Putative role of HLA polymorphism among a Brazilian HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) population.

Around ten million people are infected with HTLV-1 worldwide, and 1-4% develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), characterized by an important degeneration of the spinal cord, which can lead to death. Distinct HLA alleles have been associated with either HAM/TSP susceptibility or protection. However, these HLA alleles set may change according to the population studied. Brazil is the second country in the number of HTLV-1-infected people and there are few reports addressing the HLA influence on HTLV-1 infection as well as on disease outcome. The objective of this study was to evaluate the influence of HLA alleles as a risk factor for HAM/TSP and the proviral load (PVL) levels, clinical progression, and death outcomes in an admixed Brazilian population. The HLA-A, -B, -C, and -DRB1 were genotyped in 375 unrelated HTLV-1-infected individuals divided into asymptomatic carriers (AC) (n = 165) and HAM/TSP (n = 210) in a longitudinal cohort from 8 to 22 years of follow-up. Because locus B deviated from Hardy-Weinberg Equilibrium for the study groups, the results represented for HLA-B alleles were inconclusive. The alleles HLA-A*68 and -C*07 were related to HAM/TSP risk in multivariate analysis. The alleles HLA-A*33, and -A*36 were associated with protection against disease progression in HAM/TSP patients, while -C*12, -C*14, and -DRB1*08 were associated with increased risk of death. In the AC group, the presence of, -C*06 and -DRB1*15 alleles influenced an increased PVL, in an adjusted linear regression model, while -A*30, -A*34, -C*06, -C*17 and -DRB1*09 alleles were associated with increased PVL in HAM/TSP group compared to HAM/TSP individuals not carrying these alleles. All these alleles were also related to increased PVL associated with clinical progression outcome. Increased PVL associated with the death outcome was linked to the presence of HLA-A*30. PVL has been associated with HLA, and several alleles were related in AC and HAM/TSP patients with or without interacting with clinical progression outcomes. Understanding the prognostic value of HLA in HAM/TSP pathogenesis can provide important biomarkers tools to improve clinical management and contribute to the discovery of new therapeutic interventions.

Lack of association between single-nucleotide polymorphisms of pro- and anti-inflammatory cytokines and HTLV-1-associated myelopathy / tropical spastic paraparesis development in patients from Rio de Janeiro, Brazil.

BACKGROUND: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive neurological and inflammatory disease, associated with HTLV-1 infection. HAM/TSP neurological disease is a consequence of an inflammatory reaction, and adaptive immune responses, through the secretion of anti-inflammatory and pro-inflammatory cytokines, play an important role in the outcome of infection and disease progression. Studies addressing the association between cytokines functional single nucleotide polymorphisms and HAM/TSP development are scarce. METHODS: The genetic polymorphisms of cytokine genes were evaluated in HAM/TSP patients (n = 68) and in asymptomatic HTLV-1 positive carriers (n = 83) from Rio de Janeiro, Brazil, in a case-control study. HTLV-1 infected patients were genotyped for SNPs in five cytokine genes: TNFA-308G/A, IL6-174G/C, IFNG + 874 T/A, TGFB at the codons + 10 T/C and + 25G/C, IL10-592C/A and -819C/T, and -1082A/G and proviral load (PVL) was quantified. Associations between genotypes, haplotypes, clinical outcome and pro viral load were evaluated. RESULTS: Lack of association between the cytokine polymorphisms and disease outcome was observed. The genotypes TNFA-308GG, IL6-174GG/GC, IL10-592AA and -819CC and TGFb1 high producers phenotypes were correlated with higher PVL in HAM/TSP patients versus asymptomatic carriers. CONCLUSIONS: We did not observe association between cytokine polymorphisms and risk for HAM/TSP development in Brazilian HTLV-1 infected individuals, regardless of differences in PVL between HAM/TSP versus asymptomatic carriers in specific cytokine polymorphisms.

