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4.
Antivir Chem Chemother ; 13(4): 219-21, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12495209

ABSTRACT

This work evaluated the effect of a sulphated fucan extracted from the Laminaria abyssalis marine algae on the human T cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation. The experiments were carried out in HeLa cells cocultured with a HTLV-1-infected T cell line (C91/PL cells) in the presence of the sulphated polysaccharide at concentration below that corresponding to the ED50. The sulphated fucan inhibited almost 100% of the syncytium formation at concentration of 100 microg/mI and was still active (>95%) at a concentration of 25 microg/ml. It was also observed that the best inhibition occurred when the compound was added in the first 2 h of the cell-to-cell contact. This is the first report showing that a purified sulphated polysaccharide, extracted from marine algae, is able to inhibit the cell-to-cell contact essential for the spreading of the HTLV-1.


Subject(s)
Giant Cells/drug effects , Human T-lymphotropic virus 1/drug effects , Laminaria/chemistry , Polysaccharides/pharmacology , Cell Communication/drug effects , Cell Communication/physiology , Dextran Sulfate/pharmacology , Giant Cells/virology , HTLV-I Infections/virology , HeLa Cells , Human T-lymphotropic virus 1/physiology , Humans , T-Lymphocytes/virology , Time Factors , Tumor Cells, Cultured
5.
Int J Cancer ; 83(5): 596-600, 1999 Nov 26.
Article in English | MEDLINE | ID: mdl-10521792

ABSTRACT

Breast cancer is an important cause of morbidity and mortality in Brazil. Some studies have analyzed the potential role of organochlorine compounds in breast cancer etiology. These chemical compounds have been widely used in agriculture and in vector-control programs in Brazil. A case-control study was carried out in the main cancer hospital of the Instituto Nacional de Câncer in Rio de Janeiro to investigate exposure to organochlorinated pesticides as a risk factor for breast cancer. We investigated 177 cases of invasive breast cancer at the hospital, between May 1995 and July 1996, and 350 controls selected among female visitors at the same hospital. In addition to information obtained from interviews, blood samples were taken, to analyze residual amounts of organochlorine by gas chromatography using an electron capture detector. [1,1-Dichloro-2, 2-bis(p-chlorophenyl)ethylene] (DDE) was determined in sera of 457 women from a total of 493 participants who had serum samples available. Residues of hexachlorobenzene were found in 11 women only. No statistically significant association was found between breast cancer risk and serum level of DDE or history of exposure to pesticides. Serum levels of DDE (ng/ml) were similar in patients (median = 3.1, mean = 5.1) and controls (median = 3.1, mean = 4.8) (p = 0.93). The age-adjusted odds ratio of breast cancer for women in the upper quintile compared with those in the lowest quintile was 0.90 (95% confidence interval 0.47-1.73). When adjusted for other variables, the risk remained not statistically significant (upper quintile odds ratio = 0.79, 95% confidence interval 0.39-1.60). In our hands, exposure to organochlorinated pesticides measured by history or serum analysis was thus not a risk factor for breast cancer.


Subject(s)
Breast Neoplasms/chemically induced , Dichlorodiphenyl Dichloroethylene/analogs & derivatives , Insecticides/adverse effects , Adult , Aged , Analysis of Variance , Brazil/epidemiology , Breast Neoplasms/epidemiology , Case-Control Studies , Dichlorodiphenyl Dichloroethylene/adverse effects , Environmental Exposure/adverse effects , Female , Humans , Middle Aged , Odds Ratio
6.
Braz J Med Biol Res ; 32(4): 403-6, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10347801

