII, +3010CC, +3142GG and +3187AA genotypes were associated with lower HTLV-1 proviral load compared to respective counterpart genotypes. Findings that HLA-G has a well-recognized immunomodulatory role and that the genetic variability at HLA-G 3'-UTR may post-transcriptionally modify HLA-G production indicate a differential genetic susceptibility to: (i) the development of HTLV-1 infection, (ii) the magnitude of HTLV-1 proviral load and (iii) HAM/TSP development.

Bothrops jararaca and Bothrops erythromelas Snake Venoms Promote Cell Cycle Arrest and Induce Apoptosis via the Mitochondrial Depolarization of Cervical Cancer Cells.

Bothrops jararaca (BJ) and Bothrops erythromelas (BE) are viper snakes found in South-Southeast and Northeast regions of Brazil, respectively. Snake venoms are bioactive neurotoxic substances synthesized and stored by venom glands, with different physiological and pharmacological effects, recently suggesting a possible preference for targets in cancer cells; however, mechanisms of snakes have been little studied. Here, we investigated the mechanism responsible for snake crude venoms toxicity in cultured cervical cancer cells SiHa and HeLa. We show that BJ and BE snake crude venoms exert cytotoxic effects to these cells. The percentage of apoptotic cells and cell cycle analysis and cell proliferation were assessed by flow cytometry and MTT assay. Detection of mitochondrial membrane potential (Rhodamine-123), nuclei morphological change, and DNA fragmentation were examined by staining with DAPI. The results showed that both the BJ and BE venoms were capable of inhibiting tumor cell proliferation, promoting cytotoxicity and death by apoptosis of target SiHa and HeLa cells when treated with BJ and BE venoms. Furthermore, data revealed that both BJ venoms in SiHa cell promoted nuclear condensation, fragmentation, and formation of apoptotic bodies by DAPI assay, mitochondrial damage by Rhodamine-123, and cell cycle block in the G1-G0 phase. BJ and BE venoms present anticancer potential, suggesting that both Bothrops venoms could be used as prototypes for the development of new therapies.

Patients with systemic sclerosis present increased DNA damage differentially associated with DNA repair gene polymorphisms.

OBJECTIVE: Patients with systemic sclerosis (SSc) exhibit increased toxicity when exposed to genotoxic agents. In our study, we evaluated DNA damage and polymorphic sites in 2 DNA repair genes (XRCC1 Arg399Gln and XRCC4 Ile401Thr) in patients with SSc. METHODS: A total of 177 patients were studied for DNA repair gene polymorphisms. Fifty-six of them were also evaluated for DNA damage in peripheral blood cells using the comet assay. RESULTS: Compared to controls, the patients as a whole or stratified into major clinical variants (limited or diffuse skin involvement), irrespective of the underlying treatment schedule, exhibited increased DNA damage. XRCC1 (rs: 25487) and XRCC4 (rs: 28360135) allele and genotype frequencies observed in patients with SSc were not significantly different from those observed in controls; however, the XRCC1 Arg399Gln allele was associated with increased DNA damage only in healthy controls and the XRCC4 Ile401Thr allele was associated with increased DNA damage in both patients and controls. Further, the XRCC1 Arg399Gln allele was associated with the presence of antinuclear antibody and anticentromere antibody. No association was observed between these DNA repair gene polymorphic sites and clinical features of patients with SSc. CONCLUSION: These results corroborate the presence of genomic instability in SSc peripheral blood cells, as evaluated by increased DNA damage, and show that polymorphic sites of the XRCC1 and XRCC4 DNA repair genes may differentially influence DNA damage and the development of autoantibodies.

The variant of CD11b, rs1143679 within ITGAM, is associated with systemic lupus erythematosus and clinical manifestations in Brazilian patients.

BACKGROUND/AIMS: Immune responses mediated by complement receptors (CR) are impaired in patients with systemic lupus erythematosus (SLE). Regarding CR3 (CD11b/CD18), the CD11b subunit is encoded by the ITGAM gene and a single nucleotide polymorphism (G230A; rs1143679) in ITGAM changes an arginine to a histidine at position 77 (R77H). We assessed whether the variant R77H, rs1143679 within ITGAM, is associated with the risk to developing SLE and the clinical manifestations of Brazilian SLE patients. METHODS: The rs1143679 was genotyped by SSP-PCR in 157 patients with SLE and 147 healthy individuals. Clinical and laboratorial manifestations were obtained from the official medical records according the criteria of the American College of Rheumatology. RESULTS: The 77H variant was associated with susceptibility to SLE (OR=1.8); the frequencies of the minor allele A were 0.25 (SLE) and 0.15 (healthy) (p<0.01). In addition, the minor allele A was associated with lupus nephritis (p=0.02) and antiphospholipid antibodies (p=0.04). CONCLUSION: These results showed that the rs1143679 variant is also associated with the risk to SLE in our population and with the risk to specific clinical manifestations, as nephritis and presence of antiphospholipid antibodies. These results may have implications for discussing the association of this polymorphism with the IC deposition in SLE.

