HIV infection leads to differential expression of T-cell receptor V beta genes in CD4+ and CD8+ T cells.

OBJECTIVE: To analyse variation in T-cell receptor (TCR) V beta gene expression in T cells in HIV-infected individuals. DESIGN: Because there are very few monoclonal antibodies available for studying TCR V beta gene expression, we used polymerase chain reaction (PCR) to analyse the TCR V beta repertoire in HIV-infected individuals using specific primers for 20 distinct families of TCR V beta. METHODS: Evaluation of TCR V beta gene expression in peripheral blood from HIV-1-infected individuals [two in Centers for Disease Control (CDC) stage II, five in CDC stage III and four in CDC stage IV]. Complementary DNA was produced from CD4+ and CD8+ T cells, amplified by PCR and analysed after Southern blotting and hybridization with a C beta-specific oligonucleotide probe. RESULTS: V beta gene expression was dramatically modified, especially in AIDS patients. The CD4+ T-cell subset showed both overexpression (V beta 2) and deletions or underexpression (V beta 9-V beta 20), whereas these gene segments were expressed normally in the CD8+ subset. Only V beta 3 was deleted or underexpression in both CD4+ and CD8+ populations in AIDS patients. CONCLUSIONS: HIV-1 infection induces CD4+ T-cell deficiency, both in total numbers and by causing a paucity of select V beta gene expression in this subset. In addition, the V beta 3 gene family was deleted or underexpressed was observed in both CD4+ and CD8+ T-cell subsets from patients in CDC stage IV. These results are compatible with changes in V beta gene expression known to occur under the action of endogenous or exogenous superantigens.

Genetic diversity of GBV-C/HGV strains among HIV infected-IVDU and blood donors from Buenos Aires, Argentina.

GBV-C/HGV RNA was investigated in serum samples from 70 HIV(+) intravenous drug users (IVDU), as well as from 200 blood donors from Buenos Aires, Argentina. Viral RNA was demonstrated in 21 IVDU by reverse transcription-nested PCR of the 5' UTR. c-DNA amplified products were analyzed and their sequences compared with those downloaded from GenBank. A phylogenetic tree based on 171 sequences demonstrated the presence of three major genogroups, including two subgroups, within local samples, i.e. group 1 (n=1), 2a (n=11), 2b (n=4) and 3 (n=5). These results agreed entirely with those obtained by a novel RFLP (J. Clin. Microbiol. 37, 1340-1347, 1999) of the same 5' UTR amplicons. As expected, GBV-C/HGV RNA prevalence was significantly higher among IVDU than among blood donors (P<0.0001), although within the latter group an unexpectedly high rate was also detected, since 11 of 200 sera (5.5%) proved positive. These viral isolates were ascribed either to subgroup 2a (n=5), subgroup 2b (n=5) or genogroup 3 (n=1). Briefly, this partial view of GBV-C/HGV molecular epidemiology in Argentina shows: (i) different rates of GBV-C/HGV infection within both IVDU and blood donors; (ii) a high prevalence of viral RNA among blood donors; and (iii) a predominant circulation of genogroup 2, with minor contribution of groups 3 and 1.

Immunodominant glycoprotein 41 epitope identified by seroreactivity in HIV type 1-infected individuals.

A highly conserved gp41 epitope (amino acid sequence ELDKWA) has been described to elicit antibodies neutralizing a broad variety of HIV-1 strains. We analyzed the antibody reactivity of HIV-1-infected individuals from Argentina and Sweden to overlapping synthetic peptides covering aa647-684 of HIV-1 MN gp41. An immunodominant antigenic determinant was discovered in the C-terminal region adjacent to the ELDKWA sequence. Most of the sera from both Argentina and Sweden reacted only with peptides representing this region. Two other patterns of reactivity were also observed: some sera reacted only with peptides spanning the central region of gp41, including the ELDKWA sequence; other sera reacted with both the central and C-terminal regions. Differences in reactivity were noted between Argentinian and Swedish sera in terms of peptides covering the central region. In addition, to determine the amino acids essential for antibody binding, seroreactivity against a set of substitution peptides covering the immunodominant epitope was studied. Results obtained indicated that carboxyl amino acids WNWFDI close to the ELDKWA sequence were the most important for antibody binding. Ability of sera, tested for peptide reactivity, to neutralize HIV-1 was also analyzed. High antibody reactivity to the central region was frequently found in sera with high neutralizing titers. This observation was not seen when seroreactivity to peptides spanning the C-terminal region was analyzed. These results suggest that the immunodominant epitope C terminal to the ELDKWA sequence is not involved in inducing neutralizing antibodies.

