Increased turnover of T lymphocytes in HIV-1 infection and its reduction by antiretroviral therapy.

The mechanism of CD4(+) T cell depletion in human immunodeficiency virus (HIV)-1 infection remains controversial. Using deuterated glucose to label the DNA of proliferating cells in vivo, we studied T cell dynamics in four normal subjects and seven HIV-1-infected patients naive to antiretroviral drugs. The results were analyzed using a newly developed mathematical model to determine fractional rates of lymphocyte proliferation and death. In CD4(+) T cells, mean proliferation and death rates were elevated by 6.3- and 2.9-fold, respectively, in infected patients compared with normal controls. In CD8(+) T cells, the mean proliferation rate was 7.7-fold higher in HIV-1 infection, but the mean death rate was not significantly increased. Five of the infected patients underwent subsequent deuterated glucose labeling studies after initiating antiretroviral therapy. The lymphocyte proliferation and death rates in both CD4(+) and CD8(+) cell populations were substantially reduced by 5-11 weeks and nearly normal by one year. Taken together, these new findings strongly indicate that CD4(+) lymphocyte depletion seen in AIDS is primarily a consequence of increased cellular destruction, not decreased cellular production.

HIV-1 antigen-specific and -nonspecific B cell responses are sensitive to combination antiretroviral therapy.

We studied how combination antiviral therapy affects B cell abnormalities associated with HIV-1 infection, namely elevated circulating immunoglobulin (Ig)G antibody-secreting cell (ASC) frequencies and hypergammaglobulinemia. Within a few weeks of starting antiviral therapy, there is a marked decline in IgG-ASC frequency in both acutely and chronically infected people, whereas the hypergammaglobulinemia often present during chronic infection is more gradually resolved. These reductions are sustained while HIV-1 replication is suppressed. HIV-1 antigen-specific B cell responses are also affected by therapy, manifested by a rapid decline in circulating gp120-specific ASCs. Anti-gp120 titers slowly decrease in chronically infected individuals and usually fail to mature in acutely infected individuals who were promptly treated with antiretroviral therapy. Long-term nonprogressors have high titer antibody responses to HIV-1 antigens, but no detectable gp120-specific IgG-ASC, and normal (or subnormal) levels of total circulating IgG-ASC. Overall, we conclude that HIV-1 infection drives B cell hyperactivity, and that this polyclonal activation is rapidly responsive to decreases in viral replication caused by combination antiviral therapy.

Measuring recent thymic emigrants in blood of normal and HIV-1-infected individuals before and after effective therapy.

The role of the thymus in HIV-1 pathogenesis remains unclear. We developed an assay to quantify the number of recent thymic emigrants in blood based on the detection of a major excisional DNA byproduct (termed alpha1 circle) of T cell receptor rearrangement. By studying 532 normal individuals, we found that alpha1 circle numbers in blood remain high for the first 10-15 yr of life, a sharp drop is seen in the late teen years, and a gradual decline occurs thereafter. Compared with age-matched uninfected control individuals, alpha1 circle numbers in HIV-1-infected adults were significantly reduced; however, there were many individuals with normal alpha1 circle numbers. In 74 individuals receiving highly active antiretroviral therapy, we found no appreciable effect on alpha1 circle numbers in those whose baseline values were already within the normal range, but significant increases were observed in those with a preexisting impairment. The increases in alpha1 circle numbers were, however, numerically insufficient to account for the rise in levels of naive T lymphocytes. Overall, it is difficult to invoke thymic regenerative failure as a generalized mechanism for CD4 lymphocyte depletion in HIV-1 infection, as alpha1 circle numbers are normal in a substantial subset of HIV-1-infected individuals.

Differences in prevalence of community-associated MRSA and MSSA among U.S. and non-U.S. born populations in six New York Community Health Centers.

BACKGROUND: Staphylococcus aureus is the most common cause of Skin and Soft Tissue Infections (SSTIs) in the community in the United States of America. Community Health Centers (CHC) serve as primary care providers for thousands of immigrants in New York. METHODS: As part of a research collaborative, 6 New York City-area CHCs recruited patients with SSTIs. Characterization was performed in all S. aureus isolates from wounds and nasal swabs collected from patients. Statistical analysis examined the differences in wound and nasal cultures among immigrant compared to native-born patients. RESULTS: Wound and nasal specimens were recovered from 129 patients and tested for antibiotic susceptibility. 40 patients were immigrants from 15 different countries. Although not statistically significant, immigrants had lower rates of MRSA infections (n = 15) than did native-born participants, and immigrants showed significantly higher rates of MSSA wound cultures (n = 11) (OR = 3.5, 95% CI: 1.3, 9.7). CONCLUSIONS: In our study, immigrants were more likely to present with SSTIs caused by MSSA than US-born patients. Immigants also reported lower frequencies of antibiotic prescription or consumption in the months prior to SSTI infection. This suggests that antibiotic resistance may vary regionally and that immigrants presenting with SSTIs may benefit from a broader range of antibiotics.

