Clinical characteristics and frequency of TLR4 polymorphisms in Brazilian patients with ankylosing spondylitis.

OBJECTIVES: Innate immunity is involved in the physiopathology of ankylosing spondylitis (AS), with the participation of Gram-negative bacteria, modulation of human leukocyte antigen (HLA) B27 and the involvement of pattern recognition receptors, such as Toll-like receptors (TLRs). The aim of this study was to investigate the clinical characteristics and frequency of TLR4 polymorphisms (Asp299Gly and Thr 399Ile) in a cohort of Brazilian patients with AS. METHODS: A cross-sectional study was carried out involving 200 patients with a diagnosis of AS and a healthy control group of 200 individuals. Disease activity, severity and functional capacity were measured. The study of TLR4 polymorphisms was performed using the restriction fragment length polymorphism method. HLA-B27 was analyzed by conventional polymerase chain reaction. The IBM SPSS Statistics 20 program was used for the statistical analysis, with p-values less than 0.05 considered significant. RESULTS: Mean age and disease duration were 43.1±12.7 and 16.6±9.2 years, respectively. The sample was predominantly male (71%) and non-Caucasian (52%). A total of 66% of the group of patients were positive for HLA-B27. The sample of patients was characterized by moderate functional impairment and a high degree of disease activity. No significant association was found between the two TLR4 polymorphisms and susceptibility to AS. CONCLUSIONS: TLR4 polymorphisms 399 and 299 were not more frequent in patients with AS in comparison to the health controls and none of the clinical variables were associated with these polymorphisms.

Clinical characteristics and frequency of TLR4 polymorphisms in Brazilian patients with ankylosing spondylitis / Características clínicas e frequência de polimorfismos em TLR4 em pacientes brasileiros com espondilite anquilosante

ABSTRACT Objectives: Innate immunity is involved in the physiopathology of ankylosing spondylitis (AS), with the participation of Gram-negative bacteria, modulation of human leukocyte antigen (HLA) B27 and the involvement of pattern recognition receptors, such as Toll-like receptors (TLRs). The aim of this study was to investigate the clinical characteristics and frequency of TLR4 polymorphisms (Asp299Gly and Thr 399Ile) in a cohort of Brazilian patients with AS. Methods: A cross-sectional study was carried out involving 200 patients with a diagnosis of AS and a healthy control group of 200 individuals. Disease activity, severity and functional capacity were measured. The study of TLR4 polymorphisms was performed using the restriction fragment length polymorphism method. HLA-B27 was analyzed by conventional polymerase chain reaction. The IBM SPSS Statistics 20 program was used for the statistical analysis, with p-values less than 0.05 considered significant. Results: Mean age and disease duration were 43.1 ± 12.7 and 16.6 ± 9.2 years, respectively. The sample was predominantly male (71%) and non-Caucasian (52%). A total of 66% of the group of patients were positive for HLA-B27. The sample of patients was characterized by moderate functional impairment and a high degree of disease activity. No significant association was found between the two TLR4 polymorphisms and susceptibility to AS. Conclusions: TLR4 polymorphisms 399 and 299 were not more frequent in patients with AS in comparison to the health controls and none of the clinical variables were associated with these polymorphisms.

RESUMO Objetivos: A imunidade inata está envolvida na fisiopatologia da espondilite anquilosante (EA), com a participação de bactérias gram-negativas, modulação do antígeno leucocitário humano (HLA) B27 e o envolvimento de receptores de reconhecimento de padrões, como os receptores Toll-like (TLR). O objetivo deste estudo foi investigar as características clínicas e a frequência de polimorfismos em TLR4 (Asp299Gly e Thr399Ile) em uma coorte de pacientes brasileiros com EA. Métodos: Fez-se um estudo transversal que envolveu 200 pacientes com diagnóstico de EA e um grupo controle saudável de 200 indivíduos. Mediram-se a atividade da doença, a gravidade e a capacidade funcional. O estudo dos polimorfismos em TLR4 foi feito com o método de polimorfismo de fragmentos de restrição. O HLA-B27 foi analisado por reação em cadeia da polimerase convencional. Usou-se o programa SPSS Statistics 20 da IBM para a análise estatística e foram considerados significativos valores de p inferiores a 0,05. Resultados: A média de idade e a duração da doença foram de 43,1 ± 12,7 e 16,6 ± 9,2 anos, respectivamente. A amostra foi predominantemente do sexo masculino (71%) e de não brancos (52%). Do grupo de pacientes 66% eram HLA-B27 positivos. A amostra de pacientes foi caracterizada por uma alteração funcional moderada e um elevado grau de atividade da doença. Não foi encontrada associação estatisticamente significativa entre os polimorfismos em TLR4 e a susceptibilidade à EA. Conclusões: Os polimorfismos em TLR4 399 e 299 não foram mais frequentes em pacientes com EA em comparação com controles saudáveis e nenhuma das variáveis clínicas esteve associada a esses polimorfismos.