Role of drug injection in the spread of HIV in Argentina and Brazil.

Drug injectors' have become the second largest HIV transmission category in Argentina and Brazil, as is the case in many pattern I countries, making up more than one-quarter of all AIDS cases reported by 1991. HIV seroprevalence data suggest that the expanding proportion of AIDS cases attributable to drug injection stems from an absolute increase in the number of AIDS cases among drug injectors, and is not merely reflective of a decline in the proportion of cases reported in other transmission categories. Results of a review of studies in Argentina and Brazil indicate that HIV seroprevalence is increasing rapidly, contrary to the situation in some pattern I countries in which HIV seroprevalence among drug injectors is either stably high or increasing only slightly. Also contrary to most pattern I countries, cocaine rather than heroin is the injected drug of choice in Argentina and Brazil. Given that injectors of cocaine are more likely to be HIV infected than are heroin injectors, differences in the type of drug injected between countries may have distinct epidemiological consequences on the spread of HIV.

PIP: AIDS cases attributable to using contaminated needles in nonmedicinal drug injection have increased in Argentina from 11.3% in 1987 to 39% in 1991. A similar increase (from 1.8% in 1985 to over 30% in 1991) occurred in Brazil. To complement existing information, data searches were conducted and personal communications from current researchers were collected for a total of 24 documents from Argentina and 18 from Brazil. The median sample size was 68 in Brazil and 188 in Argentina; most studies were from outpatient facilities, males constituted more than two-thirds of the sample in half of the studies, and the median age (when reported) was between 16 and 29 years old. Analysis of data from selected studies showed that HIV seroprevalence among drug injectors in both countries has increased rapidly, with the greatest increase occurring in Brazil. This rapid increase may also be influenced by the fact that cocaine, rather than heroin, is the drug of choice. Cocaine injection involves drawing blood into the syringe before injection and also more frequent injections. The level of seroprevalence among drug injectors varies among different subpopulations. Sexually transmitted disease clinic attenders who are IV drug users show rates of 6.58% and 51.9% seropositivity. Incarcerated persons who are drug injectors were associated with seropositivity rates of 35% (adults), 60.9% (adolescents), 53.5% (adolescents in security institutes), and 18% (female inmates who volunteered for testing), with drug injection the most important risk factor in 90.4%. Among prostitute injectors, rates were reported of 20% in 1988 and 50% in 1989-90 in the same population. Street children in Rio de Janeiro who use IV drugs (68 of 3389 surveyed) had a rate of 13.2% in 1987-88. The risk factors associated with HIV infection among drug injectors are socioeconomic status and injecting and sex practices. Co-infection patterns among drug injectors have also been found, with concomitant HIV-1 and HTLV-1 reported in 20% of 85 HIV positive drug injectors. Overall, these data suggest that there is an absolute increase in the number of AIDs cases among drug injectors rather than a decline in the proportion of cases reported in other transmission categories. Drug injector transmission poses a threat to the sex partners and offspring of injectors and provides a bridge to the heterosexual infection of women. While there is no obvious quick solution to the problem of IV drug use, there are programs which can slow the spread of HIV among injectors. It is urgent to control sexual transmission and drug injection transmission of HIV in these countries.

[HIV-1 virus transmission through maternal milk]. / Transmisión del HIV-1 a través de la leche materna.

Human immunodeficiency virus type 1 (HIV-1) may be vertically transmitted during the pre, peri or postpartum period. Postnatal transmission as well as an increased risk of vertical transmission with breastfeeding has been shown for HIV-1 in several reports. Breastfeeding was here analyzed as a risk of HIV-1 transmission in a group of infants born to HIV-1 infected mothers. Among the 215 children studied in our population a significant difference was detected between those who were breastfed vs those who were bottle fed and finally became infected (p < 0.000000, R.R. = 4.29). We also report the case of a postnatal infection in a baby born to an HIV-1 seropositive father and a seronegative mother. Due to the risk of infection of the mother she had been thoroughly controlled when pregnant and after delivery. Mother and child were negative when retested at delivery, and at 10 months post-partum. At the age of 32 months the child attended the outpatient clinic with generalized lymphadenopathy and right parotitis. HIV-1 infection was then confirmed in both mother and child. At that time it was discovered that the baby had been breastfed up to the age of 24 months. This is the first reported child in Argentina whose infection may undoubtedly be attributed to breastfeeding.

