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Biomedical subjects

H C Lane

Publications and source records attributed to H C Lane.

At least 19 recordsLinked to original sources
PubMed2003

A comparison of microLC/electrospray ionization-MS and GC/MS for the measurement of stable isotope enrichment from a [2H2]-glucose metabolic probe in T-cell genomic DNA.

Measurement of the proliferation of lymphocytes and other high-turnover cell populations in vivo can be accomplished through the incorporation of an isotopically labeled DNA precursor into actively dividing cells and the subsequent determination of the isotope enrichment in the isolated genomic DNA from selected cell populations. Two published gas chromatography/mass spectrometry (GC/MS) methods were successfully modified by our laboratory whereby a postinjection methylation reaction, rather than silylation or acetylation, was used to form a volatile derivative of deoxyadenosine (dA). We also developed a second robust microcapillary liquid chromatography-electrospray ionization (microLC-ESI)/MS method that is faster and more sensitive than the GC/MS method and does not require sample derivatization. Following administration of [6,6-(2)H(2)]-glucose to human immunodeficiency virus-infected patients, peripheral blood was drawn; cells were obtained by lymphapheresis and fractionated. DNA was isolated from the desired cell subtypes and enzymatically hydrolyzed to the free deoxyribonucleosides. The digest was analyzed using both capillary GC/MS and microLC/ESI-MS to measure the levels of the dA and [(2)H(2)]-dA or their reaction products. Sample enrichments were calculated by comparison to standard curves prepared from dA and [(2)H(2)]-dA. The microLC/ESI-MS method required fewer cells, less sample preparation, shorter analysis times, and a single calibration curve. Overall, the microLC/ESI-MS method is superior to the GC/MS method in terms of precision and accuracy, while providing a 4-fold increase in sensitivity (from 20 pmol at 0.2% [(2)H(2)]-dA enrichment to 5 pmol at 0.1% [(2)H(2)]-dA enrichment).

Chromatography, Liquid↗
PubMed2001

Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV.

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = -0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.

Adult↗
PubMed2001

CD4 T cell expansions are associated with increased apoptosis rates of T lymphocytes during IL-2 cycles in HIV infected patients.

OBJECTIVE AND DESIGN: In an attempt to determine the mechanisms underlying the CD4 T cell expansions in patients receiving intermittent interleukin (IL)-2, a cohort of 10 HIV infected patients were studied during a 5-day cycle of IL-2 to measure rates of apoptosis, the expression of activation markers in CD4 and CD8 T cell subsets and the serum levels of proinflammatory cytokines. All patients were receiving highly active antiretroviral therapy. METHODS: Peripheral blood mononuclear cells were tested pre- and at the completion of IL-2 treatment with annexin V/7-AAD for the measurement of apoptosis. Phenotypic analyses of T lymphocytes were performed in parallel. Serum levels of interferon (IFN)gamma, granulocyte-macrophage colony stimulating factor, IL-6 and tumor necrosis factor (TNF)alpha were tested by enzyme-linked immunosorbent assay. RESULTS: IL-2 increased the spontaneous apoptosis rates of CD4 and CD8 T lymphocytes (P = 0.003). Expression of HLA-DR, CD38 and CD95 increased on both CD4 and CD8 T lymphocytes whereas CD25 induction was observed exclusively on CD4 T cells. Significant increases of serum IL-6 and TNFalpha levels were noted in all patients whereas viral loads remained unchanged. CONCLUSION: Administration of IL-2 for 5 days in HIV infected patients leads to enhanced apoptosis of both CD4 and CD8 T cells despite an eventual increase of the CD4 T cell count. A profound activation state with induction of activation markers on T cells and high levels of TNFalpha and IL-6 accompanies the increased apoptosis during the IL-2 cycle. These data suggest that the CD4 expansions seen in the context of intermittent IL-2 therapy are the net result of increases in both cell proliferation and cell death.

Antiretroviral Therapy, Highly Active↗
PubMed2001

Immunopathogenesis of human immunodeficiency virus: implications for immune-based therapies.

Human immunodeficiency virus (HIV) infection leads to a state of CD4 lymphopenia and generalized immune activation with subsequent development of opportunistic infections and neoplasms. The use of highly active antiretroviral treatment has dramatically improved the clinical outcome for HIV-infected patients, but the associated cost and toxicity and the eventual development of drug resistance have underscored the need for additional therapeutic strategies. Immune-based therapies, such as treatment with cytokines or immunosuppressants, adoptive immunotherapy, and therapeutic immunizations, are being intensely investigated as potential supplements to antiretroviral therapy. Although much data have been generated as a result of these efforts, to date there has been little evidence of the clinical efficacy of these strategies. Randomized clinical studies remain critical in evaluating the clinical significance and the role of immune-based therapies in the therapeutic armamentarium against HIV.