Assessment of pain in herpes zoster: lessons learned from antiviral trials.

Pain typically accompanies acute herpes zoster and, in a proportion of patients, it persists well beyond rash healing. Pain must therefore be analyzed in trials of antiviral agents in herpes zoster, but different methods have been used to analyze pain in recent published trials. These reports are reviewed and their methodological strengths and weaknesses examined. Based on this review, recommendations for the design and analysis of future trials of antiviral agents in herpes zoster are proposed. The principal recommendation is that antiviral efficacy should be evaluated both by distinguishing post-herpetic neuralgia from acute pain and by considering pain as a continuum. The primary endpoint should address both the prevalence and duration of post-herpetic neuralgia and should be examined in those patients who have post-herpetic neuralgia. Adopting the proposed recommendations in design and analysis of future trials should facilitate comparison across trials of the efficacy of antiviral agents in the treatment of herpes zoster.

Varicella-zoster virus gene 63: transcript mapping and regulatory activity.

The varicella-zoster virus (VZV) putative immediate-early (IE) protein encoded by ORF63 is the homolog of HSV-1 ICP-22. To further characterize ORF63 and its function, Northern analysis, primer extension, and S1 nuclease assays were used to map its transcripts, and transient transfection assays were performed with constructs containing ORF63 or its promoter region. Two transcripts were identified: a 0.9-kb transcript spans ORF63 alone, and a 1.8-kb transcript reads through ORF64. Two prominent transcription start sites were identified at -88 and -157 relative to the ORF63 ATG, and two potential TATA elements were identified. In transient transfection assays, the 63 promoter was weakly activated by VZV ORF4 and ORF62 under their homologous promoters, was more strongly activated by ORF62 under the control of a constitutive CMV promoter, and was synergistically activated by ORFs 4 and 62 together. ORF63, driven by its own or by a heterologous SV40 promoter, exerted minimal effects on diverse VZV putative IE and early promoters, showed no clear evidence of autoregulation, and did not directly inhibit the ORF62 promoter as had been reported previously. ORF63's behavior in transient assays suggests that it plays only a limited regulatory role in modulating VZV gene expression.

Retrovirus insertion into herpesvirus in vitro and in vivo.

Retroviruses and herpesviruses are naturally occurring pathogens of humans and animals. Coinfection of the same host with both these viruses is common. We report here that a retrovirus can integrate directly into a herpesvirus genome. Specifically, we demonstrate insertion of a nonacute retrovirus, reticuloendotheliosis virus (REV), into a herpesvirus, Marek disease virus (MDV). Both viruses are capable of inducing T lymphomas in chickens and often coexist in the same animal. REV DNA integration into MDV occurred in a recently attenuated strain of MDV and in a short-term coinfection experiment in vitro. We also provide suggestive evidence that REV has inserted into pathogenic strains of MDV in the past. Sequences homologous to the REV long terminal repeat are found in oncogenic MDV but not in nononcogenic strains. These results raise the possibility that retroviral information may be transmitted by herpesvirus and that herpesvirus expression can be modulated by retroviral elements. In addition, retrovirus may provide a useful tool to characterize herpesviral function by insertional mutagenesis.

Retrovirus insertion into herpesvirus: characterization of a Marek's disease virus harboring a solo LTR.

We recently reported the phenomenon of retroviral insertion into a herpesvirus (Isfort et al., (1992) Proc. Natl. Acad. Sci. USA 89, 991-995). We have now isolated Marek's disease virus (MDV) clones that carry retroviral inserts. Retroviral LTR insertion has the potential to activate or inactivate herpesvirus genes leading to alterations of the biological properties of the herpesviruses. The structure and the expression pattern of a virus clone carrying a solitary LTR insertion was characterized in detail. The LTR insertion is accompanied by deletions in the herpesvirus genome at each of the junctions of the short unique region (US) and its inverted repeats (RS). This results in a significantly truncated RS region. Despite these structural rearrangements, the virus is replication-competent and demonstrates an enhanced growth rate in vitro. While the MDV homologs of herpes simplex US genes are retained in this virus, at least two open reading frames are deleted and one transcript observed in wild-type virus is disrupted. In addition to defining several regions of MDV that are nonessential for in vitro growth, this clone illustrates one way in which retroviral information can be stably transmitted by a herpesvirus.