ABSTRACT

Apoptosis is a well-known specific process of cell death that normally occurs in physiological situations such as tissue or organ development and involution. During tumor growth there is a balance between proliferation and cell death which involves apoptotic mechanisms. In the present study genomic DNAs from 120 breast tumor biopsies were analyzed by agarose gel electrophoresis and none of them presented the fragmentation pattern characteristic of the apoptosis process. However, 33% of the 105 breast cancer patients clearly showed the apoptotic pattern when DNA from blood cells was analyzed. None of the DNAs from healthy volunteer blood cells showed any trace of apoptosis. Since the breast cancer patients were not receiving chemo- or hormone therapy, the possible relationship between blood cortisol levels and the apoptotic pattern found in patient blood cells was investigated. Using a chemoluminescence immunodetection assay, similar cortisol levels were observed in breast cancer patient sera presenting or not apoptotic blood cells and in healthy volunteer sera. Analysis of the clinical data obtained from 60 of these patients showed that patients bearing tumors of smaller size (under 20 mm) were more susceptible to the apoptotic effect in blood cells. According to the Elston grade, it was observed that 7 of 12 patients with grade III tumors (58%) presented apoptotic peripheral blood cells, in contrast to 10 of 48 patients with grade I and grade II tumors. These observations may reflect the immunosuppression characteristic of some breast cancer patients, which may contribute to tumor growth. Therefore, further studies are necessary to elucidate the factor(s) involved in such massive blood cell death.


Subject(s)
Apoptosis , Blood Cells/physiology , Breast Neoplasms/physiopathology , DNA , Electrophoresis, Agar Gel , Humans , Hydrocortisone/blood
7.
Braz. j. med. biol. res ; 32(4): 403-6, Apr. 1999. ilus, tab
Article in English | LILACS | ID: lil-231729

ABSTRACT

Apoptosis is a well-known specific process of cell death that normally occurs in physiological situations such as tissue or organ development and involution. During tumor growth there is a balance between proliferation and cell death which involves apoptotic mechanisms. In the present study genomic DNAs from 120 breast tumor biopsies were analyzed by agarose gel electrophoresis and none of them presented the fragmentation pattern characteristic of the apoptosis process. However, 33 per cent of the 105 breast cancer patients clearly showed the apoptotic pattern when DNA from blood cells was analyzed. None of the DNAs from healthy volunteer blood cells showed any trace of apoptosis. Since the breast cancer patients were not receiving chemo- or hormone therapy, the possible relationship between blood cortisol levels and the apoptotic pattern found in patient blood cells was investigated. Using a chemoluminescence immunodetection assay, similar cortisol levels were observed in breast cancer patient sera presenting or not apoptotic blood cells and in healthy volunteer sera. Analysis of the clinical data obtained from 60 of these patients showed that patients bearing tumors of smaller size (under 20 mm) were more susceptible to the apoptotic effect in blood cells. According to the Elston grade, it was observed that 7 of 12 patients with grade III tumors (58 per cent) presented apoptotic peripheral blood cells, in contrast to 10 of 48 patients with grade I and grade II tumors. These observations may reflect the immunosuppression characteristic of some breast cancer patients, which may contribute to tumor growth. Therefore, further studies are necessary to elucidate the factor(s) involved in such massive blood cell death.


Subject(s)
Humans , Apoptosis , Blood Cells/physiology , Breast Neoplasms/physiopathology , DNA , Electrophoresis, Agar Gel , Hydrocortisone/blood
8.
J Submicrosc Cytol Pathol ; 30(3): 393-7, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9723200

ABSTRACT

Several studies carried out in Brazil have shown a high incidence of HTLV-I infection among the general population and in different groups, such as blood donors, hemophiliacs, hematological and neurological patients. Cases of adult T-cell leukemia/lymphoma as well as tropical spastic paraparesis/HTLV-I associated myelopathy have already been described. Therefore, it is important to characterize Brazilian HTLV strains using different technical approaches. The purpose of this paper is to characterize and recognize HTLV particles employing routine and immunoelectron microscopy in lymphocyte cocultures. Ultrastructural analysis showed typical large and small virus particles in close relation with the lymphocyte membrane. Immunoelectron microscopy, carried out with HTLV positive sera, allowed the identification of the virus as a type C oncovirus, group HTLV-BLV. The first interaction events between virus and lymphocyte membrane have also been analysed and structures related to the endocytic route for HTLV entrance were pointed out.


