Quantitative evaluation of the recombinant HIV-1 phenotype to protease inhibitors by a single-step strategy.

OBJECTIVE: To develop and optimize a fast and quantitative recombinant strategy for evaluating the HIV-1 phenotype to protease inhibitors (PI). DESIGN AND METHODS: A non-replicative HIV-1 molecular vector (designated pdelta prodelta env) capable of expressing exogenous HIV-1 protease-encoding sequences was developed in this study. The HIV-1 protease sequences were amplified from either viral isolates or plasma samples (both from 21 HIV-1-infected individuals, 19 of whom were failing different anti-HIV-1 combination treatments) and cloned in the pdelta prodelta env backbone. The HIV-1 recombinant phenotype to PI was determined directly after transfection of viral chimeric clones by measuring protease activity and calculating a percentage sensitivity index (SI%; the ratio between the results from each clone and those from a PI-sensitive reference strain). RESULTS: The SI% values obtained from the recombinant clones paralleled the IC50 results of the viral isolates and documented different degrees of resistance and cross-resistance to PI, compatible, with few exceptions, with the respective genotype. Interestingly, an inverse correlation between SI% values and the presence of primary mutations for resistance to PI (P = 0.0038 and P = 0.0414, for indinavir and ritonavir, respectively) and a difference in SI% between samples harbouring an increasing number of mutations (indinavir, P = 0.022; ritonavir, P = 0.0466) were observed. CONCLUSION: The data substantiate the reliability of the novel strategy for a fast (5 day) quantitative evaluation of HIV-1 phenotype to PI, and indicate that this method may contribute to the understanding of mechanisms of virus resistance to PI.

Prevalence of transmitted nucleoside analogue-resistant HIV-1 strains and pre-existing mutations in pol reverse transcriptase and protease region: outcome after treatment in recently infected individuals.

We retrospectively studied 38 Italian recently HIV-1-infected subjects who seroconverted from 1994 to 1997 to investigate: (i) the prevalence of nucleoside reverse transcriptase inhibitors (NRTI)-related mutations at primary infection; (ii) the proportion of naturally occurring mutations in reverse transcriptase (RT) and protease regions of patients naive for non-nucleoside RT inhibitors (NNRTIs) and protease inhibitors (PIs); (iii) the drug-susceptibility to NRTIs and PIs in subjects with NRTI- and/or PI-related mutations; and (iv) the outcome of seroconverters treated with various NRTIs or NRTI/PI regimens. Baseline HIV-1 plasma viraemia and absolute CD4 count at baseline could not be used to distinguish patients with NRTI- and/or PI-related pre-existing mutations from those with wild-type virus (P = 0.693 and P = 0.542, respectively). The frequency of zidovudine-related mutations was 21% in the study period. The response to treatment was not significantly different in subjects with or without genotypic zidovudine-related mutations at primary infection (P = 0.744 for HIV-1 RNA and P = 0.102 for CD4 cells). Some natural variation (2.6%) was present within regions 98-108 and 179-190 of RT involved in NNRTI resistance. The high natural polymorphism in the protease region present in our patients was similar to that reported by others. In our study some PI-associated substitutions, thought to be compensatory in protease enzymatic function, could confer intermediate to high PI-resistance. As discrepancies between genotypic and phenotypic results may exist in recent seroconverters, our data suggest that the role of transmitted NRTI- and PI-resistant variants remain to be fully elucidated in vivo.

Identification of two distinct subsets of long-term nonprogressors with divergent viral activity by stromal-derived factor 1 chemokine gene polymorphism analysis.

Stromal-derived factor (SDF)-1, the natural ligand for CXCR4, is present in a common polymorphic variant defined by a G-->A transition in the 3' untranslated region of the gene. In persons infected with human immunodeficiency virus type 1 (HIV-1), the homozygous genotype (SDF1-3'A/3'A) has been postulated to interfere with the appearance of T-tropic syncytium-inducing strains. The polymorphism of SDF1 was correlated with HIV-1 phenotype, plasma viremia, and unspliced and multiply spliced specific transcripts in 158 virologically characterized HIV-1-infected patients (39 recent seroconverters, 75 typical progressors, and 44 AIDS patients) and in 42 HIV-1-infected long-term nonprogressors (LTNPs). Analysis of SDF1 allele distribution revealed that SDF1-3'A/3'A status is associated with low CD4 cell count (P=.0449) but not with a specific HIV-1 phenotype. In LTNPs, SDF1-+/+ condition defined a subset of persons with lower HIV-1 replication than in heterozygous subjects. The low viral activity in SDF1-+/+ LTNPs suggests that other factors play a major role in vivo in determining the course of HIV-1 infection.