Estudo da influência dos alelos do HLA classe I e II e do polimorfismo dos genes de citocinas na infecção pelo HTLV-1 / Study of the influence of hla class i and ii alleles and the polymorphism of cytokines genes in htlv-1 infection

O vírus linfotrópico para células T humanas (HTLV-1) é o principal agente causador da Paraparesia Espástica Tropical / Mielopatia associada ao HTLV-1 (PET/MAH) e da Leucemia da célula T do Adulto (LTA). [...]Fatores da interação HTLV-1/ hospedeiro estão envolvidos no risco de desenvolver doença. A lesão neurológica na PET/MAH parece ser consequência de uma reação inflamatória, desencadeada pelo reconhecimento de células infectadas por linfócitos T citotóxicos, com consequente liberação de citocinas e lesão medular. OBJETIVO: Identificar marcadores genéticos, que possam ajudar no prognóstico e tratamento dos pacientes portadores do HTLV-1. MÉTODOS: Nas amostras de 117 portadores do HTLV-1 assintomáticos e 171 pacientes com acometimento neurológico em acompanhamento na cidade do Rio de Janeiro, foram realizadas as tipificações dos genes do HLA Classe I e II, dos polimorfismos dos genes das citocinas -308TNF-α,-174IL-6, +874IFN-γ, códon 10 e 25TGF-β1 e -1082 - 819-592IL-10, e a quantificação da carga proviral. Os dados foram organizados em um banco de dados no programa SPSS. As frequências alélicas e genotípicas foram obtidas por contagem direta. O equilíbrio de Hardy-Weinberg foi avaliado para os polimorfismos das citocinas no sitio http://bioinfo.iconcologia.net/ubbweb/SNPStats_web, em relação ao HLA foram utilizadas as ferramentas disponíveis no sítio “Los Alamos HIV database tools”. As comparações entre os grupos foram realizadas através de tabelas de contingência 2x2 (quiquadrado, exato de Fisher e odds ratios), valores de p≤0,05 foram considerados significantes...

The human T cell lymphotropic vírus (HTLV-1) is the main causingagent of Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (HAM/TSP) aswell as of Adult T Cell Leukemia (ATL). [...] Factorsrelated to the HTLV-1/host interaction may be involved in the risk of developing thediseases. The neurological lesion in HAM/TSP may be the consequence of aninflammatory reaction, triggered by the recognition of infected cells by cytotoxic Tlymphocytes, followed by the release of cytokines and central nervous system lesion.OBJECTIVE: This work aims to identify genetic markers, which may help in theprognosis and treatment of HTLV-1 patients. Methods: The polymorphism of the HLAClass I and II genes, as well as the TNF-α, IL6, IFN-γ, TGF-β and IL-10 cytokinegenes, and the proviral load were analysed in 117 asymptomatic HTLV-1 carriersand 171 HTLV-1 symptomatic carriers from Rio de Janeiro city. Data were organizedinto a database using SPSS. The Hardy-Weinberg equilibrium was evaluated forcytokine polimorphisms using the site http://bioinfo.iconcologia.net/ubbweb/SNPStats_web. The tools available in the site “Los Alamos HIV database tools” were used toanalyze the HLA polimorphisms. Comparisons between groups were made using 2x2contingency tables (Fisher Exact test/ χ2 and odds ratios), p values p≤0,05 wereconsidered significant...

Neurological manifestations of coinfection with HIV and human T-lymphotropic virus type 1.

HIV-individuals are at risk for human T-lymphotropic virus (HTLV) coinfection and neurological diseases. Little is known about the impact of HAART among coinfected patients. In this study, 47 out of 428 HIV individuals were coinfected with HTLV (10.9%). Coinfection was an independent variable associated with neurological outcome (odds ratio 8.73). Coinfection was associated with myelopathy [chi square (X(2)) = 93, P < 0.001], peripheral neuropathy (X(2) = 6.5, P = 0.01), and hepatitis C virus infection (X(2) = 36.5, P < 0.001). HAART did not appear to protect against neurological diseases and had no impact on HTLV proviral load.

Multiplex real-time PCR for the detection and quantitation of HTLV-1 and HTLV-2 proviral load: addressing the issue of indeterminate HTLV results.