Retroviral insertions into a herpesvirus are clustered at the junctions of the short repeat and short unique sequences.

We previously described the integration of a nonacute retrovirus, reticuloendotheliosis virus (REV), into the genome of a herpesvirus, Marek disease virus (MDV), following both long-term and short-term coinfection in cultured fibroblasts. The long-term coinfection occurred in the course of attenuating the oncogenicity of the JM strain of MDV and was sustained for > 100 passages. The short-term coinfection, which spanned only 16 passages, was designed to recreate the insertion phenomenon under controlled conditions. We found that REV integrations into MDV were common and could occur within the first passage following coinfection. Now we have mapped the integration sites. After 5-16 passages in vitro, 17 out of 19 REV junction sites are clustered in two 1-kilobase regions at the junctions of the short unique and short repeat region of MDV. In the long-term cocultivation experiment, 6 out of 10 insertions also mapped in this region. In both cases, integrated proviruses are unstable and undergo subsequent recombinative deletion, often leaving a solitary long terminal repeat. The long terminal repeat sequences are, however, stably maintained for many rounds of passaging in vitro. This clustering of insertions presumably is influenced by selection for viable and fast-growing viruses, and occurs in a region of the MDV genome which shows significant size heterogeneity in several strains.

Localized MR 1H spectroscopy reveals alterations of susceptibility in bone marrow with hemosiderosis.

A noninvasive investigation of the structure of hemopoietic bone marrow is based on the determination of the magnetic field distribution within small volume elements in vertebral bodies by localized 1H MR spectroscopy. In patients with hematological diseases the status of the bone marrow was found to considerably influence the homogeneity of the magnetic field in trabecular bone in vivo. The line widths of the 1H signals were evaluated in follow-up studies during initial chemotherapy of eight patients with leukemia. Intraindividual comparison revealed significant broadening of the field distribution after a few weeks of cytotoxic treatment in five of the patients. Additionally, 19 patients after bone marrow transplantation showed significantly broader field distributions in the lipid signals than 13 matched healthy volunteers. These alterations of the microscopic field homogeneity were not caused by trabecular density effects. Iliac crest biopsies revealed high amounts of hemosiderin in the cases with broadened line widths. Ten of the 19 patients after bone marrow transplantation showed high amounts of hemosiderin and broad lines in the spectra. The content of hemosiderin of the other patients was not significantly increased.

Hematopoietic reconstitution after bone marrow transplantation: assessment with MR imaging and H-1 localized spectroscopy.

Magnetic resonance (MR) studies were performed in 14 patients as early as possible (21-110 days) after bone marrow transplantation (BMT). MR characteristics of lumbar vertebral bone marrow were studied with T1-weighted spin-echo imaging, water- and fat-selective imaging with a frequency-selective excitation technique, and point-resolved spatially localized proton spectroscopy. Signals from water and fat protons and their T1 and T2 values were analyzed. Water proton signal intensity correlated well with cellularity within bone marrow, as determined with parallel iliac crest biopsies. The fraction of signal from water in red bone marrow of patients with allogeneic transplants from siblings (four cases) was significantly higher than in four patients with autologous transplants. The latter showed very low cellularity in the period of about 4 weeks after BMT because of the cytotoxic pretreatment of the bone marrow. The MR results in six patients with allogeneic transplants from unrelated donors ranged widely, depending on the complications after BMT. Analysis of data obtained with the different techniques showed that water- and fat-selective MR imaging and spectroscopic methods are useful for noninvasive monitoring of hematopoietic reconstitution after BMT.

Virologic and immunologic effect of antiretroviral therapy on HIV-1 in gut-associated lymphoid tissue.

