Journal of human virology最新文献

Objective: To define the usefulness of molecular parameters in patients with chronic hepatitis C who are undergoing antiviral therapy. Anti-hepatitis C virus (HCV) treatment was monitored by determination of serum HCV load and by presence of HCV RNA in peripheral blood mononuclear cells (PBMCs).

Study design/methods: Fifty-one patients with chronic hepatitis C undergoing antiviral therapy with interferon-alpha plus ribavirin were studied. Serum HCV RNA load was tested with a quantitative assay (Amplicor HCV Monitor Test) before, during, and up to 12 months after end of treatment. If HCV RNA was not detectable, serum samples were subsequently tested with a qualitative assay (Cobas Amplicor HCV Test) and corresponding ethylenediaminetetraacetic acid (EDTA)-treated blood was checked for presence of HCV RNA in peripheral blood mononuclear cells (PBMCs). Sustained virologic response was defined by loss of HCV RNA 12 months after the end of treatment.

Results: Four patients (7.8%) were found to be sustained virologic responders, 17 (33.3%) were transient virologic responders, and 30 (58.8%) were virologic nonresponders. No significant difference was found in the median pretreatment serum HCV RNA load between sustained virologic responders, transient virologic responders, and virologic nonresponders. At 1 month after start of therapy, HCV RNA was not detectable with both the serum and the PBMC assay in 12 (23.5%) of 51 patients. Four remained HCV RNA-negative until 12 months after the end of treatment. In 14 of 17 transient virologic responders, reappearance of HCV RNA was detected earlier in PBMCs than in serum.

Conclusions: Based on these results in 51 patients, quantitation of baseline serum HCV RNA does not appear to be a decisive factor to the management of the individual patient. Early assessment of serum HCV RNA level after start of anti-viral treatment seems to be of major importance to identify virologic nonresponders. Reappearance of HCV RNA may be demonstrated earlier in PBMCs than in serum.

Objective: In previous studies we have shown that removal of the spleen in HIV-infected people during the asymptomatic phase of disease results in slower time to AIDS and may also result in improved survival. In this paper, we examine whether splenectomy affects lymphocyte counts, T-cell subsets, and HIV plasma viremia in a manner that could explain the clinical benefits associated with this intervention.

Methods: 10 HIV-infected patients who underwent splenectomy and 23 HIV-infected controls with idiopathic thrombocytopenia purpura who did not undergo splenectomy were studied. These groups were compared for changes in cell subpopulations and HIV plasma viremia.

Results: Splenectomy resulted in increases in absolute lymphocyte numbers with rises in both CD4 and CD8 counts, whereas CD4 and CD8 percentage levels remained unchanged. In controls, absolute and percentage CD4+ T-cell counts declined with time from date of HIV infection. Plasma viremia decreased more than threefold, the limit of biologic variation, after splenectomy in 4 of 9 subjects and in only 1 of 18 controls. The proportion of subjects exhibiting reduced viremia following splenectomy was greater than that in HIV-infected patients that did not undergo splenectomy (chi 2 test, P = .015).

Conclusions: Improved survival and time to AIDS in splenectomized HIV-infected patients is associated with temporary reduction of plasma viremia and increase in absolute CD4 and CD8 counts. These effects could not be attributed to antiretroviral therapy because subjects were either untreated or treated with antiretroviral monotherapy during the observation period. These observations may have importance in the understanding of T-cell dynamics and the potential for splenectomy as an HIV reservoir-debulking procedure.

Objectives: To analyze HIV-1 nef gene mutations in a cohort of Italian and Swedish long-term nonprogressors (LTNP) and to investigate whether particular amino acid substitutions are associated with LTNP.

Study design/methods: nef alleles from 21 LTNP and 8 progressor controls were amplified by polymerase chain reaction (PCR) and sequenced. The amino acid sequences were compared with the previously reported sequences of 16 North American LTNP and of 28 patients with progressive infection.

Results: An untruncated intact open reading frame was observed as major sequence in all LTNP and controls. None of the amino acid substitutions in known biologically functional sites was linked to LTNP. A valine/isoleucine at the variable position 11 was associated with both European (P = .0001) and American (P = .001) LTNP. The interpatient nef variation was lower among European LTNP (P = .002) than in European progressor controls.

Conclusions: Nef amino acid heterogeneity is lower among LTNP, probably reflecting the lower HIV-1 replication rate. Nef gene defects appear uncommon in both Swedish and Italian LTNP, although the presence of a valine/isoleucine at position 11 is statistically associated with a lower probability to progress to disease.

