Structural and immunogenicity analysis of chimeric B-cell epitope constructs derived from the gp46 and gp21 subunits of the envelope glycoproteins of HTLV-1.

B-cell epitopes were selected from the gp21 and gp46 subunits of the envelope glycoprotein of human T-cell lymphotropic virus type 1 (HTLV-1) by computer-aided analyses of protein antigenicity. Molecular modeling was used to design and synthesize the epitopes as chimeric constructs with promiscuous T-helper epitopes derived either from the tetanus toxoid (amino acids 947-967) or measles virus fusion protein (amino acids 288-302). Circular dichroism measurements revealed that the peptides had a secondary structure that correlated well with the crystal structure data or predicted structure. The chimeric peptides were then evaluated for their immunogenicity in rabbits or mice. Antibodies against one of the epitopes derived from the gp21 subunit were found to be neutralizing in its ability to inhibit the formation of virus-induced syncytia. These studies underscore the importance of the gp21 transmembrane region for the development of vaccine candidates. The applicability of a chimeric approach is discussed in the context of recent findings regarding the role of gp21 transmembrane region in the viral fusion process.

High expression of p40(tax) and pro-inflammatory cytokines and chemokines in the lungs of human T-lymphotropic virus type 1-related bronchopulmonary disorders.

STUDY OBJECTIVE: Human T-lymphotropic virus type 1 (HTLV-1) is closely associated with the development of certain pulmonary diseases, such as bronchiolitis, although the pathologic mechanism remains unclear. To elucidate the pathogenesis of HTLV-1-associated bronchopulmonary disorders, we analyzed the relationship between expression of p40(tax), a regulatory component of HTLV-1 that stimulates various host genes, and synthesis of pro-inflammatory cytokines and chemokines by cells in BAL fluid (BALF) obtained from HTLV-1-infected patients. DESIGN: Reverse transcription-polymerase chain reaction was used to compare the expression of p40(tax) and pro-inflammatory cytokines and chemokines messenger RNA (mRNA) in BALF of 10 HTLV-1 carriers and 7 healthy subjects. We also studied the correlation between these parameters and the proportion of lymphocytes in BALF. RESULTS: The expression levels of pro-inflammatory cytokines (interferon [IFN]-gamma, interleukin-2) and chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein [MIP]-1alpha, IFN-gamma-inducible protein-10 [IP-10]) were significantly higher in BALF of patients than of healthy subjects. The expression of IFN-gamma and MIP-1alpha mRNA correlated with that of p40(tax). IFN-gamma and IP-10 mRNA expression correlated with the proportion of lymphocytes in BALF. The percentage of lymphocytes in BALF increased with higher expression levels of p40(tax) mRNA, although the correlation was not significant. CONCLUSION: Our results suggested that p40(tax) seems be involved in the development of HTLV-1-associated bronchopulmonary disorders at least in part through the local production of pro-inflammatory cytokines and chemokines.

Feasibility of human T-lymphotropic virus type I screening using pooled sera.

BACKGROUND AND PURPOSE: The human T-cell lymphotropic virus type I (HTLV-I) seroprevalence rate among volunteer blood donors in Taiwan is low. To study the feasibility of HTLV-I enzyme immunoassay (EIA) screening using pooled sera, we prospectively compared its sensitivity to that of the routine test for each donor. METHODS: HTLV-I EIA tests for each serum sample and a pooled-sera test with 50 samples from voluntary donated blood samples were performed concurrently to assess the effectiveness and cost savings of this screening method. RESULTS: Of 135,606 blood samples from volunteer donors tested for HTLV-I infection, 60 samples (0.044%) were HTLV-I EIA-positive using the routine method. Among these, the positive results were confirmed by Western blot in 22 samples (36.7%). In the pooled-sera test, 17 of 2,713 pooled samples were EIA-positive and these results were all confirmed by Western blot. Five of the 22 (22.7%) EIA-positive samples had a false-negative result in the pooled-sera test. Serial dilution in these five cases revealed that the maximum dilution before loss of sensitivity was 8-fold for two specimens and 16-fold for three specimens. CONCLUSION: In this study, the 50-pooled sera test had higher specificity (100%), but lower sensitivity (77.3%), than the routine HTLV-I screening. Our results suggest that use of a pooling method with five samples would leave a reasonable safety margin and be feasible for HTLV-I mass screening in areas with low seroprevalence for HTLV-I infection.

