Prevalence of HIV Drug Resistance Mutations in HIV Type 1 Isolates in Antiretroviral Therapy Naïve Population from Northern India.

Objective. The increased use of antiretroviral therapy (ART) has reduced the morbidity and mortality associated with HIV, adversely leading to the emergence of HIV drug resistance (HIVDR). In this study we aim to evaluate the prevalence of HIVDR mutations in ART-naive HIV-1 infected patients from northern India. Design. Analysis was performed using Viroseq genotyping system based on sequencing of entire protease and two-thirds of the Reverse Transcriptase (RT) region of pol gene. Results. Seventy three chronic HIV-1 infected ART naïve patients eligible for first line ART were enrolled from April 2006 to August 2008. In 68 patients DNA was successfully amplified and sequencing was done. 97% of HIV-1 strains belonged to subtype C, and one each to subtype A1 and subtype B. The overall prevalence of primary DRMs was 2.9% [2/68, 95% confidence interval (CI), 0.3%-10.2%]. One patient had a major RT mutation M184V, known to confer resistance to lamivudine, and another had a major protease inhibitor (PI) mutation D30N that imparts resistance to nelfinavir. Conclusion. Our study shows that primary HIVDR mutations have a prevalence of 2.9% among ART-naive chronic HIV-1 infected individuals.

Continuing medical education on antiretroviral therapy in HIV/AIDS in India: needs assessment and impact on clinicians and allied health personnel.

BACKGROUND: Clinicians and associated health professionals charged with prescribing antiretroviral therapy (ART) deal with continuously evolving new drugs and combinations. To meet the needs of clinicians in India for ongoing education in this field, continuing medical education (CME) programmes on ART for HIV/AIDS were developed, conducted, evaluated and revised. Over a 2-year period, 2005-2007, 3 CME programmes for ART were conducted for physicians and a fourth (predominantly) for paediatricians. METHODS: Both 1- and 2-day CME programmes on various aspects of ART were held on weekends for professionals treating patients with AIDS in Delhi and adjacent states. Topics included characteristics of ART drugs, their dosages, monitoring and toxicity management, adherence, complications of therapy, dealing with treatment failure and HIV co-infections. These topics were addressed in lectures and group discussions and via case presentations. Programmes were evaluated by anonymous response to questionnaires, by a 1-year follow up of participants and by informal discussions with participants and faculty. Detailed analyses and a recommended format for these programmes are presented. RESULTS: The CMEs were attended primarily by clinicians (physicians and paediatricians). Nurses, laboratory scientists, and others involved in the treatment of AIDS also attended the programmes. An interactive workshop format was evolved with substantial time devoted to discussions and case analyses. One-day programmes such as the one included here can be comprehensive and effective. The educational needs of healthcare professionals who provide care and support to patients receiving ART were similar to those of the prescribing doctors. Because of new drugs being made available and with continued clinical experience, updated programme content was required each year. Participants preferred case-based interactive discussions rather than didactic lectures. Participants suggested that there should be more time for discussion after each talk. CONCLUSION: Annual CME programmes focused on ART are required to meet the professional needs of clinicians in India for providing quality care management to patients with AIDS.

Impaired response to HAART in HIV-infected individuals with high autonomic nervous system activity.

Neurotransmitters can accelerate HIV-1 replication in vitro, leading us to examine whether differences in autonomic nervous system (ANS) activity might promote residual HIV-1 replication in patients treated with highly active antiretroviral therapy. Patients who showed constitutively high levels of ANS activity before highly active antiretroviral therapy experienced poorer suppression of plasma viral load and poorer CD4(+) T cell recovery over 3-11 months of therapy. ANS activity was not related to demographic or behavioral characteristics that might influence pathogenesis. However, the ANS neurotransmitter norepinephrine enhanced replication of both CCR5- and CXCR4-tropic strains of HIV-1 in vitro via chemokine receptor up-regulation and enhanced viral gene expression, suggesting that neural activity may directly promote residual viral replication.

Idiopathic CD4+ T cell lymphocytopenia evolving to monoclonal immunoglobulins and progressive renal damage responsive to IL-2 therapy.

Idiopathic CD4+ T cell lymphocytopenia was unexpectedly detected in a 33-year-old, otherwise healthy young woman with no HIV or other viral infection, autoimmune, or neoplastic disease or increased susceptibility to infection. CD4+ T cell levels were 60-140/microl over a 3.5-year period. Following an uneventful pregnancy, the patient developed anemia and interstitial nephritis associated with a plasma cell dyscrasia with a monoclonal IgA gammopathy and a shifting immunoglobulin pattern that included IgG and IgA monoclonal proteins and increased urinary light chains. Osteolytic lesions were never detected and bone marrow aspirations revealed up to 10% atypical plasma cells. Various therapies often used in treating multiple myeloma only temporarily controlled the increasing renal damage. IL-2 therapy of 600,000 to 1 million units subcutaneously daily resulted in increased CD4+ T cells to normal levels, a decrease in the gammopathy, a return of renal function, energy, and weight gain, and apparently normal health status sustained for 2 years. The findings are compatible with a potentially fatal but nonmalignant immunoregulatory disorder that can be controlled by IL-2 administration.