Clinical impact of an angiotensin I-converting enzyme insertion/deletion and kinin B2 receptor +9/-9 polymorphisms in the prognosis of renal transplantation.

There is a consensus in the scientific literature that supports the importance of the kallikrein kinin and renin angiotensin systems in renal physiology, but few studies have investigated their importance after renal transplantation. The aim of this study was to investigate the clinical effects of the insertion/deletion polymorphism in the angiotensin I-converting enzyme (ACE) gene and the +9/-9 polymorphism in the kinin B2 receptor (B2R) gene in kidney-transplanted patients (n=215 ACE, n=203 B2R) compared with 443 healthy individuals. Demographic results showed that there is a higher frequency of the D allele (high plasma ACE activity) and +9 allele (lower B2R expression) in transplant patients compared with control individuals. We also observed a higher frequency of these alleles in patients who had an elevated level of plasma creatinine. At day 7 post-transplantation, we found a higher prevalence of individuals with the DD genotype with elevated plasma creatinine level. Furthermore, individuals with the DD genotype had a higher chronic allograft dysfunction and graft loss compared with the II patient genotype, which showed no loss of graft. Taken together, our data suggest that the DD genotype is an indicator of an unfavorable prognosis following renal transplantation and could be related to kinin modulation.

Detection of chicken anemia virus and infectious bursal disease virus co-infection in broilers / Detecção do virus da anemia das galinhas em coinfecção com o vírus doença infecciosa bursal em frangos

This survey aimed to investigate chicken anemia virus (CAV) in broilers flocks experimenting retarded growth and increasing mortality since the fourth day of age. Clinically, chickens presented depression, paleness, depigmentation and retarded growth. At necropsy, chickens presented CAV-compatible lesions. Samples from liver, spleen and thymus were tested by PCR for a 675-bp fragment of the CAV VP-1 gene, and all tested samples were positive. Serological and molecular techniques did not detect other pathogens, such as adenovirus, reovirus, astrovirus, infectious bursal disease and avian infectious bronchitis virus. These results showed that chicken anemia virus (CAV) may occur since the first few days of life in broilers - a fact not as yet reported -, associated with high pathogenic Infectious Bursal Disease Virus (IBDV) vaccine strain may induce a persistent growth retarded for several weeks in broilers.

Este estudo investigou a manifestação do vírus da Anemia Infecciosa das Aves (VAIA) em lotes de frangos que apresentavam retardo no crescimento e aumento da mortalidade observado a partir do quarto dia de idade. Clinicamente, as aves apresentavam depresão, palidez, despigmentação e retardo de crescimento. À necropsia, as aves apresentavam lesões compatíveis com a infecção pelo vírus da Anemia infecciosa das aves (VAIA). Amostras de fígado, baço e timo foram examinadas por PCR que amplifica um frangmento de 675 pb do gene VP-1 do VAIA. Todos os órgãos examinados foram positivos para o vírus da Anemia Infecciosa das Aves. Os demais patógenos, como adenovírus, reovírus, astrovírus, vírus da doença infecciosa bursal e coronavírus aviário não foram detectados pelas diferentes técnicas laboratoriais, como sorologia, PCR ou PAGE. Os resultados mostraram que o vírus da Anemia Infecciosa das Aves (VAIA) pode manifestar-se clinicamente nos primeiros dias de vida dos frangos – um fato ainda não reportado – associado ao vírus vacinal da doença infecciosa bursal (DIB) cepa forte pode induzir um persistente retardo de crescimento, por várias semanas, em frangos.

Expression of TLR-4 and -2 in peripheral mononuclear cells in renal transplant patients with TLR-4 gene polymorphism.