Polymorphic sites at the 3' untranslated region of the HLA-G gene are associated with differential hla-g soluble levels in the Brazilian and French population.

HLA-G molecule has well-recognized tolerogenic properties, and the encoding gene shows lower frequency of polymorphism at the coding region but higher variability at regulatory 5' and 3' untranslated (3'UTR) regions. At least three 3'UTR polymorphic sites have been associated with HLA-G mRNA regulation, including the 14 base pair (14bp) Insertion/Deletion, +3142C-G and +3187A-G. We studied the association of polymorphic sites at 3'UTR (sequencing analysis, encompassing the 14bp Ins-Del/+3003T-C/+3010C-G/+3027C-A/+3035C-T/+3142C-G/+3187A-G/+3196C-G polymorphic sites) with plasma soluble HLA-G levels (sHLA-G, detected by ELISA) in 187 French and 153 Brazilian healthy individuals. Allele and genotype frequencies were closely similar in both populations; however, Brazilians showed a higher HLA-G 3'UTR haplotype diversity. Considering sHLA-G levels in both populations altogether, individuals presenting 14bp Del/Del showed higher levels compared to 14bpIns/Ins genotype (P <0.05); those presenting +3010C/G showed higher levels compared to the +3010C-C genotype (P< 0.05); those presenting +3027C-C showed higher levels than the +3027A-A genotype (P< 0.05); and those bearing +3035C-C showed higher levels compared to the +3035C-T (P < 0.01) and +3035T-T (P < 0.05) genotypes. The analyses of 3'UTR haplotypes showed that UTR-1 (DelTGCCCGC) was associated with higher expression of sHLA-G, whereas UTR-5 (InsTCCTGAC) and UTR-7 (InsTCATGAC) with lower expression and other UTRs (UTR-2/3/4/6) exhibited intermediate levels. Since the differential expression of HLA-G may be beneficial or harmful depending on the underlying condition, the identification of individuals genetically programmed to differentially express HLA-G may help on defining novel strategies to control the immune response against the underlying disorder.

Insights on the HLA-G evolutionary history provided by a nearby Alu insertion.

The AluyHG element belongs to the AluYb8 subfamily. It is a polymorphic insertion, located approximately 20 kb from the HLA-G 3'-untranslated region (3'-UTR), which has been used for evolution studies because it exhibits identity for descendants and it is still polymorphic in the human genome. To understand the evolutionary mechanisms acting on HLA-G, we evaluated the presence or absence of the AluyHG element, associating this variable site with others observed at HLA-G coding, 3'-UTR, or both regions in four distinct populations (Brazilian, French, Congolese, and Senegalese). The results were compared with the 1000Genomes Consortium data. The worldwide AluyHG frequencies showed an increment, starting lower in Africa and increasing following distance and time of human dispersion out of Africa. The same haplotype pattern was observed in all populations, indicating that most of the HLA-G haplotypes already detected were originated earlier in Africa, before Homo sapiens dispersion. The AluyHG insertion was associated with the G*01:01:01:01/UTR-1 haplotype, with rare recombinants. Despite its high frequency in worldwide populations, the G*01:01:01:01/UTR-1 haplotype should be very recent. The low frequency of recombinants indicates that the rate of recombination at the HLA-G gene is very low.

HLA-G expression in the skin of patients with systemic sclerosis.

OBJECTIVE: To determine HLA-G expression in skin biopsies from patients with systemic sclerosis (SSc), and its association with epidemiological, clinical, and laboratory variables and survival. METHODS: Paraffin-embedded skin biopsies obtained from 21 SSc patients (14 limited SSc, 7 diffuse SSc) and from 28 healthy controls were studied. HLA-G expression was evaluated by immunohistochemistry. RESULTS: HLA-G molecules were detected in 57% of skin biopsies from patients with SSc (9 from limited SSc, 3 from diffuse SSc), whereas no control sample expressed HLA-G (p=0.000004). In patients, HLA-G molecules were consistently observed within epidermal and some dermal cells. HLA-G expression was associated with a lower frequency of vascular cutaneous ulcers (p=0.0004), telangiectasias (p=0.008), and inflammatory polyarthralgia (p=0.02). After a 15-year followup, SSc patients who exhibited HLA-G survived longer than patients who did not. CONCLUSION: HLA-G is expressed in skin biopsies from patients with SSc, and this is associated with a better disease prognosis. This suggests a modulatory role of HLA-G in SSc, as observed in other skin disorders.