Antigenic Peptides Capable of Inducing Specific Antibodies for Detection of the Major Alterations Found in Type 2B Von Willebrand Disease.

Von Willebrand disease (VWD) is an inherited hemorrhagic disorder promoted by either quantitative or qualitative defects of the von Willebrand factor (VWF). The disease represents the most common human coagulopathy afflicting 1.3% of the population. Qualitative defects are subdivided into four subtypes and classified according to the molecular dysfunction of the VWF. The differential diagnosis of the VWD is a difficult task, relying on a panel of tests aimed to assess the plasma levels and function of the VWF. Here, we propose biochemical approaches for the identification of structural variants of the VWF. A bioinformatic analysis was conducted to design seven peptides among which three were representatives of specific amino acid sequences belonging to normal VWF and four encompassed sequences found in the most common VWD subtype 2B. These peptides were used to immunize mice, after which, peptide-specific immunoglobulins were purified. This resulted in four Ig preparations capable of detecting alterations in the subtype 2B VWD plus additional three antibody fractions targeting the normal VWF. The panel of antibodies could serve many applications among them (1) assessment of VWF: antigen interaction, (2) VWF multimer analysis, and (3) production of monoclonal antibodies against VWF for therapeutic purposes as in thrombotic thrombocytopenic purpura.

Thrombospondin-1 expression in human T-lymphotropic virus 1 asymptomatic carriers and patients with HTLV-associated myelopathy/tropical spastic paraparesis / Expressão de Trombospondina-1 em indivíduos infectados pelo HTLV-1 assintomáticos e pacientes portadores da mielopatia associada ao HTLV/Paraparesia Espástica Tropical

O Vírus Linfotrópico de células T humanas tipo 1 (HTLV-1) está associado a uma mielopatia (chamada mielopatia associada ao HTLV - HAM/TSP). A trombospondina-1 (TSP-1) é uma proteína da matriz que interfere com a adesão, a motilidade, e a proliferação celular. Níveis deexpressão de RNA mensageiro (mRNA) da trombospondina-1 foram avaliados em indivíduos infectados por HTLV-1: 11 pacientes assintomáticos, 18 com mielopatia ou oligossintomáticos, e 13participantes não-infectados. O RNA de células mononucleares do sangue periférico foi submetido à análise de RT-PCR para trombospondina-1. O número de indivíduos que expressaram esta proteína foi maior no grupo com mielopatia/sintomas (14/18, p igual 0,007). Em geral, a tendência para valores mais elevados de mRNA de trombospondina-1 foi observada no grupo de infectados pelo vírus (p igual 0,062). Os níveis mais elevados de expressão do mRNA foram detectados no início dos sintomas clínicos da HAM/TSP. Estudos adicionais com maior número de amostras são necessários para elucidar melhor o papel desta proteína da matriz na rede inflamatória relacionada à HAM/TSP.

Long-term serological follow-up of blood donors with an HTLV-indeterminate western blot: antibody profile of seroconverters and individuals with false reactions.

The high proportion of indeterminate results of the screening test for human T-lymphotropic virus (HTLV) infection has been a challenge worldwide. In this study, 60 persons with seroindeterminate results for HTLV were followed until their serological status was defined. At least two independent serological tests (EIA and WB) from sequential samples were performed at an average interval of 4.4 years, totaling 141 serum samples tested. Seroconversion occurred in 12 individuals (reactive by EIA, positive by WB and PCR), and 48 were classified as false reactions (non-reactive EIA and negative PCR, but indeterminate WB). The seroconverter group had epidemiological features similar to those seen in HTLV-1 carriers, and the average time of follow-up for seroconversion was 4 years. In the group with false reactions, the most frequent indeterminate WB pattern in the samples was the presence of p24 alone. This pattern was absent in the seroconverter group, suggesting that p24 alone is an indicator of false reactivity. In contrast, the presence of p19 and p24 seems to be an indicator of true reactivity, since this pattern was frequent (66.7%) among the seroconverters and much less common (10.4% of the first samples) among the individuals with false reactions (P = 0.0001). Thus, HTLV infection may be suspected when reactivity to p19 and p24 is observed. Individuals with an indeterminate WB pattern should be followed-up and retested. The improvement of the HTLV algorithm screening of blood donors has been necessary to reduce inconclusive results and to avoid unnecessary follow-up to define the status of infection.

Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors.