[Molecular analysis of the principal neutralization epitope (V3 loop) of human immunodeficiency virus type 1 in Argentina]. / Análisis molecular del principal epítope de neutralización ("V3 loop") del virus de la inmunodeficiencia humana tipo 1 de la Argentina.

The main goal of the present paper was to analyze the molecular diversity of the principal neutralizing domain (V3 loop) of the HIV 1 gp120 in samples from patients of Argentina. The study was carried out on a total of 30 HIV 1 positive blood samples, obtained during 1991-1992, belonging to 15 intravenous drug users (group A), 5 homosexual men (group B), 8 children born to HIV 1 positive mothers (group C) and 2 AIDS patients (group D). By using extracted DNA from peripheral blood lymphocytes and from infected cells of the viral isolates in the case of the 2 AIDS patients, the V3 loop region was amplified by means of polymerase chain reaction. Direct sequencing by Sanger methodology was then performed on DNA fragments and nucleotide sequences obtained were translated into the correspondent amino acids. Consensus sequences for each group and a general consensus sequence were established (Table 1). Its alignment with V3 loop amino acid sequences of the major HIV 1 strains isolated worldwide is showed in table 2. Homology analysis between each sequence of the study population and sequence of different HIV 1 isolates showed that most of these samples share high homology with SF2 and BH10 strains. In contrast a low homology was found with JH3 and MN isolated (table 3). The presence of highly conserved amino acid residues as substitutions and insertions was determined in the Argentinian V3 loop sequences giving them a local pattern. The present paper is of great importance for our country, considering that the V3 loop is the main neutralizing domain becoming a major target in the development of HIV 1 vaccine. To our knowledge, this is the first report on the sequencing of the principal neutralizing domain of the Human Immunodeficiency Virus type 1 in Latin America.

Regression of AIDS-related Kaposi's sarcoma following combined antiretroviral treatment.

The aim of the study was to assess regression of Kaposi's sarcoma (KS) in AIDS patients in Argentina. Eighteen male AIDS patients with human immunodeficiency virus (HIV)-associated Kaposi's sarcoma at different clinical stages received KS specific treatment and/or anti-retroviral therapy. Triple anti-retroviral therapy was given to most of the patients with the exception of four who received zidovudine (ZDV) in combination with another nucleoside analogue but no protease inhibitors. Plasma viral load and CD4+ T lymphocyte number were measured in two blood samples (before and after treatment). Complete remission was found in all patients (five) at KS stage I, three out of eight patients at stage II but in none at stages III and IV. Two out of three patients at KS stage IV did not respond to treatments at all. Three patients at KS stages I and II showed complete remission of sarcoma with only anti-retroviral therapy suggesting that anti-retroviral therapy and non-KS specific chemotherapy can successfully control KS.

[Molecular characterization of HTLV-II from an intravenous drug addict with AIDS in Argentina]. / Caracterización molecular de HTLV-II de un paciente drogadicto endovenoso con SIDA de la Argentina.

Ten intravenous drug user AIDS patients were studied to determine the presence of HTLV-I/II infection. Sera were screened by particle agglutination test and by an in house indirect immunofluorescence assay using MT2 (HTLV-I) and C19 (HTLV-II) producing cell lines. Out of the ten sera, one was confirmed HTLV-II positive by Western blot. Peripheral blood mononuclear cells were obtained from this patient, separated through a Ficoll-Hypaque gradient and DNA was extracted and amplified by semi-nested PCR. The amplified product obtained was cloned and sequenced. Results demonstrated the presence of HTLV-II proviral DNA and comparison of the obtained sequence with different HTLV-II prototypes led to classify the virus into subtype a. This is the first molecular characterization of HTLV-II in an intravenous drug user in Argentina.

[Evaluation of indirect immunofluorescence as a supplementary test for the diagnosis of HIV-1 infection]. / Evaluación de la inmunofluorescencia indirecta como prueba suplementaria para el diagnóstico de la infección por HIV-1.

In order to be used as an alternative or complementary test to confirm HIV-1 infection, the efficiency of indirect immunofluorescence assay (IFA) was compared with Western blot (WB) in 362 samples from persons with high and low risk behaviour. A panel of sera with 220 WB positive, 122 WB negative and 20 WB indeterminate sera were tested by an "in house" IFA. The sensitivity of IFA was found to be 98.63% and the specificity 98.36%. Therefore, IFA appeared to be an efficient alternative method to WB, since the cost of testing by IFA is less than 10% of WB testing. We observed a direct relationship between WB protein reactivity and IFA results. In 15 samples with coincident indeterminate results for WB and IFA, antibody reactivity to p24 and gp160 presented the highest frequency. On the other hand, antibodies to viral glycoproteins were always present in IFA weak positive samples, showing their high predictive value.