Subject(s)
Human T-lymphotropic virus 1/ultrastructure , Adult , Brazil , Cell Membrane/ultrastructure , Cell Membrane/virology , Cells, Cultured , HTLV-I Infections/virology , Human T-lymphotropic virus 1/isolation & purification , Human T-lymphotropic virus 1/physiology , Humans , Lymphocytes/ultrastructure , Lymphocytes/virology , Microscopy, Electron , Microscopy, Immunoelectron , Organelles/ultrastructure
9.
J Neurol Sci ; 139(1): 7-14, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8836966

ABSTRACT

Brazilian patients with HTLV-1 myelopathy present a significant spontaneous lymphocyte proliferation (SLP), and an increased response to IL-2 exogenous stimulation, in both peripheral blood lymphocytes and in whole blood proliferative assays, when compared to the control group. High antibody titers against HTLV-I antigens were also observed in comparison to healthy seropositive individuals. IL-6 was detected in cerebrospinal fluid (CSF) of 50% of the patients (10 out of 20) and TNF-alpha in four out of nineteen individuals. No correlation was found between the presence of levels of cytokines IL-6 and TNF-alpha and duration or severity of disease. The addition of cyclosporin A (CsA) significantly inhibited SLP suggesting that this therapeutic agent should be studied in HTLV-1 myelopathy. Brazilian patients with HTLV-I myelopathy present the same immunological abnormalities described in other endemic regions. The whole blood assay reflects the same results of separated blood cells and, due to its rapid execution may be used as an assay to follow clinical trials.


Subject(s)
Cyclosporine/pharmacology , Cytokines/cerebrospinal fluid , HTLV-I Infections/immunology , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , Paraparesis, Tropical Spastic/immunology , Adolescent , Adult , Aged , Brazil , Cells, Cultured , Family , Female , HTLV-I Infections/cerebrospinal fluid , Humans , Interleukin-2/pharmacology , Interleukin-6/cerebrospinal fluid , Male , Middle Aged , Paraparesis, Tropical Spastic/cerebrospinal fluid , Reference Values , Tumor Necrosis Factor-alpha/cerebrospinal fluid , Urban Population
10.
Article in English | MEDLINE | ID: mdl-8797701

ABSTRACT

Brazil, the largest Latin American country, is highly heterogeneous, both demographically and socioeconomically. The overall human T-cell lymphotropic virus type I (HTLV-I) seroprevalence among blood donors is approximately 0.45%. These rates are highly variable, from 0 to 1.8%. Since 1989, many series of tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) cases have appeared in the literature, with high variation in the prevalence of HTLV-I among TSP patients (14.7-57%). The main clinical features of Brazilian TSP/HAM are similar to those in other endemic countries, but sensory signs are more frequent. Recently, the presence of peripheral nerve and muscular involvement has been characterized. The first nationwide study on the disease has been recently completed: it enrolled 163 patients and concluded that TSP/HAM is common in Brazil, mainly in the northeast and southeast regions; it predominates among women and whites; the most important risk factors for infection are sexual promiscuity and blood transfusion; and, although a remarkably uniform disease throughout the country, some statistically significant differences were detected, such as a higher proportion of females over males in the northeast region, a higher proportion of whites the southeast and the south and mulattos in the northeast, and, finally, a high rate of venereal diseases in the southeast region and of intravenous drug use in the south. Brazil seems to be a perfect setting for future epidemiologic, clinical, basic, and therapeutic studies on TSP/HAM.


Subject(s)
HTLV-I Infections/epidemiology , Paraparesis, Tropical Spastic/epidemiology , Blood Donors , Blood Transfusion , Brazil , Female , HTLV-I Infections/diagnosis , HTLV-I Infections/transmission , Humans , Male , Paraparesis, Tropical Spastic/diagnosis , Paraparesis, Tropical Spastic/transmission , Seroepidemiologic Studies , Sexually Transmitted Diseases/virology , Substance Abuse, Intravenous/virology
11.
J Neurol Sci ; 129(2): 147-51, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7608729