[Hypercalcemia due to adult T-cell lymphoma in a man from Surinam]. / Hypercalciëmie door adult T-cellymfoom bij een Surinaamse man.

A 48-year-old male patient from Surinam presented with anorexia, nausea and weight loss. An extreme hypercalcaemia of 5.08 mmol/l was found. Further diagnostic investigations showed that this patient had a HTLV-1 positive adult T-cell leukaemia/lymphoma (ATL/L). This is often associated with multilobularly nucleated lymphocytes, bone destruction and hypercalcaemia. Skin localisations are frequently observed. The combination of cytomorphology, immunophenotyping, HTLV-1 seropositivity and clinical findings points to the diagnosis. The patient was treated with 6 courses of chemotherapy consisting of cyclophosphamide, doxorubicin, teniposide, prednisone, vincristine and bleomycin. Upon inquiry it appeared that he had died elsewhere. Chemotherapy induces a short-lived remission in a minority of ATL/L patients. Antiviral therapy (alpha interferon, zidovudine) might offer new possibilities.

Crusted (Norwegian) scabies in a patient with smoldering adult T-cell leukemia.

Crusted (Norwegian) scabies is described in a patient with smoldering adult T-cell leukemia (ATL). The patient is an 84-year-old Japanese woman who presented with widespread scaling over the trunk and limbs and crusted lesions on the scalp and ears. Microscopical examination of scrapings from the scalp and ears showed extraordinarily large numbers of scabies mites. The white blood cell count was 5.1 x 10(9)/L with 6% abnormal lymphocytes with mature nuclei that showed convolution and lobulation. Anti-HTLV-I antibodies were positive. Southern blot analysis revealed that the cellular DNA extracted from this patient's peripheral blood cells, digested with Pst I, showed the same pattern of provirus genome as the DNA from ATL patients. A diagnosis of crusted scabies with smoldering ATL was made. It is possible that crusted scabies could be an opportunistic infection and a pre-diagnostic sign of ATL.

[Evaluation of commercial equipment used in blood banks in Córdoba for the detection of anti-HTLV-I/II antibodies]. / Evaluación de los equipos comerciales utilizados en los bancos de sangre de Córdoba para la detección de anticuerpos anti HTLV-I/II.

In order to assess the efficiency of currently used screening tests, Abbott HTLV-I/HTLV-II EIA, Vironostika HTLV-I/II Organon Teknika, Particle Agglutination (PA) assay Serodia Fujirebio Inc. (Tokyo, Japan) for HTLV-I/II antibody detection in blood donors samples, a panel of 100 sera from different blood banks of Córdoba city were studied. An "in house" indirect immunofluorescence assay (IFA) was used as reference test. The correlation rates were: 66% for Abbott HTLV-I/HTLV-II EIA, 97% for Vironostika HTLV-I/II Organon Teknika EIA and 99% for PA Serodia. Vironostika HTLV-I/II Organon Teknika EIA and PA Serodia assay proved to be more reliable for HTlV-I/II antibody screening in blood donors from Córdoba, yielding a very low rate of false positive results as compared with Abbot HTLV-I/HTLV-II EIA.

Leucemia linfoma T del adulto en Chile: estudio clínico patológico y molecular de 26 pacientes / Adult T cell leukemia lymphoma in Chile: a clinical-pathological and molecular study of 26 patients