Acute Disease↗
PubMed2001

Interleukin-2 induced immune effects in human immunodeficiency virus-infected patients receiving intermittent interleukin-2 immunotherapy.

To characterize the immunological effects of intermittent IL-2 therapy, which leads to selective increases in CD4+ T lymphocytes in HIV-infected patients, 11 patients underwent extensive immunological evaluation. While IL-2 induced changes in both CD4+ and CD8+ cell number acutely, only CD4+ cells showed sustained increases following discontinuation of IL-2. Transient increases in expression of the activation markers CD38 and HLA-DR were seen on both CD4+ and CD8+ cells, but CD25 (a chain of the IL-2 receptor) increased exclusively on CD4+ cells. This increase in CD25 expression was sustained for months following discontinuation of IL-2, and was seen in naive as well as memory cells. IL-2 induced cell proliferation, but tachyphylaxis to these proliferative effects developed after 1 week despite continued IL-2 administration. It thus appears that sustained CD25 expression selectively on CD4+ cells is a critical component of the immunological response to IL-2, and that intermittent administration of IL-2 is necessary to overcome the tachyphylaxis to IL-2-induced proliferation.

ADP-ribosyl Cyclase↗
PubMed2001

Immunotherapy of HIV-infected patients with intermittent interleukin-2: effects of cycle frequency and cycle duration on degree of CD4(+) T-lymphocyte expansion.

The ability of IL-2 to induce expansion of the CD4(+) T lymphocyte pool has made it the most studied cytokine in the treatment of HIV infection. The majority of trials have used an empirical regimen of 5-day IL-2 cycles given every 8 weeks--a regimen based upon early pharmacodynamic studies and patient preference. To better define optimal duration and frequency of cycles, a randomized trial was conducted in which patients who received this "standard" regimen were compared to patients who received cycles of variable duration (based on individual patterns of cell cycle progression) and to patients who received cycles of variable frequency (based on individual CD4(+) T lymphocyte responses to previous cycles). Twenty-two patients with HIV-1 infection and CD4(+) T lymphocyte counts > 200 cells/mm(3) were randomized to one of three treatment groups for 32 weeks of study. Eight participants received four 5-day IL-2 cycles (controls) every 8 weeks; 7 participants received four cycles of longer duration (mean 7.7-days); and 7 participants received an increased frequency of 5-day cycles (every 4.1 weeks on average). All three groups experienced significant increases in mean CD4(+) T lymphocytes. However, there were no statistically significant differences in CD4(+) T lymphocyte increases between the group that received longer cycles (median increase 239 cells/mm(3), P = 0.78) or between the group that received more frequent cycles (median increase 511 cells/mm(3), P = 0.54) and the control group (median 284 cells/mm(3)). HIV-1 viral loads decreased during the study period in all three groups. Our inability to demonstrate a significant advantage of increased frequency or duration of IL-2 administration provides corroborating experimental evidence for the use of an IL-2 regimen consisting of 5-day cycles administered no more frequently than every 8 weeks in future clinical trials aimed at expanding the CD4(+) T lymphocyte pool.

Adult↗
PubMed2001

Amino acid deletion at codon 67 and Thr-to-Gly change at codon 69 of human immunodeficiency virus type 1 reverse transcriptase confer novel drug resistance profiles.

The potential roles of an amino acid deletion at codon 67 (Delta67) and a Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase of human immunodeficiency virus (HIV) type 1 in drug sensitivity and relative replication fitness were studied. Our results suggest that the Delta67 and T69G changes can be categorized as mutations associated with multidrug resistance. The combination of both mutations with an L74I change (Delta67+T69G/L74I) leads to a novel 3'-azido-3'-deoxythymidine resistance motif and compensates for impaired HIV replication.

Amino Acid Motifs↗
PubMed2000

Human immunodeficiency virus type 1 quasi species that rebound after discontinuation of highly active antiretroviral therapy are similar to the viral quasi species present before initiation of therapy.

In an effort to identify the sources of the viruses that emerge after discontinuation of therapy, analyses of human immunodeficiency virus (HIV) quasi species were done for 3 patients with sustained levels of HIV RNA of <50 copies/mL for 1-3 years. The sequences found in the rebounding plasma virus were closely related to those of the actively replicating form of viruses present before the initiation of combination therapy. All quasi species found in the rebounding plasma virus were also present in proviral DNA, cell-associated RNA in peripheral blood mononuclear cells (PBMC), and virion RNA derived from PBMC coculture during periods when plasma HIV RNA levels were <50 copies/mL. These findings suggest that the rapid resurgence of plasma viremia observed after discontinuation of therapy and the viruses cocultured from PBMC are derived from a relatively stable pool of the replicating form of virus rather than from activation of a previously latent pool.