[Diagnostic value of ERTL4: a screening test of language disorders in 4-year-old children]. / Valeur diagnostique de ERTL4: un test de repérage des troubles du langage chez l'enfant de 4 ans.

BACKGROUND: Language disorders in children are frequent and may sometimes severely affect their development. ERTL4 (screening test for language disorders of children aged 4) is the first test for screening language disorders to be used by doctors. It has not yet been evaluated. AIM: The objective of this study is to assess and optimize the diagnostic value of the ERTL4 test and to evaluate its potential interest as a screening test. MATERIAL AND METHODS: A sample of 370 children was randomly constituted among children aged between 3 years 9 months and 4 years 6 months from schools in Nancy, France and surrounding areas. These children undertook both the ERTL4 test and a standardized examination of language and speech as a reference. RESULTS: Among the 325 children included in the analysis, 34.1% presented with disorders requiring speech and language therapy. ERTL4 sensitivity and specificity were 88.6% and 66.8%, respectively. Varying thresholds for disorder diagnoses and proposals for a modified test led to a sensitivity of 72.9% and a specificity of 91.0%. The proportion of well-classified subjects improved from 73.8% to 85.3% (P < 10(-3)). The positive predictive value was 78.7% and the negative predictive value was 88.1% (with the observed frequency in the sample). CONCLUSION: ERTL4 is a good test for doctors to screening of children's language disorders and their management.

Lack of in vitro anti-gp160 antibody production is a correlate of nonprogression among HIV type 1-infected individuals.

The aim of our study was to investigate the possible correlation of in vitro antibody production (IVAP) directed to the gp160 protein of HIV-1 with CD4+ slopes, plasma viremia, and disease progression in long-term nonprogressors (LTNPs). Nineteen subjects with a long-term nonprogressive HIV-1 infection were studied and followed for 2 years. During the follow-up, in vitro anti-gp160 producers showed negative CD4+ slopes in the majority of cases (9 of 12), whereas 5 of 7 nonproducers showed positive CD4+ slopes. Plasma viremia values, which were not significantly different in the two groups at baseline, became significantly higher in anti-gp160 producers when compared with nonproducers during the follow-up (p = 0.012). Finally, a trend toward progression was observed in the group of producers but not in nonproducers. These findings suggest that the in vitro production of anti-gp160 antibodies by peripheral B cells is not a correlate of protection, and may represent an early predictor of progression in LTNPs.

Patterns of in vitro anti-human immunodeficiency virus type 1 antibody production in long-term nonprogressors.

With the aim of evaluating the specific pattern of in vitro antibody production (IVAP) in human immunodeficiency virus type 1 (HIV-1)-infected long-term non-progressors (LTNPs), we tested 20 subjects who had remained asymptomatic for more than 8 years with a CD4+ cell count higher than 500/microliter and 59 patients at different stages of HIV-1 infection as controls. In cell cultures, IVAP was detected in 14 out of 20 LTNPs (70%), in 5 out of 6 recent seroconverters (83%), and in all the other control patients. Anti-p24 antibody production was significantly lower in LTNPs than in asymptomatic patients with a more recent infection. Recent seroconverters and patients with AIDS did not produce anti-p24 antibodies (P = 0.02). Anti-gp160 antibodies were produced by peripheral blood mononuclear cells from LTNPs in 12/20 cases. CD4+ cell count was significantly higher in IVAP-negative than in IVAP-positive LTNPs (P = 0.013), while the viral load was not significantly different. Specific anti-HIV-1 antibody production did not seem to be a correlate of long-term nonprogression.

Pentoxifylline improves cell-mediated immunity and reduces human immunodeficiency virus (HIV) plasma viremia in asymptomatic HIV-seropositive persons.