Background: Adult T cell leukemia lymphoma is a lymphoproliferative syndrome etiologically associated to human T cell lymphotropic virus type I. Aim: To describe the clinical and laboratory features of 26 caucasian chilean patients, with HTLV-I positive adult T-cell leukemia lymphoma (ATLL). Material and methods: Diagnostic criteria included clinical features, cell morphology, immunophenotype, HTLV-I serology and/or DNA analysis by southern blot or PCR. Results: According to the clinical presentation, 12 cases had the acute ATLL form, 6 had a lymphoma, 4 the chronic form and 4 had smoldering ATLL. The median presentation age was 50 years, younger than the Japanese patients, but significantly older than patients from other south american countries (eg Brasil, Jamaica, Colombia). The main clinical features: lymphadenopathy, skin lesions and hepatosplenomegaly, were similar in frequency to those of patients from other countries, except for the high incidence of associated neurological disease. Tropical spastic paraparesis (TSP) in our series of ATLL, was seen in one third of the patients (8/26). A T-cell immunophenotype was shown in all 26 cases and HTLV-I serology was positive in 25/26 patients. Molecular analysis on the seronegative patient showed clonal integration of proviral HTLV-I DNA into the lymphocytes DNA, and thus he may have been a poor responder to the retroviral infection. Proviral DNA integration was also demonstrated in 15/16 patients being clonal in 10, polyclonal in 3 (all smoldering cases) and oligoclonal in one. Conclusions: ATLL in Chile has similar clinical and laboratory features than the disease in other parts of the world, except for a younger age than japanese patients but older than those from other latin american countries and a high incidence of patients with associated TSP. Detailed morphological and immunophenotypic analysis of the abnormal circulating lymphocytes, together with the documentation of HTLV-I by serology and/or DNA analysis are key tests for the identification of this disease

[Seroprevalence of human T-cell lymphotropic virus type I in Curaçao and predictive variables for that infection]. / Seroprevalentie van humaan T-cellymfotroop virus type I op Curaçao en voorspellende variabelen voor die infectie.

OBJECTIVE: To determine the seroprevalence of human T-cell leukaemia virus (HTLV) type I and predictive variables in Curaçao. DESIGN: Descriptive. SETTING: St. Elisabeth Hospital, Curaçao. METHODS: A total of 2531 sera were randomly collected from a total population of approximately 145,000 over a period of three months (of seven the sex was not known). An initial ELISA test was performed to detect anti-HTLV-I antibodies. If this test was positive an ELISA re-test (in duplicate) was performed. If one of these re-tests was found positive a western blot confirmation test was performed. The association with age, sex, social class and history of syphilis were analysed with multiple logistic regression models and adjusted for confounding. RESULTS: The estimated prevalence of HTLV-I was 1.9% (49/2524). No significant sex differences were observed (odds ratio (OR): 1.13; 95% confidence interval (95% CI): 0.62-2.05). Increasing age (p for trend = 0.0003) and lower social class (OR: 1.86; 95% CI: 1.03-3.38) were important predictive factors for HTLV-I infection. Members of the lower social classes and persons 50 years or older were at relatively high risk (OR: 3.91; 95% CI: 2.21-6.94). CONCLUSION: HTLV-I infection is endemic in the island of Curaçao, as in other Caribbean islands. The estimated prevalence is 1.9%. Age and lower social class were important predictive factors for HTLV-I infection.

[Progressive myelopathy caused by human T-cell lymphotropic virus type I (HTLV-I) in 3 patients in The Netherlands]. / Progressieve myelopathie veroorzaakt door humaan T-cellymfotroop virus type I (HTLV-I) bij 3 patiënten in Nederland.

One man and two women (aged 30, 44 and 46, respectively) were seen between 1987 and 1992 with a myelopathy caused by the human T-cell lymphotropic virus type I (HTLV-I). The first symptoms were impaired gait in the man and micturition disorders in the women. Diagnosing took 2 to 4 years, possibly due to the fact that many Dutch physicians are unfamiliar with the disease. The diagnosis was based on originating from an endemic area (i.e. Surinam and the Caribbean basin), the clinical picture and the presence of antibodies against HTLV-I in blood and CSF. The disease in a number of years leads to spastic paraparesis, incontinence for urine and dependence on a wheelchair.

[Tropical spastic paraparesis due to HTLV-I. 4 case reports and literature review]. / La paraparésie spastique tropicale à HLTV-I. Etude de 4 cas et revue de la littérature.

We describe the clinical, radiological, neurophysiological and biological characteristics of 4 patients suffering from tropical spastic paraparesis due to a HTLV-I infection. Patient 1 immigrated in Europe at the age of 16 from Caribbean islands. Patient 2 was a White male Caucasian who lived during several years with a prostitute also native of Caribbean islands. Patient 3 was a Black male Zaïrian and Patient 4 was a female of mixed race who lived many years in Zaäire and in Senegal. All the 4 patients presented with walking difficulties, a spastic paraparesis, urinary disturbances and often, severe constipation. The diagnosis was based on a positive ELISA test for HTLV-I antibodies, confirmed in 2 cases each by Western blots and detection of the viral genome by the Polymerase Chain Reaction. In all cases, CSF pleocytosis and CSF-restricted oligoclonal IgG bands were observed. MRI of the spinal cord showed a global atrophy in 3 cases. Cerebral MRI revealed unusual extensive, progressive and symmetric lesions of the white matter in Patient 1, and some patchy periventricular lesions in Patient 4; this examination was normal in the two other cases. Sensory evoked potentials indicated abnormalities of central conduction velocities at the level of the posterior columns. The pathogenesis of tropical spastic paraparesis is briefly discussed.