OBJECTIVES: We evaluated virologic and immunologic responses to antiretroviral therapy in gut-associated lymphoid tissue (GALT) compared with those found in peripheral blood. METHODS: Eight HIV-1-infected individuals were treated with three reverse transcriptase inhibitors and one protease inhibitor. Endoscopic biopsies were performed at baseline, and at months 1, 2, and 6. We measured the level of cell-associated multiply spliced and unspliced HIV-1 mRNA in GALT and in peripheral blood mononuclear cells. Immunologic responses were assessed by flow cytometry. RESULTS: Levels of multiply spliced HIV-1 mRNA declined in parallel fashion both in peripheral blood and GALT. After 6 months of therapy, unspliced HIV-1 mRNA in the GALT was below assay detection although it persisted in peripheral blood mononuclear cells in 4 study subjects. Although the percentage of CD4+ lymphocytes increased significantly in peripheral blood, only modest increases occurred in GALT. The percentage of activated CD8+ T cells decreased significantly in peripheral blood whereas only modest reductions occurred in GALT. CONCLUSIONS: Antiretroviral therapy effectively suppressed HIV-1 replication in GALT. The percentage of CD4+ T cells in peripheral blood uniformly increased in all study subjects, whereas it was more variable in the GALT.

Open-label phase II trial of amprenavir, abacavir, and fixed-dose zidovudine/lamivudine in newly and chronically HIV-1--infected patients.

A Phase II clinical trial was designed to evaluate the efficacy and tolerability of twice-daily abacavir, amprenavir, and zidovudine (ZDV)/lamivudine (3TC) in HIV-1-infected study subjects naive to protease inhibitors and 3TC. Plasma and cerebrospinal fluid (CSF) HIV-1 RNA levels and T-cell subsets were measured. In all, 27 newly diagnosed and 12 chronically HIV-1-infected study subjects are included in the analysis. Week 48 plasma HIV-1 RNA levels were <500 copies/ml in 100% of study subjects, and <50 copies/ml in 80% of chronically infected and 100% of newly infected study subjects. The mean change in CD4 was (+)150 cells/microl (newly infected, p <.001), and (+)155 cells/microl (chronically infected, p <.001). At Week 48, evidence of cellular activation persisted in both cohorts. A twice-daily regimen of amprenavir, abacavir, and ZDV/3TC affords potent viral suppression and significant increases in total CD4(+) cells in HIV-1--infected study subjects. Patient intolerance may limit the efficacy of this combination.

Placebo-controlled trial of vaccination with recombinant glycoprotein D of herpes simplex virus type 2 for immunotherapy of genital herpes.

Immunotherapy of chronic viral diseases with vaccines is an important but unproven concept. We investigated the effect of a vaccine containing recombinant glycoprotein D (gD2) of herpes simplex virus type 2 (HSV-2) on the frequency of symptomatic outbreaks in patients with genital herpes. 98 patients with documented genital herpes who reported 4-14 recurrences per year were enrolled in a double-blind, placebo-controlled trial. Subjects received injections of either 100 micrograms gD2 in alum or alum alone (placebo) at 0 and 2 months, and recurrences were documented for 1 year. The vaccine was well tolerated. gD2 recipients reported fewer recurrences per month than placebo recipients (mean 0.42 [SE 0.05] vs 0.55 [0.05]; p = 0.055), had fewer virologically confirmed recurrences per month (0.18 [0.03] vs 0.28 [0.03]; p = 0.019), and had a lower median number of recurrences for the study year (4 [range 0-17] vs 6 [0-15]; p = 0.039). Neither genital recurrence nor the placebo vaccine had any discernible effect on HSV-2-specific antibody responses, but gD2 vaccine boosted neutralising antibodies to HSV-2 fourfold and gD2-specific titres sevenfold over baseline levels. These results inspire optimism about the potential use of vaccine for the treatment of chronic, recurring viral diseases.

Immunotherapy of recurrent genital herpes with recombinant herpes simplex virus type 2 glycoproteins D and B: results of a placebo-controlled vaccine trial.

To determine the safety, immunogenicity, and efficacy of a recombinant herpes simplex virus type 2 glycoprotein D and B vaccine in the treatment of recurrent genital herpes, a randomized, placebo-controlled trial was held at two referral centers. Healthy patients with 4-14 recurrences per year received injections of both glycoproteins in MF59 adjuvant or of MF59 alone at 0, 2, 12, and 14 months. For 18 study months, the rate and number of recurrences, the duration and severity of the first confirmed recurrence, vaccine immunogenicity, and rates of local and systemic reactions were determined. The monthly rate of recurrences was not significantly improved, but the duration and severity of the first study outbreak was reduced significantly by vaccination. Glycoprotein-specific and neutralizing antibodies were boosted by vaccination for the duration of the study. This vaccine is safe and immunogenic and ameliorated an observed first postvaccination genital recurrence, but it does not reduce recurrence frequency.