Objective: The functions of the Epstein-Barr virus (EBV)-encoded small RNAs (EBER-1 and EBER-2) are unknown. We examined their fine intranuclear localization as the first step to investigate their function.

Methods: We analyzed EBER-1 and EBER-2 by in situ hybridization combined with two-color immunofluorescence tagging.

Results: EBER-1 was visualized as fine dots, mainly in the euchromatin. Ribosomal protein L-22 also formed fine dots, similar to those formed by EBER-1, in the nuclei of EBV-carrying cells and colocalized with the latter by double staining. EBER-2 was predominantly found in the nucleoli and was also present in the euchromatin. The two EBERs were similarly expressed in B-cell lines of the different phenotypes examined. The EBERs showed no colocalization by double staining. Image analysis indicated that the level of their expression was not correlated.

Conclusion: The differential localization and expression of the EBER-1 and EBER-2 suggests that they may play different functional roles.

Objectives: Definition of the cellular constituents of breast milk and infant saliva that are involved in milk-borne transmission of HIV infectivity.

Study design/methods: Productively infected cells in colostrum and milk of HIV-1-seropositive mothers were identified by in situ hybridization and immunocytochemistry. Additionally, normal cells from mature milk were infected in vitro to determine which cell types were capable of supporting productive HIV-1 infection. Cellular interactions and transfer of HIV-1 in saliva-milk mixtures were studied to monitor the viability of milk cells and the potential for transfer of infectious virus during ingestion of milk.

Results: Colostrum and early milk from HIV-1-seropositive mothers contained 0.1% to 1% productivity infected macrophages and T cells. Macrophages and epithelial cells from mature milk were susceptible to productive HIV infection in vitro. When milk was mixed with saliva, milk cells became disrupted or were bound and endocytized by salivary epithelial cells.

Conclusions: Productively infected milk cells may contribute directly to transmission of HIV infectivity in breastfed infants during both early and late lactation. Macrophages are the principal cellular carriers of productive HIV-1 infection in milk. Cellular complexes produced during milk-saliva interactions may play a key role in oral transmission of HIV.

Objective: To use HIV-1 vectors to mediate stable gene transfer into hematopoietic stem/progenitor cells.

Study design/methods: Purified human CD34+ cells were transduced with HIV-1 vectors pseudotyped with VSV-G and subjected to colony-forming assays and differentiation in liquid culture. Transduction was determined by DNA-polymerase chain reaction (PCR) for the transgene. GFP reporter gene expression and phenotypes of progeny cells were assessed by microscopy and flow cytometry.

Results: The HIV-1 vector transduced CD34+ cells with high efficiency. Transduction did not interfere with CD34+ cells differentiation in vitro. Transduced genes are expressed in different subsets of progeny cells, including those with normal dendritic cells (DC) morphology and phenotypes (HLADR+/CD1a+/CD86+/CD14-).

Conclusions: We have demonstrated efficient transduction of hematopoietic progenitor cells by HIV-1 vectors. The transgenes are expressed in different subsets of progeny cells, which suggests stable integration. The generation of DCs stably expressing HIV antigens provides a new approach for vaccine development.

Objective: To identify a lipopolysaccharide (LPS) that retains the capacity to induce beta-chemokine secretion without the concomitant activation of pyrogenic cytokines.

Methods: LPS was extracted from strain MLK986 (mLPS), an htrB1::Tn10, msbB::ocam mutant of Escherichia coli that is defective for lipid A synthesis, and from wild-type parent E coli strains, W3110 (wtLPS). The capacity of these LPS preparations to induce tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory proteins 1 alpha (MIP-1 alpha) and MIP-1 beta was assessed using a human peripheral blood mononuclear cell (PBMC) activation assay.

Results: Stimulation of PBMCs with mLPS did not induce measurable levels of pyrogenic cytokines TNF-alpha and IL-1 beta, whereas wtLPS induced high levels of these cytokines. Furthermore, mLPS antagonized the induction of TNF-alpha secretion by wtLPS. Nonetheless, mLPS retained a discrete agonist activity that induced MIP-1 alpha and MIP-1 beta secretion by PBMCs. This latter agonist activity appears to be unique to mLPS, since two previously documented LPS antagonists, Rhodobacter sphaeroides diphosphoryl lipid A and synthetic lipid IVA, did not induce MIP-1 alpha and MIP-1 beta secretion. Furthermore, synthetic lipid IVA was an antagonist of MIP-1 alpha and MIP-1 beta induction by mLPS.

Conclusion: These results show that mLPS exhibits a novel bipartite activity, being an effective antagonist of TNF-alpha induction by wtLPS, while paradoxically being an agonist of MIP-1 alpha and MIP-1 beta secretion.