Polimiositis primaria asociada a HTLV-I / Primary polymyositis associated to HTLV-I

We report a 68 years old male with a polymyositis associated to HTLV-I. Diagnosis was based on clinical picture, an increased creatin-phosphokinase levels, electromyography and muscle biopsy. The patient had positive HTVL-I antibodies, measured by particle agglutination test, indirect immunofluorescence and polymerase chain reaction in lymphocytes. Skin biopsy showed a mycosis fungoides. Schirmer test and minor salivary gland biopsy showed a dacryosialoadenitis. There was no central nervous system involvement. This patient is the only with positive HTLV-I antibodies, among 18 patients with polymiositis in whom these antibodies were measured

Inhibitory activity in saliva of cell-to-cell transmission of human T-cell lymphotropic virus type 1 in vitro: evaluation of saliva as an alternative source of transmission.

Human T-cell lymphotropic virus type 1 (HTLV-1) is known to be transmitted vertically through breastfeeding and horizontally by blood transfusion and sexual contact. Our intervention study has suggested the presence of additional alternative maternal transmission pathways. To explore the possibility of transmission through saliva, we used PCR to quantify the HTLV-1 provirus in saliva samples from 18 carrier mothers and 10 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis. The provirus was detected in 60 and 90%, respectively, of the samples, with estimated copy numbers in the range of 10 to 10(4)/ml. However, the saliva, regardless of the presence or absence of antibodies to the virus, showed a strong tendency to inhibit the cell-to-cell transmission of HTLV-1 in vitro, as examined by a syncytium inhibition assay. The natural inhibitory activity in saliva of seronegative volunteers was heat sensitive, and most of the activity was recovered by ultrafiltration in the fraction of macromolecules with a molecular weight of more than 100,000. In addition to this natural activity, saliva of HTLV-1-infected individuals contained immunoglobulin G molecules capable of neutralizing syncytium formation. These results strongly suggested that HTLV-1-infected cells in the carriers' saliva, which contains neutralizing antibodies in addition to the natural activity inhibiting cell-to-cell viral infection, barely transmit the virus. Transmission of HTLV-1 through the saliva would thus seem to be rare, if it occurs at all.

A novel magnetic particle agglutination in microtiter plates for rapid detection of human T-lymphotropic virus type I antibody.

We have developed a novel magnetic particle agglutination (MPA) method for rapid detection of antibody to human T-lymphotropic virus type I (HTLV-I). This method is suitable for screening determination. The assay uses purified viral antigen coated on magnetic particles, which are novel artificial particles made of gelatin, arabic gum, and ferrite. MPA uses a special magnet and inclined plate holder. In MPA, the specimen is incubated with magnetic particles in the well; magnetic particles are then magnetically attracted to the bottom of the well. Then the plate is inclined and read by the naked eye. In a positive specimen, agglutination particles stay at the bottom of the well. On the other hand, non-agglutination particles with negative sample run down the side of the well. The total assay time of MPA was estimated at 8 min/microtiter-plate. The results obtained by correlating MPA for the detection of HTLV-I antibody with other method emphasized the precision of MPA.

[Detection of markers of the human T-cell leukemia virus in patients with T-cell lymphoma and Kaposi's sarcoma]. / Obnaruzhenie markerov virusa T-kletochnogo leikoza cheloveka u bol'nykh T-kletochnoi limfomoi i sarkomoi kaposhi.

Nine patients with Kaposi's sarcoma and five suffering from T-cell lymphoma have been examined. Antibodies to HTLV-1 were not found in these patients. The primary cellular cultures were isolated from blood and lymph nodes of the patients. Viral particles (type C) were found in the culture obtained from the patient with lymphoma of T-cell origin. DNAs from the primary cellular cultures from patients with lymphoma or sarcoma contained the sequences homologous to gag-gene of HTLV-1. The data suppose the patients with T-cell lymphoma and Kaposi's sarcoma to carry HTLV-1 virus.