ABSTRACT

HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is apparently a disease with a chronic evolution without spontaneous remissions. The real profile of its natural history and of the progression of the neurological disability, however, awaits confirmation. We devised the present study to evaluate the progression profile of the neurological disability of HAM/TSP in a series of 43 patients who have never received any kind of previous immune therapy. Patients were divided into different groups according to the duration of their disease. Age, gender and the Kurtzke's disability status scale (DSS) at the time of the first examination were compared. There were no statistically significant differences among groups with different disease duration. The present study suggests that the evolution of the neurological disability in HAM/TSP occurs mainly during the first year of the disease and becomes relatively stable after that. Therefore we speculate that the variable therapeutic success rates observed in many series of the literature could be due to the timing in the beginning of the pharmacological immunosuppression. Probably the therapeutic window in HAM/TSP lies within the first year of the disease. Thus it might be of utmost importance that future therapeutical trials take into consideration the duration of the disease since this factor can play an important role in the results of the trial.


Subject(s)
Paraparesis, Tropical Spastic/physiopathology , Adolescent , Adult , Aged , Disease Progression , Female , Humans , Male , Middle Aged
12.
Clin Infect Dis ; 20(3): 696-9, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7756498

ABSTRACT

Human T-cell lymphotropic virus type I (HTLV-I) can be associated with either adult T-cell leukemia or HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive immune-mediated myelopathy. Skin manifestations such as xerosis and erythema may be associated with HAM/TSP. Infective dermatitis due to Staphylococcus aureus or beta-hemolytic Streptococcus has recently been described as a marker for HTLV-I infection and as a probable risk factor for the development of adult T-cell leukemia and lymphoma in Jamaican children. We report a case of folliculitis decalvans, a rare chronic follicular inflammatory process of bacterial origin that is extremely resistant to treatment, in a patient with HAM/TSP. This case suggests the possibility that the disturbance of the immune system that was observed in patients with HAM/TSP can play a role in the persistence of this severe skin lesion. In addition, the findings of our case cast doubt on the hypothesis that the cause of infective dermatitis in persons infected with HTLV-I is immunosuppression due to congenital or perinatal infection of the immature immune system.


Subject(s)
Folliculitis/complications , Paraparesis, Tropical Spastic/complications , Staphylococcal Infections/complications , Adult , Base Sequence , DNA Probes , Folliculitis/drug therapy , Folliculitis/microbiology , Humans , Male , Molecular Sequence Data , Paraparesis, Tropical Spastic/immunology , Prednisone/therapeutic use , Staphylococcal Infections/drug therapy , Staphylococcal Infections/immunology
13.
Braz J Med Biol Res ; 28(1): 51-7, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7581028

ABSTRACT

Some Brazilian regions are considered to be endemic for human T-cell leukemia/lymphoma virus type I (HTLV-I) infection. Several studies have shown a high prevalence of HTLV-I infection among different groups such as blood donors, hemophiliacs and patients suffering from hematological and neurological diseases. Cases of adult T-cell leukemia/lymphoma as well as tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) have already been described in Brazil. We report the isolation of an HTLV-I strain from cultured lymphocytes of a TSP/HAM patient. An IL-2-dependent HTLV-I-infected T-cell line (ROB) expressing viral antigens was established and reverse transcriptase activity could be detected in the culture supernatant. Ultrastructural analysis showed immature and mature HTLV retrovirus particles. Finally, HTLV-I provirus type I was demonstrated by the polymerase chain reaction. This is the first isolation completely carried out in Latin America. The molecular analysis of viral strains, now in progress, should clarify the molecular epidemiology of HTLV-I in Brazil.


Subject(s)
Human T-lymphotropic virus 1/isolation & purification , Lymphocytes/virology , Paraparesis, Tropical Spastic/epidemiology , Adult , Base Sequence , Brazil/epidemiology , Genome, Viral , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 1/ultrastructure , Humans , Male , Molecular Sequence Data , Paraparesis, Tropical Spastic/virology
14.
Acta Neurol Scand ; 88(1): 59-62, 1993 Jul.
Article in English | MEDLINE | ID: mdl-8372632