BACKGROUND: Routine diagnosis of Human T Lymphotropic virus (HTLV) infection is primarily serologically based; however the proportion of unresolved and indeterminate Western blot results range from 0.02% to 50% in endemic areas. OBJECTIVES: To validate a sensitive in-house quantitative multiplex real-time assay (mqRT-PCR), capable of detecting and quantifying HTLV-1 and HTLV-2, and use it to differentiate unresolved serological profiles, and monitor infection in HTLV-1 infected patients. STUDY DESIGN: The mqRT-PCR was designed as a single-tube assay. Quantitative results were reported as copy number of HTLV provirus per 10(6) cells and the numbers of cells were calculated based on the quantitative result for albumin, of which there are 2 copies/cell. Assay standards were amplified from HTLV-1 infected MT-2 cells and HTLV-2 transfected CEM cells. Blood samples were obtained from HTLV seropositive former blood donors. RESULTS: The mqRT-PCR assay was efficient (98.8-101.2%), reproducible (coefficient of variance<5%) and sensitive to 1 copy for HTLV-1, HTLV-2 and Albumin. The assay resolved the infection profile in 16/17 patients, with undetermined subtype, all of which were reassigned as HTLV-1 infections. In addition, the average PVL detected in patients suffering from HTLV-1 associated HAM/TSP (n=23, 13,450 copies/10(6) cells) was significantly higher than those detected in asymptomatic carriers (n=21, 6665 copies/10(6) cells). CONCLUSIONS: We propose a new testing algorithm for the laboratory diagnosis of HTLV infection, which includes HTLV specific mqRT-PCR for resolving HTLV serological results. Furthermore, quantitation of PVL load by real-time PCR may be useful in assessing the link between infection and disease, and in monitoring patients undergoing therapy.

High frequencies of functionally competent circulating Tax-specific CD8+ T cells in human T lymphotropic virus type 2 infection.

Human T lymphotropic virus type 2 (HTLV-2) is characterized by a clinically asymptomatic persistent infection in the vast majority of infected individuals. In this study, we have characterized for the first time ex vivo specific CTL responses against the HTLV-2 Tax protein. We could detect CTL responses only against a single HLA-A*0201-restricted Tax2 epitope, comprising residues 11-19 (LLYGYPVYV), among three alleles screened. Virus-specific CTLs could be detected in most evaluated subjects, with frequencies as high as 24% of circulating CD8(+) T cells. The frequency of specific CTLs had a statistically significant positive correlation with proviral load levels. The majority of virus-specific CD8(+) T cells exhibited an effector memory/terminally differentiated phenotype, expressed high levels of cytotoxicity mediators, including perforin and granzyme B, and lysed in vitro target cells pulsed with Tax2((11-19)) synthetic peptide in a dose-dependent manner. Our findings suggest that a strong, effective CTL response may control HTLV-2 viral burden and that this may be a significant factor in maintaining persistent infection and in the prevention of disease in infected individuals.

Subacute progression of human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis.

Although human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is usually described as a chronic disabling disease, a rapid course over months or even weeks has been reported in some patients. The authors describe the clinical features of HAM/TSP in a Brazilian cohort and evaluate the prevalence of patients with a subacute progression of the disease. This was defined as the requirement of a wheelchair during the first 2 years after the onset of symptoms. Patients with this subacute course and patients with the chronic clinical course were compared in terms of their HTLV-I proviral loads (PLs) using real-time polymerase chain reaction (PCR). Seven out of 88 patients (7.9%) had a subacute progression. All patients were women and 5/7 acquired HTLV-I through sexual contact. There was no significant difference in the real-time PLs between the group with subacute evolution (mean 8.5 copies/100 cells, range 6.03 to 12.09) and those patients with a typical course of disease (mean 11.34 copies/100 cells, range 0.4 to 67.72) (P = .68), suggesting that factors other than the number of infected cells are implicated in the development of such an aggressive course of disease. Early recognition of this subgroup is important because immunosuppressive treatment might be beneficial if instituted promptly.

Human T lymphotropic virus type 1 (HTLV-1) proviral load in asymptomatic carriers, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other neurological abnormalities associated with HTLV-1 infection.

Recent reports have demonstrated that human T lymphotropic virus type 1 (HTLV-1) is associated with other neurological abnormalities in addition to HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It has been well established that high HTLV-1 proviral loads are associated with the development of HAM/TSP. We now demonstrate, for the first time, to our knowledge, that HTLV-1 proviral loads in patients with other neurological abnormalities are also significantly higher than in asymptomatic HTLV-1 carriers.