INTRODUCTION: HTLV-1/2 screening among blood donors commonly utilizes an enzyme-linked immunosorbent assay (EIA), followed by a confirmatory method such as Western blot (WB) if the EIA is positive. However, this algorithm yields a high rate of inconclusive results, and is expensive. METHODS: Two qualitative real-time PCR assays were developed to detect HTLV-1 and 2, and a total of 318 samples were tested (152 blood donors, 108 asymptomatic carriers, 26 HAM/TSP patients and 30 seronegative individuals). RESULTS: The sensitivity and specificity of PCR in comparison with WB results were 99.4% and 98.5%, respectively. PCR tests were more efficient for identifying the virus type, detecting HTLV-2 infection and defining inconclusive cases. CONCLUSIONS: Because real-time PCR is sensitive and practical and costs much less than WB, this technique can be used as a confirmatory test for HTLV in blood banks, as a replacement for WB.

Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors / Análise do uso da PCR em tempo real para HTLV-1 e 2 como teste confirmatório na triagem de doadores de sangue

INTRODUCTION: HTLV-1/2 screening among blood donors commonly utilizes an enzyme-linked immunosorbent assay (EIA), followed by a confirmatory method such as Western blot (WB) if the EIA is positive. However, this algorithm yields a high rate of inconclusive results, and is expensive. METHODS: Two qualitative real-time PCR assays were developed to detect HTLV-1 and 2, and a total of 318 samples were tested (152 blood donors, 108 asymptomatic carriers, 26 HAM/TSP patients and 30 seronegative individuals). RESULTS: The sensitivity and specificity of PCR in comparison with WB results were 99.4 percent and 98.5 percent, respectively. PCR tests were more efficient for identifying the virus type, detecting HTLV-2 infection and defining inconclusive cases. CONCLUSIONS: Because real-time PCR is sensitive and practical and costs much less than WB, this technique can be used as a confirmatory test for HTLV in blood banks, as a replacement for WB.

INTRODUÇÃO: A triagem para HTLV-1/2 em doadores de sangue geralmente utiliza imunoensaio enzimático, seguido de um método confirmatório como Western blot quando o EIA é positivo, mas este algoritmo mostra alta taxa de resultados inconclusivos, e elevado custo. MÉTODOS: Dois ensaios qualitativos de PCR em tempo real foram desenvolvidos para detectar HTLV-1 e 2 e um total de 318 amostras foram testadas por PCR (152 de doadores de sangue, 108 de portadores assintomáticos, 26 de pacientes HAM/TSP e 30 de indivíduos soronegativos). RESULTADOS: A sensibilidade e especificidade das PCR em relação aos resultados de WB foram de 99,4 por cento e 98,5 por cento, respectivamente. As PCR foram mais eficientes em identificar o tipo viral, a infecção pelo HTLV-2 e úteis para definir casos inconclusivos. CONCLUSÕES: Por serem sensíveis, práticas e de custo muito inferior ao do WB, as técnicas de PCR em tempo real podem ser usadas como teste confirmatório do HTLV em bancos de sangue, em substituição ao WB.

HTLV-2 horizontal and vertical transmission in a family from a Brazilian urban area: seroepidemiological, clinical and molecular study.

Human T cell lymphotropic virus type 2 (HTLV-2) has been shown to be endemic in Brazilian Indians and among intravenous drug users in urban areas, but transmission of this infection seems to be infrequent in the general population living in urban areas in Brazil. Six persons in three generations of a Brazilian family were evaluated to assess HTLV-2 transmission and its molecular features in the positive cases. The index was detected during screening (HTLV EIA) of donated blood in Fundação Hemominas, Belo Horizonte, Brazil. Confirmatory serological test and viral typing were performed by Western blotting and polymerase chain reaction. The family consisted of husband, wife (index case), three daughters, and the mother of the index case. The husband and one daughter were found positive, thus pointing to horizontal and vertical transmission. The husband was a truck driver, who reported casual sex during frequent traveling. The positive daughter was breast-fed for 3 months, as opposed to the remaining two (seronegative), who breast-fed for 1 month. The index case's mother was negative. To identify HTLV-2 subtype(s), phylogenetic analysis of the noncoding long terminal repeat region and part of the env and tax coding regions was performed. These new isolates from Belo Horizonte are related to subtype IIa but present a molecular variant with extended tax, previously reported in subtype IIc. Analyzing both LTR and env regions, the family's sequences clustered with isolates of Brazilian intravenous drug users and transfusion transmitted virus.