The effects of pentoxifylline on immunologic and virologic parameters were evaluated in 10 human immunodeficiency virus-infected patients not receiving antiretroviral treatment. Patients were asymptomatic, had 300-500 CD4 cells/microL, and received pentoxifylline (1200 mg/day orally) for 4 months. Peripheral blood mononuclear cells were tested before and at five time points during therapy. A transient increase in CD4 cells was observed in 8 of 9 patients, and CD8 cells increased in 7 of 9 patients. These increases were negatively correlated with susceptibility to in vitro mitogen-stimulated apoptotic cell death. Pentoxifylline had a temporary effect on mitogen-stimulated cytokine production; thus, interferon-gamma, interleukin (IL)-2, tumor necrosis factor-alpha, and lymphotoxin increased more than IL-10. Pentoxifylline also potentiated antigen-stimulated IL-2 production and proliferation in 8 of 9 patients and induced significant but transient decreases in plasma viremia in 7 of 9 patients. These preliminary findings suggest that pentoxifylline in vivo has an interesting but temporary influence on both immunologic and virologic parameters.

Comparable biological and molecular determinants in HIV type 1-infected long-term nonprogressors and recently infected individuals.

Human immunodeficiency virus type 1 (HIV-1) isolability, rate of replication, phenotype, plasma viremia, and specific intracellular transcripts were cross-sectionally analyzed in 61 HIV-1-seropositive individuals to evaluate the correlations between the virological and molecular correlates of protection and progression in different clinical subsets: recently infected subjects (RIs), long-term nonprogressors (LTNPs), late progressors (LPs), and typical progressors (TPs). Comparison of the major virological and molecular features of HIV-1 infection has defined distinct profiles for different subsets of patients. LTNPs or RIs, as well as LPs or TPs, exhibited similar titers of coculture p24 antigen; the differences between the former and the latter were statistically significant at all the time points tested (p = 0.0001; 0.0003 and 0.0001). Whereas LTNPs and RIs revealed comparable low levels of indexes of viral replication, LPs and TPs showed higher genome and mRNA copy numbers (p = 0.0004 and p = 0.0008, respectively). We demonstrated close biological and molecular similarities between RIs and LTNPs on the one hand, and LPs and TPs on the other. In LTNPs both viral biological properties and viral load are important determinants of the course of the disease.

Plasma viremia and virus phenotype are correlates of disease progression in vertically human immunodeficiency virus type 1-infected children.

OBJECTIVE: To analyze the relationships among HIV-1 plasma viremia, phenotype and CD4 T cell counts in vertically infected children. METHODS: Plasma viremia was quantified in 37 vertically infected children at different stages of the disease by a standardized molecular assay. Virus isolation and non-syncytia-inducing or syncytia-inducing (SI) HIV-1 phenotype evaluation were performed in parallel. RESULTS: HIV-1 RNA genomes were found to be significantly different in CDC clinical classes N, A, B and C (P = 0.0135) and in immunologic classes 1, 2 and 3 (P = 0.0110). None of the children in Class N or A harbored HIV-1 isolates with SI phenotype, whereas SI primary isolates were detected in 2 of 7 (29%) and 7 of 10 (70%) Class B and C children, respectively. Similarly SI variants were present in only 9 of 13 children in immunologic Class 3 (70%). When stratified according to the increasing severity of virologic status, the children showed a significant difference (P = 0.0458) in viral burden. CONCLUSIONS: Clinical symptoms, the most dramatic being reduction in the number of CD4 lymphocytes, and the highest plasma viremia levels were observed in the children in whom fast replicating, highly cytopathic SI variants were isolated. These data extend the virologic characterization of vertically HIV-1 infected children and suggest that both the plasma viremia levels and phenotype of primary isolates are viral correlates of disease progression in vertically infected children.

Long-term resistance to HIV infection in vertical HIV infection: cytokine production, HIV isolation, and HIV phenotype define long-term resistant hosts.