Adult↗
PubMed2000

Impact of HIV-1 infection and highly active antiretroviral therapy on the kinetics of CD4+ and CD8+ T cell turnover in HIV-infected patients.

To evaluate the effects of HIV infection on T cell turnover, we examined levels of DNA synthesis in lymph node and peripheral blood mononuclear cell subsets by using ex vivo labeling with BrdUrd. Compared with healthy controls (n = 67), HIV-infected patients (n = 57) had significant increases in the number and fraction of dividing CD4(+) and CD8(+) T cells. Higher percentages of dividing CD4(+) and CD8(+) T cells were noted in patients with the higher viral burdens. No direct correlation was noted between rates of T cell turnover and CD4(+) T cell counts. Marked reductions in CD4(+) and CD8(+) T cell proliferation were seen in 11/11 patients 1-12 weeks after initiation of highly active antiretroviral therapy (HAART). These reductions persisted for the length of the study (16-72 weeks). Decreases in naive T cell proliferation correlated with increases in the levels of T cell receptor rearrangement excision circles. Division of CD4(+) and CD8(+) T cells increased dramatically in association with rapid increases in HIV-1 viral loads in 9/9 patients 5 weeks after termination of HAART and declined to pre-HAART-termination levels 8 weeks after reinitiation of therapy. These data are consistent with the hypothesis that HIV-1 infection induces a viral burden-related, global activation of the immune system, leading to increases in lymphocyte proliferation.

Antiretroviral Therapy, Highly Active↗
PubMed2000

Longitudinal changes in CD4+ T cell antigen receptor diversity and naive/memory cell phenotype during 9 to 26 months of antiretroviral therapy of HIV-infected patients.

Although skewing of the CD4+ TCR repertoire in advanced HIV infection is well documented, increases in polyclonality during antiretroviral therapy have been less consistently observed. Ten patients, each with documented abnormalities within the CD4+ TCR repertoire, were studied by CDR3 spectratyping, semiquantitative PCR, and SSCP during 9-26 months of therapy. Naive and memory cell phenotypes were analyzed by flow cytometry. Six of 10 patients showed increased polyclonality of their TCR repertoires, 1 showed no change, and 3 showed increased TCR skewing, despite suppressed viral replication. Overall, there was no significant change in the percentage of abnormal BV subfamilies (from a mean of 25.5 to 17.1%) or the percentage of naive CD4+ T cells (from a mean of 18 to 25%). Further, progression of TCR repertoire disruptions was observed in some patients even with suppression of plasma viral RNA below 500 copies/ml. Although a spectrum of changes may be seen within the CD4+ TCR repertoire in the setting of antiretroviral therapy, increases in polyclonality are observed in some patients.

Anti-HIV Agents↗
PubMed2000

A randomized, controlled 24-week study of intermittent subcutaneous interleukin-2 in HIV-1 infected patients in Thailand.

OBJECTIVES: To assess the immunological and virological effects, safety profile and feasibility of subcutaneous interleukin-2 (scIL-2) therapy in an HIV-infected Thai population. DESIGN: Seventy-two patients with baseline CD4 cell count of > or = 350 x 10(6)/l and no history of opportunistic infection were randomized to receive antiretroviral therapy plus scIL-2 (scIL-2 group) or antiretroviral therapy alone (control group). scIL-2 was administered at one of three doses for at least 24 weeks. The main measure of treatment efficacy was change in CD4 cell count. RESULTS: The time-weighted mean change in CD4 cell count from baseline to week 24 was + 252 x 10(6)/l for the scIL-2 group compared with + 42 x 10(6)/l for the control group (P< 0.0001). Changes in plasma HIV RNA were not significantly different between the groups over the same time period: there was a 0.83 log10 copies/ml decrease for the scIL-2 group and a 0.70 log copies/ml decrease for the control group (P= 0.362). CONCLUSIONS: This study provides the most extensive experience of scIL-2 therapy in HIV-1 infected women and Asians, and demonstrates the immunological efficacy, tolerability and feasability of scIL-2 therapy in this population. Data from this study were instrumental in guiding the selection of the scIL-2 dosing regimen for ongoing phase III trials.

Adult↗
PubMed2000

Effects of intermittent interleukin-2 therapy on plasma and tissue human immunodeficiency virus levels and quasi-species expression.