Troublesome diagnosis in two children born to HIV-1 infected mothers.

In order to assess early HIV infection vertically transmitted in children it is necessary to use techniques that directly detect HIV. Positive results were found in some rare cases who later seemed to have cleared the infection. We communicate two cases of children with troublesome diagnosis. Two girls born to HIV-1 infected mothers were followed-up since birth up to 40 months old, with viral culture, polymerase chain reaction (PCR), p24 antigen detection and serologic techniques. PCRs were positive in three opportunities at 2, 8 and 30 months of age in the first child and confirmatory tests for specific antibodies remained indeterminate up to the age of 34 months. Positive viral DNAs were detected in two opportunities in the second child at 2 and 4 months of age. Western Blots were negative since 25 months. No virus was recovered nor was p24 antigen detected during the whole period of study in either child. Sequestration of the virus in lymphatic tissue, low replicative ability of the virus and/or immunological tolerance can be postulated in the first case. In patient 2, it could be hypothesized that infection, if any, had cleared up.

Human immunodeficiency virus bDNA assay for pediatric cases.

Techniques to quantify plasma HIV-1 RNA viral load (VL) are commercially available, and they are adequate for monitoring adults infected by HIV and treated with antiretroviral drugs. Little experience on HIV VL has been reported in pediatric cases. In Argentina, the evaluation of several assays for VL in pediatrics are now being considered. To evaluate the pediatric protocol for bDNA assay in HIV-infected children, 25 samples from HIV-infected children (according to CDC criteria for pediatric AIDS) were analyzed by using Quantiplex HIV RNA 2.0 Assay (Chiron Corporation) following the manufacturer's recommendations in a protocol that uses 50 microliters of patient's plasma (sensitivity: 10,000 copies/ml). When HIV-RNA was not detected, samples were run with the 1 ml standard bDNA protocol (sensitivity: 500 HIV-RNA c/ml). Nine samples belonged to infants under 12 months of age (group A) and 16 were over 12 months (group B). All infants under one year of age had high HIV-RNA copies in plasma. VL ranged from 30,800 to 2,560,000 RNA copies/ml (median = 362,000 c/ml) for group A and < 10,000 to 554,600 c/ml (median = < 10,000) for group B. Only 25% of children in group B had detectable HIV-RNA. By using the standard test of quantification, none of the patients had non detectable HIV-RNA, ranging between 950 and 226,200 c/ml for group B (median = 23,300 RNA c/ml). The suggested pediatric protocol could be useful in children under 12 months of age, but 1 ml standard protocol must be used for older children. Samples with undetectable results from children under one year of age should be repeated using the standard protocol.

[Detection of the viral load and its use as a virological marker in the follow-up of HIV-1-positive patients]. / Detección de la carga viral y su uso como marcador virológico en el seguimiento de pacientes HIV-1 positivos.

The evaluation of viral load as virological marker and its clinical and immunological correlation are presented. The first viral load studies were performed during 1996 at the National Reference Center for AIDS in Argentina in HIV-1 positive patients derived from different Hospitals in Buenos Aires. The study included 216 HIV-1 positive patients, 49 females and 167 males. Plasma was used for evaluating viral load and a second sample was obtained in 25 of the 216 patients for their monitoring. Viral load was performed using bDNA technique (Quantiplex HIV RNA assay 2.0, Chiron Corporation, USA). Other parameters such as CD4 count determined by flow cytometry and clinical stages according to CDC classification were obtained in order to correlate clinical and immunological status of the patients. When CD4 count was compared with viral load, the results showed a trend of viral RNA increase in plasma along with a decrease in CD4+ lymphocytes. This trend was also observed to correlate with the progression to AIDS disease. In all groups of patients, considering either CD4 counts or clinical status, ranges of viral load values were broad. Thus, as shown by percentiles 25 and 75, patients with CD4 counts < 200/ml, presented viral load values between 18,395 c/ml to 215,425 c/ml and patients with > 200/ml viral RNA showed values from < 10,000 to 35,180 c/ml. Patients with CDC's A and B stages presented values from < 10,000 to 45,160 c/ml and 87,000 c/ml respectively, while patients classified as C had 10,582 to 215,000 c/ml. Results of two consecutive samples in the 25 patients showed the usefulness of this technique for monitoring antiretroviral therapy. Nevertheless, despite the tendency of viral load to increase along with the progression of the disease, the broad range of values suggested the importance of using both virological and immunological parameters for the management of HIV infected patients.