We analyzed immunologic (CD4 and CD8 slopes; interferon-gamma, interleukin-2, interleukin-10, and chemokines production; concentration of IgE; beta 2-microglobulin) and virologic (p24; HIV isolability and phenotype; plasma viremia) parameters in HIV vertically infected children > or = 8 years of age without disease progression or mild symptoms and an absolute CD4+ count > or = 500/microliter with CD4+ percentage > or = 25%. The results were compared to those of two control groups: (1) slow progressors, children > or = 8 years of age with moderate symptomatology and/or moderate CD4 depletion, and (2) progressors, children > or = 8 years of age with severe clinical disease and/or severe CD4 depletion. Pediatric long-term resistant hosts were characterized by higher production of interleukin-2 and interferon-gamma and lower production of interleukin-10, normal concentration of IgE, HIV isolates with a non-syncytium-inducing phenotype, and lower plasma viremia. This condition was not associated with the concentration of beta 2-microglobulin, p24, and chemokines, or with HIV isolability. The IL-10/IL-2 ratio best correlated with both CD4 counts and disease progression. Thus, vertically infected children showing resistance to disease progression are immunologically and virologically distinct from those in whom progressive HIV infection is observed.

HIV type 1 phenotype correlates with the stage of infection in vertically infected children.

A cohort of 39 vertically infected children (class N, A, B, and C of the CDC HIV classification for pediatric infection) was studied by virus isolation and non-syncytium inducing (NSI)/syncytium inducing (SI) HIV-1 phenotype evaluation. The HIV-1 isolates were recovered from PBMCs and the MT-2 cell line was used to perform the syncytium assay. HIV-1 could be isolated in 34 of 39 (87%) infected children, regardless of the clinical and immunological stage of the disease. Class N and A subjects harbored exclusively NSI strains, whereas the SI phenotype was detected in two of eight class B and five of nine class C patients. All of the SI variants were observed in severely CD4-depleted children (class 3 patients). The capability of pediatric HIV-1 isolates to induce a cytopathic effect is associated with the clinical status and the degree of CD4 depletion. These data suggest that the biological properties of HIV-1 isolates in children do not differ from those observed in adults, and that viral phenotype strictly correlates with disease progression in vertically infected children.

Virologic and immunologic markers of disease progression in pediatric HIV infection.

Correlates of progression of human immunodeficiency virus (HIV) infection to AIDS include the reduction in CD4+ T cells and the emergence of syncytium-inducing (SI) HIV variants. It has been suggested that progressive defects in interleukin 2 (IL-2), IL-12, and IFN- gamma production (type 1 cytokines), and increased production of IL-4 (and of IL-4-driven hyper-IgE), IL-6, and IL-10 (type 2 cytokines), could provide another correlate of disease progression. To determine the possible association among these markers, viral phenotype, cytokine production, IgE serum concentration, and rate of CD4 depletion were analyzed in a cohort of vertically HIV-infected children. We report that significantly higher production of type 2 cytokines and augmented IgE concentration are observed in children in whom HIV SI is isolated. In addition, we observed that the isolation of HIV SI and the production of high quantities of type 2 cytokines are correlated with increased loss of CD4 T cells in the 12 months preceding the determinations. These data suggest that the virologic and immunologic parameters characteristic of advanced HIV infection may be associated in pediatric HIV infection, and indicate a virologic-immunologic pathogenesis leading to the appearance of AIDS.

[Function of the pituitary-thyroid axis after partial resection of the thyroid]. / Funzionalità dell'asse ipofisio-tiroideo dopo asportazione parziale della tiroide.

The TSH, T4 and T3 responses to TRH were measured in 48 patients who underwent selective goitre resection (hemithyroidectomy, ablation of single nodule); moreover 3 groups of healthy, hypothyroid and hyperthyroid persons served as reference groups. The main object was to detect eventual onset of preclinical hypothyroidism. This condition was observed after hemithyroidectomy only in 12.5%, and never after ablation of single nodule. Were detected too, among operated patients, 2 subjects with light and 1 with clear hyperthyroidism. Therapeutic implications of such a finding were taken into consideration.

[The TRH test in simple goiter]. / Il TRH test nel gozzo semplice.

The TSH response to TRH and the increase in serum T3 after TRH infusion were measured in 25 euthyroid patients with non toxic goiter. On the ground of anatomic characters we divided the goitrous patients into 3 groups: subjects with diffuse goiter, with multinodular and with uninodular goiter; delta TSH and delta T3 did not differ significantly in the three groups. On the contrary, either absent hormonal increase (preclinic hyperthyroidism), or exceeding response (preclinic hypothyroidism), or normal response (real euthyroidism) were found in the different patients of a same group. Therapeutic implications of such a finding were taken into consideration.