To characterize the effects of intermittent interleukin (IL)-2 therapy on human immunodeficiency virus (HIV), 11 patients underwent detailed virological evaluation during a year of IL-2 therapy. Six patients showed a >0.5 log increase in plasma HIV during at least 1 IL-2 cycle, with 2 experiencing an increase in >50% of cycles. Three of the remaining 5 patients had a >0.5 log decrease during at least 1 IL-2 cycle, and the remaining patients exhibited <0.5 log changes. No changes in lymphoid (tonsil) levels of HIV were seen during the year. Quasi-species analysis in a separate cohort demonstrated that the virus induced by IL-2 most commonly resembled pre-IL-2 plasma quasi species. Thus, intermittent IL-2 does not result in sustained increases in either plasma or tissue levels of HIV and does not result in sustained expression of a previously silent quasi species.

Adult↗
PubMed2000

Rapid activation of lymph nodes and mononuclear cell HIV expression upon interrupting highly active antiretroviral therapy in patients after prolonged viral suppression.

OBJECTIVE: To compare the architecture and HIV-1 RNA and Gag p24 protein expression in lymph nodes (LN) excised from individuals during chronic highly active antiretroviral therapy (HAART) with LN removed from the same patient after plasma virus rebound following the interruption of HAART. MATERIALS AND METHODS: Six HIV-1-infected patients on HAART, with CD4 cell counts greater than 350 cells/microl, and plasma HIV-1 RNA less than 50 copies/ml, underwent inguinal LN excision upon discontinuation of HAART, and again after rebound of plasma virus. Lymph nodes were evaluated by immunohistochemical staining for Gag p24 antigen and Ki67, in-situ hybridization for HIV-1 RNA and H3-histone, and transmission electron microscopy (TEM). RESULTS: LN at baseline were quiescent to mildly hyperplastic and generally contained more primary than secondary follicles. Only one LN had detectable follicular dendritic cell (FDC)-associated p24 antigen, none had HIV RNA. Few mononuclear cells (MNC) expressed RNA or p24 antigen. Plasma virus at the second biopsy ranged from 329 to 3.2 x 10(6) copies/ml. CD4 cell count decline ranged from 5 to 51% during drug hiatus, and was greatest in patients with highest viral rebound. Four of six of the second LN were more hyperplastic than the initial LN, two showed paracortical hyperplasia. MNC expression of HIV RNA in the second LN paralleled the level of plasma viremia. Increased Ki67 and H3-histone signal occurred in the second LN. CONCLUSION: Quiescent LN from individuals on HAART rapidly become hyperplastic and activated within 1-2 months after treatment interruption. As in acute HIV infection, virus expression by LN MNC parallels the rebound in plasma viremia and fall in CD4 cell count.

Antiretroviral Therapy, Highly Active↗
PubMed2000

Pooled analysis of 3 randomized, controlled trials of interleukin-2 therapy in adult human immunodeficiency virus type 1 disease.

We collected human immunodeficiency virus (HIV) disease progression, survival, most recent CD4 cell count, and plasma HIV RNA levels from patients (n=157) who participated in randomized clinical trials of interleukin (IL)-2 that commenced before 1995. Data were available for 155 (99%) patients. Statistical analyses were based on the intention-to-treat principle. Median follow-up was 28 months and 30 months for control and IL-2 patients, respectively. Twenty-five (16%) patients developed AIDS or died during follow-up (16 control patients vs. 9 IL-2 patients; R2=0.57; P=.22). Mean change from baseline CD4 cell count was significantly higher in patients randomized to receive IL-2 (368 vs. 153 cells/microL; P=.003). Mean change from baseline plasma HIV RNA was significantly lower in patients randomized to receive IL-2 (-0.98 vs. -0.63 log copies/mL; P=.004). Significant improvements in CD4 cell count and plasma HIV RNA in recipients of IL-2 relative to control patients were associated with a nonsignificant trend toward improved clinical outcome.

Adult↗
PubMed2000

Evaluation of CD4+ T cell function In vivo in HIV-infected patients as measured by bacteriophage phiX174 immunization.

Bacteriophage phiX174 immunization was used to measure CD4(+) T cell function in vivo in human immunodeficiency virus (HIV)-infected patients across all disease stages. Function was evaluated by measuring the ability of T cells to provide help to B cells in antibody production, amplification, and isotype switching. A total of 33 patients and 10 controls received 3 bacteriophage phiX174 immunizations 6 weeks apart. The patients' responses regarding bacteriophage-specific total antibody titers and IgG titers were quantitatively and qualitatively inferior to the controls' responses. Overall, 7 of 33 patients had normal T cell function. Baseline CD4 counts provided the strongest correlation with total antibody and IgG titers. HIV RNA had a weaker association with responses but had some predictive power among patients with a CD4 count >200 cells/microL. Bacteriophage phiX174 immunization seems to be a useful tool for measuring immune function in vivo, which suggests that most HIV-infected patients may have abnormal CD4(+) T cell function despite adequate antiretroviral treatment.

Adolescent↗