INTRODUCTION: TLR-4 has also been identified as a receptor for endogenous alarmins, which are increased post transplantation. TLR-4 has also been associated with a polymorphism that could impact graft outcome. OBJECTIVE: To assess the expression of TLR-4 in kidney transplant patients carrying or not a polymorphism. METHODS: TLR-4 polymorphism (A299G/T399I) was studied in 200 renal transplant patients. Healthy volunteers were also enrolled as control group. The polymorphism analysis was performed using restriction enzymes technique (RFLP). Functionality of TLR-4 polymorphism was assessed in samples from controls by quantification of TNF-α after LPS stimulus. TLR-4 and -2 expressions were also analyzed by flow cytometry. RESULTS: TLR-4 polymorphism was present in 8.5% of renal transplant patients. This polymorphism was associated with impairment in TNF-α secretion. In general, in renal transplant patients, TLR-4 expression in monocytes and in neutrophils was lower than in health volunteers. TLR-2 and TLR-4 expressions in healthy volunteers with A299G/T399I TLR-4 polymorphism was higher than in wild-type genotype healthy volunteers (p<0.01 and p<0.05, respectively), and also higher than A299G/T399I TLR-4 polymorphism renal transplant patients (p<0.05). TLR-2 expression on neutrophils in wild-type genotype renal transplant patients was higher compared to wild-type genotype healthy volunteers, and was also higher in relation to A299G/T399I kidney transplanted patients (p<0.01). CONCLUSION: Stable renal transplant patients with TLR-4 polymorphism have a lower expression of TLR-4 and TLR-2 receptors in peripheral mononuclear cells, which ultimately indicate a less responsiveness for alarmins.

Toll-like receptors-related genes in kidney transplant patients with chronic allograft nephropathy and acute rejection.

INTRODUCTION: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. METHODS: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. RESULTS: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. CONCLUSION: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN.

Clinical correlations of human cytomegalovirus strains and viral load in kidney transplant recipients.

Little is known about clinical differences associated with cytomegalovirus (CMV) infection by distinct strains in renal transplant patients. Different clinical pictures may be associated with specific viral genotypes, viral load, as well as host factors. The objective of this study was to identify CMV strains to determine viral load (antigenemia), and their correlation with clinical data in renal transplant recipients. Seventy-one patients were enrolled, comprising 91 samples. After selection, polymorphonuclear cells were used to amplify and sequence the gB region of CMV DNA. The sequences were analyzed to ascertain the frequency of different genotypes. Additionally, the results of this study showed that the gB coding gene presents a great variability, revealing a variety of patterns: classical gB1 (1.4%), gB1V (46.4%), classical gB2 (35.2%), gB2V (2.8%), gB3 (1.4%), classical gB4 (4.9%) and gB4V (4.9%). The mean viral load in kidney transplant patient was 75.1 positive cells (1-1000). A higher viral load was observed in patients with genotype 4 infection. Statistically significant differences were detected between gB1 and gB4 (p=0.010), and between gB2 and gB4 (p=0.021). The average numbers of positive cells in relation to clinical presentation were: 34.5 in asymptomatic, 49.5 in CMV associated syndrome and 120.7 in patients with invasive disease (p=0.048). As a group, gB1 was the most frequent strain and revealed a potential risk for developing invasive disease. Viral load also seemed to be important as a marker associated with clinical presentation of the disease.

The use of sirolimus in ganciclovir-resistant cytomegalovirus infections in renal transplant recipients.

BACKGROUND: The widespread use of prophylactic ganciclovir and anti-lymphocyte/thymocyte therapies are associated with increased induction of ganciclovir-resistant cytomegalovirus (CMV) strains. The use of sirolimus has been associated with a lower incidence of CMV infection in transplant recipients. We questioned whether it could also be effective as a therapeutic treatment of resistant CMV infection. METHODS: Patients with ganciclovir-resistant CMV infections determined clinically and by DNA sequencing analysis were enrolled. Antigenaemia and DNA sequencing were used to diagnosis and follow the mutations. RESULTS: Nine transplant patients were given sirolimus plus mycophenolate mofetil (n = 4) or a calcineurin inhibitor (n = 5). Seven out of nine recipients were CMV IgG negative before transplantation. We observed a rapid decrease in antigenaemia levels, reaching zero in eight out of nine (88.9%) patients within a median of 20.3 +/- 10.1 d. Graft function remained stable and no patient presented acute rejection or recurrence of the CMV infection. CONCLUSIONS: This suggests that the use of sirolimus plus ganciclovir therapy could be useful in ganciclovir-resistant CMV infections.