ABSTRACT

In Rio de Janeiro (RJ) most cases of paraparesis of obscure origin are associated with the human T-cell lymphotropic virus type I (HTLV-I). Thirty-four consecutive patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) from RJ were evaluated. Most patients came from low socio-economic levels. There was no difference in terms of gender. The main affected racial group was white. A history of sexually transmitted diseases was a major risk factor for HAM/TSP and a positive serology for syphilis was found in 26.5% of the patients. The major clinical findings were of a spastic paraparesis with generalized brisk tendon jerks and bilateral Babinki's sign. Sensation was abnormal in 25 patients (73.5%) and five (14.7%) had a sensory level. Three patients (8.8%) had optic atrophy. The cerebrospinal fluid showed a lymphocytic pleocytosis with a mean total protein content of 0.4 g/litre, and an increased intrathecal IgG synthesis in 59.4% of patients. HAM/TSP and multiple sclerosis (MS) occur indigenously in RJ and some HAM/TSP cases can be sometimes confused with MS. Therefore we propose that, in places where MS coexist with HAM/TSP, HTLV-I antibodies should be sought routinely in those MS suspected cases with prominent spastic paraparesis.


Subject(s)
Leukemia-Lymphoma, Adult T-Cell/diagnosis , Paraparesis, Tropical Spastic/diagnosis , Adolescent , Adult , Brazil/epidemiology , Cerebrospinal Fluid/chemistry , Cerebrospinal Fluid/immunology , Ethnicity , Female , Fluorescent Antibody Technique , HTLV-I Antibodies/isolation & purification , Humans , Leukemia-Lymphoma, Adult T-Cell/complications , Male , Neurologic Examination , Paraparesis, Tropical Spastic/complications , Paraparesis, Tropical Spastic/epidemiology , Paresthesia/etiology , Reflex, Babinski , Risk Factors , Sex Factors , Sexual Behavior , Sexually Transmitted Diseases/blood , Sexually Transmitted Diseases/complications , Syphilis/blood
15.
J Neurol Sci ; 116(2): 165-9, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8336163

ABSTRACT

In order to find clinical findings that could significantly discriminate between HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and HTLV-I negative spastic paraparesis of obscure origin (SPOneg) prior to serological testing, and to find risk factors significantly associated with HAM/TSP we devised a case-control study. Sixty consecutive SPO patients were studied without previous knowledge of their HTLV-I serological status. Thirty-four (56.7%) turned out to be HTLV-I positive and 26 (43.3%) HTLV-I negative. HTLV-I infected patients tended to have more commonly motor and bladder disturbances at the beginning of their illness and a disease that was still in progression at the time of the examination. Bladder dysfunction, constipation and penile impotence, and more widespread pyramidal signs, were also more frequent during the whole course of their illness. Likewise, an increased intrathecal synthesis of IgG was found more often in the HTLV-I positive group. The only risk factor for HTLV-I infection significantly associated to HAM/TSP was a prior history of sexually transmitted diseases. These results suggest that, at least in RJ, HAM/TSP might be mainly sexually acquired.


Subject(s)
HTLV-I Antibodies/analysis , Muscle Spasticity/diagnosis , Paraparesis, Tropical Spastic/diagnosis , Adolescent , Adult , Aged , Brazil , Diagnosis, Differential , Female , Humans , Immunoglobulin G/biosynthesis , Male , Middle Aged , Muscle Spasticity/etiology , Muscle Spasticity/immunology , Paraparesis, Tropical Spastic/etiology , Paraparesis, Tropical Spastic/immunology , Risk Factors , Sexually Transmitted Diseases/complications
17.
Lancet ; 336(8721): 987-90, 1990 Oct 20.
Article in English | MEDLINE | ID: mdl-1977015

ABSTRACT

In a series of fourteen patients with adult T-cell lymphoma-leukaemia (ATLL) in Brazil the main features were lymphadenopathy, hepatosplenomegaly, hypercalcaemia, and high leucocyte counts, with abnormal lymphoid cells which had irregular nuclei. The series included the youngest patient with ATLL so far (18 months). Analysis with monoclonal antibodies showed a mature T-cell phenotype (CD4+, CD8-). Antibodies to HTLV-I and/or integration of HTLV-I proviral DNA were found in eleven patients. In the other three HTLV-I DNA could not be demonstrated even by means of the polymerase chain reaction; they therefore had HTLV-I-negative ATLL. This report of ATLL in Brazil corroborates serological reports that HTLV-I may be endemic in some parts of that country. Follow-up studies are required to identify precisely the main route of transmission of HTLV-I in South America and the risk factors for the development of ATLL in carriers.