Estabelecimento e caracterização de linhagens celulares infectadas pelo vírus linfotrópico de célula T humana do tipo I (HTLV-I)

O vírus linfotrópico de célula T humana do tipo I (HTLV-I) está associado etiologicamente ao desenvolvimento da leucemia da célula T do adulto (LTA) e da paraparesia espástica tropical/mielopatia associada ao HTLV-I (PET/MAH). O objetivo deste trabalho foi estabelecer linhagens celulares infectadas pelo HTLV-I e caracterizá-las quanto a: a) morfologia, b) tipo celular, c) proliferação contínua sem adição de fatores de crescimento, d) demonstrar a presença do DNA proviral integrado ao genoma das células das linhagens, e) caracterizar esta integração, f) verificar a expressão de partículas virais, g) expressão de citocinas e finalmente, h) avaliar o efeito de imunossupressores na proliferação celular. Estabelecemos três linhagens infectadas pelo HTLV-I a partir de células mononucleares de pacientes portadores de LTA. Não foi utilizada a técnica de co-cultivo com células não infectadas. As culturas apresentam células dismórficas, bi e multinucleadas com figuras de mitose, uma das linhagens manteve o fenótipo de célula T, enquanto que nas duas outras não foi possível caracterizar o tipo celular predominante, tendo havido perda de todos os marcadores de superfície, e não se conseguiu demonstrar rearranjo da cadeia g do TCR. Caracterizou-se a cepa de HTLV-I em relação à seu subtipo, sendo uma cepa Cosmopolita B, mais freqüente no Japão, local de origem do paciente doador das células e as duas outras do subtipo Cosmopolita A, cepa comum em várias regiões do Globo. Demonstrou-se haver dois clones celulares nas linhagens através do estudo do local de integração do DNA proviral, entretanto não podemos afastar a possibilidade de ser um único clone com dois DNAs provirais integrados. A expressão tanto de proteínas como de partículas virais não foi significativa, somente em uma linhagem conseguiu-se mostrar partículas virais por contrastação negativa em microscopia eletrônica. As linhagens expressam de forma constitutiva três citocinas IL-10, IL-15 e IFN-g com intensidade variável. E finalmente, as três linhagens foram resistentes à ação da Ciclosporina A. O estabelecimento destas linhagens possibilitará o estudo das vias de ativação alteradas pela infecção, assim como servirão de modelo experimental terapêutico para o ensaio in vitro de novas drogas.

The human T-cell type lymphotropic virus human type I (HTLV-I) is etiologically associated to two main diseases: a malignant one, the adult T-cell leukemia (ATL) and to a progessive myelopathy, tropical spastic paraparesis/HTLV-I- associated myelopathy (TSP/HAM). The objectives of this work were the establishment of HTLV-I-infected cell lines and the analysis of these lines in the following aspects: a) morphology, b) cellular phenotype, c) continuous proliferation without exogenous growth factors, d) presence of integrated HTLV-I DNA provirus; e) the type of integration (mono, oligo or policlonal) as well as the HTLV-I subtype, f) expression of viral proteins as well as viral particles, g) expression of cytokines; and finally h) evaliation of Cyclosporin A effect on the proliferation of the lines. We established three HTLV-I transformed cell lines. We established three HTLV-I transformed cell lines. The cells are large and dimorphic, showing two or multiple nucleus; one cell line remainde with the T cell phenotype. It was not possible to characterize the two others, due to the loss of their surface markers and, it was not possible to demonstrate the TCR g chain rearrangement. Concerning the HTLV-I subtype strain, one of the cell lines harbours the Cosmopolitan B subtype, which is more frequent in Japan, the place of origin of the cells’ donor. The other two cell lines are the Cosmopolitan A subtype, common in many regions around the world. Concerning provirus integration, the cell lines are oligoclonal. We were able to demonstrate two cellular clones in the lines by study of the local site of integration, but can not exclude the possibility to be just one clone with two proviral DNAs. The viral proteins as well as viral particles were not significantly expressed. Only one cell line showed viral particles in the negative staining technique on electronic microscopy. The three cell lines constitutively express, with variable intensity, the following citokines: IL-10, IL-15 e IFN-g. The infected cells proliferated even in the presence of 1 mg/ml of Cyclosporine A. The establishment of these cells will provide a helpful tool for the study of the activation pathways altered by the infection as well as an experimental model for therapeutic drugs assays.