Títulos de anticuerpos anti-VTLV-I en individuos infectados seropositivos / Anti HTLV-I antibody titers in seropositive infected individuals

The aim of this study was to determine anti HTLV-I antibody titers in seropositive symptomatic and asymptomatic infected subjects. One hundred seven infected subjects (47 with spastic paraparesis and 60 asymptomatic) were studied. HTLV-I antibodies were determined using indirect immunofluorescence in cells infected with the retrovirus. The mean titer was 1/234 in asymptomatic subjects and 1/2138 in symptomatic patients (p<0,001). These results suggest an association between HTLV-I antibody titers and clinical stage of infected subjects

Prevalência de anticorpos anti-HTLVI/II em doadores de sangue da Fundaçäo Pró-Sangue Hemocentro de Säo Paulo / Prevalence of anti-HTLVI/II antibodies in blood donors of Fundaçäo Pró-Sangue Hemocentro de Säo Paulo

O teste ELISA ant-HTLVI/II foi introduzido na triagem sorlógica de doadores de sangue na Fundaçäo Pró-sangue Hemocentro de Säo Paulo (FPS/HSP) em julho de 1991. NO período compreendido entre julho de 1991 e julho de 1994 foram submetidos à triagem serológica 597.727 doadores. Destes, 7682 foram recusados por terem apresentado reatividade no teste ELISA anti-HTLVI/II. A positividade observada, para o referido teste, foi diminuindo com o correr do tempo: 2,12 por cento em 1991; 1,6 por cento em 1992; 0,8 por cento em 1993 e 1,0 por cento em 1994, sendo esse fato atribuido a melhora da especialidade e reprodutividade dos kits comerciais. Foi utilizado o teste suplementar de Western Blot para confirmar os resultados dos testes ELISA. Em 249 amostras de soros de doadores, com resultado repetidamente positivo (RRP) no teste ELISA (hemobio), o poder confirmatório do Western Blot (Cambridge Biotech) foi de 24.9 por cento (IC/90 por cento: 20,4 por cento-29,39 por cento). Baseados nesses dados, considera-se uma expectativa de prevalência de indivíduos infectados pelo HTLVI/II, na populaçäo de doadores de sangue da FPS/HSP de 0,142 por cento (IC/90 por cento; 0,116 por cento-0,167 por cento). Em 437 amostras de soro de doadores que retornaram ao Banco de Sangue, para confirmar o resultado inicial e apresentaram RRP no teste ELISA, o poder confirmatório do Western Blot foi de 34,55 por cento (IC/90 por cento: 30,82 por cento-38,28 por cento)...

[Prevalence of human T-cell lymphotropic virus type I among Dutch blood donors]. / De prevalentie van humaan T-lymfotroop virus type I onder Nederlandse bloeddonors.

OBJECTIVE. To establish the prevalence of human T-lymphotropic virus (HTLV) type I among Dutch blood donors. DESIGN. Study to confirm screening results. SETTING. Central Laboratory of the Blood Transfusion Service of the Dutch Red Cross, section Virus Diagnostics, Amsterdam. METHOD. The majority (550,000) of the Dutch blood donors were tested in the course of 1993 for presence of HTLV antibodies. For serological confirmation using the Western Blot test, 20 of the 22 Dutch blood banks sent in blood samples of 714 donors found positive at HTLV screening. Material of 36 of these donors was suitable for testing using the polymerase chain reaction (PCR) for DNA of HTLV types I and II. RESULTS. The Western Blot confirmation test confirmed HTLV infection of 10 of the 714 blood donors; eight of these samples, examined with the PCR test, proved to be positive for DNA of HTLV I. In 537 of the 714 ELISA-reactive donors, the Western Blot test gave doubtful results. The PCR test for HTLV I/II DNA was negative in 26 of these samples, but the possibility could not be excluded that there were HTLV carriers among the other donors with a doubtful Western blot test result. With inclusion of the four donors infected with HTLV I that had been detected earlier, the prevalence of HTLV I among Dutch blood donors is found to be 1:39,000.