Tim-3 expression in human kidney allografts.

BACKGROUND: Tim-3 was recently described as a Th1-specific molecule, participating in the regulation of immune responses and in the induction of allograft tolerance. Here, we studied Tim-3 mRNA expression together with molecular markers of T-cell activation and cytotoxicity, in rejected human kidney grafts. METHODS: Twenty human kidney grafts that had undergone nephrectomy due to an irreversible acute rejection episode were studied. We quantified intragraft expression of Tim-3, granzyme B, perforin, IFN-gamma and Fas-ligand mRNA by real-time RT-PCR, with probes and primers TaqMan. Protocol biopsies were studied as controls. Statistical analyses were performed to compare groups, and to investigate the potential association with gene transcripts measures and rejection. RESULTS: All molecules studied were up-regulated in the rejection group compared with controls (p<0.001). Acute rejection type III (Banff 97) profiles were associated with higher values, where granzyme B and perforin presented the highest (5672.51+/-9002.16 and 1866.59+/-2426.38, respectively) and Tim-3 had the lowest ones (166.62+/-174.94). Tim-3 had also a lower expression in those patients that did not respond to anti-rejection therapy. There was a positive correlation between Tim-3 and IFN-gamma mRNA expression levels (r(2)=0.73; p<0.001). CONCLUSIONS: Our results corroborate the concept that acute rejection is an active process, where inflammatory as well as regulatory factors have their roles. Severe episodes of acute rejection were associated with higher expression of cytotoxic molecules and lower expression of potential regulatory molecule.

Genome sequencing analysis of Brazilian chicken anemia virus isolates that lack MSB-1 cell culture tropism.

Specific amino acid (aa) substitutions in VP1, VP2 and VP3 genes were reported as a distinctive feature of the American CIA-1 strain, characterized as having a variable rate of growth and tropism for different MSB-1 cell sublines [Renshaw RW, Soiné C, Weinkle T, O'Connell PH, Ohashi K, Watson S, et al. A hypervariable region in VP1 of chicken anemia virus mediates rate of spread and cell tropism in tissue culture. J Virol 1996;70(12):8872-8]. DNA sequencing of 878 nucleotides from twelve Brazilian CAV, eight of which tested for in vitro isolation in three different sources of MDCC-MSB1 cell line and identified as lacking capacity to propagate in any of these cells, were compared to sequence data available for CAV strains propagated or not in cell culture. Alignment of the deduced aa resulted in a lack of singled out amino acid substitutions in the partial genomic sequences of Brazilian isolates that would entirely contrast them to viruses propagated in MSB-1 cells, indicating that the combined VP1, VP2 and VP3 substitutions observed may not entirely account as sole determinants of CAV isolation and propagation in MDCC-MSB-1 cells.

The emergence of cytomegalovirus resistance to ganciclovir therapy in kidney transplant recipients.

Transplant recipients that have not been previously exposed to the cytomegalovirus (CMV) are highly susceptible to viral diseases while under immunosuppression therapy. CMV disease requires prolonged therapy, facilitating the emergence of resistant strains. Persistence of positive antigenemia represents clinical evidence of the presence of resistant strains, although its frequency is unknown. These strains may present amino acid deletions or substitutions in conserved regions of the UL97 protein, point mutations in the DNA polymerase (UL54), or both. In this study we aimed to analyze the prevalence of mutations associated with ganciclovir resistance in transplant recipients. Fifteen kidney transplant recipients and four kidney-pancreas transplant recipients, with a positive and oscillating CMV viremia detected by sequential antigenemia test, were enrolled. The UL97 gene was amplified by Nested-PCR and enzymatically digested in samples of these patients in order to detect mutations in the most common codons, such as 460 (M460V), 594 (A594V) and 595(L595S/F). The end-product fragments were further sequenced. Nine (47.4%) out of 19 patients presented with mutations in UL97 at codons L595S (55.6%), A594V (11.1%), A595F/A594V (11.1%) and L595S/A594V (22.2%). None presented with mutation at the M460V codon. Renal transplant patients with oscillation in viral load for more than 2 weeks might have developed viral resistance to anti-drug therapy. Its detection might aid physicians in their clinical plan of tapering the patient's immunosuppression.