Subject(s)
Leukemia-Lymphoma, Adult T-Cell/epidemiology , Adult , Antibodies, Viral/analysis , Blotting, Southern , Brazil/epidemiology , DNA, Viral/analysis , Female , Human T-lymphotropic virus 1/genetics , Humans , Infant , Leukemia-Lymphoma, Adult T-Cell/ethnology , Leukemia-Lymphoma, Adult T-Cell/immunology , Leukemia-Lymphoma, Adult T-Cell/microbiology , Leukemia-Lymphoma, Adult T-Cell/mortality , Male , Middle Aged , Polymerase Chain Reaction
18.
Int J Cancer ; 44(3): 389-93, 1989 Sep 15.
Article in English | MEDLINE | ID: mdl-2777403

ABSTRACT

The prevalence of antibodies to HTLV-I and HIV-I in Brazil was determined by testing sera from: (a) 119 members of an isolated Amazonian community of African origin; (b) 100 voluntary blood donors in Rio de Janeiro; (c) 215 patients treated at the Hematology Service, National Cancer Institute, Rio de Janeiro, and (d) 44 Cebus apella New World monkeys, wild-caught in Amazonia. Anti-HTLV-I was detected in 1 (0.84%) of 119 Amazonians, in 8 (3.72%) of the 215 patients and in none of the blood donors or monkeys. The high prevalence found in patients included 4 (5.79%) of 69 with non-Hodgkin lymphoma, 2 (5.88%) of 34 with Hodgkin lymphoma, 1 (16.66%) of 6 patients with diagnosis of anemia and 1 (20%) of 5 with HIV-I infection. Anti-HIV-I was found in 7 (14.89%) of 47 patients and in none of the other groups. The high incidence of HTLV-I infection in the patient group suggests that this retrovirus is endemic in parts of Brazil.


Subject(s)
HIV Antibodies/analysis , HTLV-I Antibodies/analysis , Adult , Aged , Animals , Blood Donors , Brazil , Cebus , Child , Child, Preschool , Female , Humans , Male , Middle Aged
19.
Immunol Lett ; 18(1): 15-8, 1988 May.
Article in English | MEDLINE | ID: mdl-2897948

ABSTRACT

The prevalence of human T lymphotropic virus type I (HTLV-I) and human immunodeficiency virus (HIV) antibodies was evaluated in Brazil among 116 aboriginal Indians living in a pre-Amazonian region, and in 44 patients with haematological malignant disorders being treated in Rio de Janeiro. Screening for the presence of antibodies to HIV was performed routinely for 17,224 blood donors at the National Cancer Institute, Rio de Janeiro, from January 1986 to May 1987. The results demonstrated that HIV infection was not endemic among Brazilian Indians, as none of them had antibodies to HIV, in contrast with the population of Rio de Janeiro, which showed a high prevalence (0.34%) of positivity among normal individuals. In a small group of patients with haematological disease only one with acute lymphoblastic leukaemia proved to be HIV-positive, the infection having been acquired through previous blood transfusion. None of the serum samples reacted with HTLV-I, including those of 17 non-Hodgkin's lymphoma patients. HTLV-I infection does not seem to be endemic in this country, but further large scale studies are necessary, especially in patients with haematological disorders, homosexual individuals and drug users.


Subject(s)
Deltaretrovirus Infections/epidemiology , HIV Seropositivity/epidemiology , Adolescent , Adult , Antibodies, Viral/analysis , Blood Donors , Brazil , Deltaretrovirus/immunology , Deltaretrovirus Infections/complications , Deltaretrovirus Infections/immunology , Female , HIV/immunology , HIV Antibodies , HIV Seropositivity/immunology , Humans , Indians, South American , Lymphoproliferative Disorders/complications , Male , Middle Aged
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