Análise molecuar de cepas de citomegalovírus provenientes de pacientes transplantados / Molecular analysis of cytomegalovirus strains from transplant recipient patient

A infecçao pelo citomegalovírus é uma importante complicaçao em pacientes transplantados em geral, e uma causa comum de morbidade e mortalidade em outros pacientes imunocomprometidos. Com o aumento do uso de antiviral para o tratamento de infecçoes por HCMV tem sido relatado a emergência de vírus resistentes, dificultando a terapêutica. A resistência a drogas pode ser resultado de deleçao ou inserçao de nucleotídeos num gene viral, na quinase que monofosforila a droga ou uma mutaçao pontual no gene UL54 que codifica para a DNA polimerase viral. Mutaçoes na DNA polimerase viral podem coexistir com mutaçoes na UL97, resultando em dupla infecçao a drogas. A glicoproteína B é abundantemente expressa na superfície dos vírions e células infectadas. Diferentes cepas, poderiam variar em virulência. Como pouco se sabe sobre as diferenças funcionais que podem existir entre as diversas cepas de HCMV, os objetivos deste trabalho foram; 1) identificar cepas de HCMV em pacientes transplantados; 2) análisar resistência em alguns pacientes. Foram estudados pacientes submetidos a transplante no período de Janeiro de 1999 a Maio de 2001. A identificaçao da infecçao pelo HCMV foi realizada através do ensaio de antigenemia. Foram estudados 86 pacientes que geraram 109 amostras, sendo que 04 amostras eram negativas. As amostras separadas para o estudo foram amplificadas por Nested-PCR para as três regioes estudadas: gB, UL54 e UL97 e depois seqüênciadas. A regiao do gene da gB foi estudada com o objetivo de analisar genotipicamente as amostras. Para a análise de resistência foram estudadas as regioes da DNA polimerase (UL54 - que foi dividida em duas partes), e UL97. Após as amostras serem seqüênciadas, foram alinhadas e analisadas por programas de análise filogenéticas. Das 109 amostras estudadas para a regiao da gB, 0,9 por cento pertencia ao genótipo gB1, 47,7 por cento ao genótipo gBlV, 36,8 por cento ao genótipo gB2, 1,9 por cento ao gB2V, 0,9 por cento ao genótipo gB3, 4,8 por cento ao genótipo gB4 e 7,3 por cento ao genótipo gB4V. 17 pacientes tiveram mais de uma amostra analisada. 29,4 por cento apresentaram diferentes cepas em amostras diferentes, e 17,6 por cento apresentaram mais de uma cepa ao mesmo tempo. Foram estudadas entre 14, 17 e 15 amostras...(au)

Nutritional assessment and surgical risk makers in children submitted to cardiac surgery

Introduçäo e Objetivos: A desnutriçäo é freqüente em crianças cardiopatas e pode associar-se a uma maior morbidade no período pós-operatório. Com o objetivo de avaliar o estado nutricional de crianças portadoras de cardiopatias congênitas e o papel dos parâmetros de avaliaçäo nutricional em predizer complicaçöes pós-operatórias. Material e Métodos: Foram estudadas 50 crianças admitidas para cirurgia cardíaca eletiva, classificadas como de alto ou de baixo risco cirúrgico. Antes da cirurgia cada paciente era submetido a uma avaliaçäo nutricional, compreendendo parâmetros antropométricos e dosagem das proteínas plasmáticas. Resultados: A prevalência global de desnutriçäo de acordo com o critério de Waterlow foi de 78 por cento sendo 90 por cento no grupo de alto risco e 60 por cento no de baixo risco cirúrgico. Nas crianças de alto risco cirúrgico, medidas antropométricas do braço situadas abaixo do percentil 5 mostraram associaçäo significante com complicaçöes pós-operatórias gerais (circunferência braquial p=0,0019); circunferência muscular braquial, p=0,0419). A relaçäo de peso esperado para a estatura e as concentraçöes séricas de albumina e de transferrina näo tiveram papel prognóstico para morbidade pós-cirúrgica. O valor médio de pré-albumina foi significantemente inferior nas crianças de alto risco que desenvolveram infecçäo no pós-operatório quando comparado ao das que näo tiveram infecçäo (p < 0,01). Conclusäo: Embora a classificaçäo de risco nutricional tenha se mostrado um bom método para identificar subgrupos de pacientes com um risco adicional de morbidade pós-operatória, säo necessários testes mais sensíveis e específicos que permitam identificar individualmente essas crianças