Thalidomide in low intermittent doses does not prevent recurrence of human immunodeficiency virus-associated aphthous ulcers.

A multicenter, double-blind, randomized, placebo-controlled study was conducted to determine the safety and efficacy of thalidomide in reduced, intermittent doses for preventing recurrences of oral and esophageal aphthous ulcers in patients with human immunodeficiency virus (HIV) infection. Forty-nine HIV-infected patients whose ulcers previously had healed as a result of thalidomide therapy were randomly assigned to receive either 100 mg of oral thalidomide or placebo 3 times per week for 6 months. Ulcers recurred in 14 (61%) of 23 thalidomide-randomized patients, compared with 11 (42%) of 26 placebo-randomized patients, with no significant difference in the median time to recurrence of ulcers (P=.221). There were no changes in plasma levels of HIV RNA, tumor necrosis factor (TNF)-alpha, and soluble TNF receptor II at the time of ulcer recurrence. Adverse events among patients treated with thalidomide included neutropenia (5 patients), rash (5 patients), and peripheral sensory neuropathy (3 patients). Thalidomide in lower intermittent doses is ineffective at preventing recurrence of aphthous ulcers in HIV-infected persons.

Cytokine gene expression occurs more rapidly in stimulated peripheral blood mononuclear cells from human immunodeficiency virus-infected persons.

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV-) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV- and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P<0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-alpha]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV- PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-alpha mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P<0.05) and TNF-alpha (P<0.005), compared to HIV- PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-alpha) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.

Need for an external proficiency testing program for cytokines, chemokines, and plasma markers of immune activation.

An external evaluation program for measuring the performance of laboratories testing for cytokines and immune activation markers in biological fluids was developed. Cytokines, chemokines, soluble cytokine receptors, and other soluble markers of immune activation (CSM) were measured in plasma from a healthy human immunodeficiency virus (HIV)-seronegative reference population and from HIV-seropositive individuals as well as in supernatant fluids from in vitro-stimulated human immune cells. The 14 components measured were tumor necrosis factor (TNF) alpha, gamma interferon, interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, Rantes, MIP-Ia, MIP-Ibeta, soluble TNF receptor II, soluble IL-2 receptor alpha, beta(2)-microglobulin, and neopterin. Twelve laboratories associated with the Adult and Pediatric AIDS Clinical Trial Groups participated in the study. The performance features that were evaluated included intralaboratory variability, interlaboratory variability, comparison of reagent sources, and ability to detect CSM in the plasma of normal subjects as well as the changes occurring in disease. The principal findings were as follows: (i) on initial testing, i.e., before participating in the program, laboratories frequently differed markedly in their analytic results; (ii) the quality of testing of a CSM in individual participating laboratories could be assessed; (iii) most commercial kits allowed distinction between normal and abnormal plasma CSM levels and between supernatants of stimulated and unstimulated cells; (iv) different sources of reagents and reference standards frequently provided different absolute values; (v) inexperienced laboratories can benefit from participating in the program; (vi) laboratory performance improved during active participation in the program; and (vii) comparability between analyses conducted at different sites can be ensured by an external proficiency testing program.

Distinct categories of immunologic changes in frail elderly.

Immune changes and their relationships in a frail elderly population (N=116, age 70-103, median 86 years) were defined in comparison to a healthy younger group. Previous immune studies in the elderly have generally focused on one or few parameters without correlation analyses. Furthermore, the study populations have been active elderly in relatively small numbers. A total of 33 immune parameters representing many aspects of the immune system were quantified. Most changes in the frail elderly were parallel to those reported in active elderly. A classification tree analysis revealed that increased plasma activation markers (neopterin and sTNF-R) and increased CD28 expression on CD8 T cells and proliferative response separated the aged and control populations. Statistical procedures utilizing principal components analyses, partial correlations and exploratory factor analyses all indicated that immunologic parameters in frail elderly are grouped in three major clusters of immunologic results. These involved (a) increased plasma levels of neopterin and sTNF receptor indicating elevated IFNgamma and TNF cytokine activity; (b) increased proportion of mature (CD45RO) versus naïve (CD45RA) T cells; and (c) a diverse group of related changes including impaired proliferative response, reduced T cells, CD28 and CD25 expression, B cell percentage and lower CD4:CD8 ratios and increased HLA-DR expression. These findings emphasize that several different groups of immune parameters but not 33 independent immune changes, occurred in the aged population.

Increased natural killer-cell mobilization and cytotoxicity during marital conflict.

Natural killer (NK) cells are reproducibly mobilized into the circulation in response to intense physical exercise or acute psychological stress, and altered expression of adhesion molecules potentially contributes to NK-cell mobilization. Studies of leukocyte mobilization during acute stress have used psychological stressors which facilitate tight experimental control but have limited applicability to everyday life. We therefore used a laboratory model of marital conflict as an experientially meaningful acute stressor to elucidate relationships among conflict, cardiovascular reactivity, and altered leukocyte phenotype and function. Forty-one ethnically diverse, nondistressed, healthy married couples were asked to discuss a specific problem in their marriage for 15 min. Blood pressure and heart rate were measured before, during, and after the discussion, and blood was remotely drawn at the same time points to quantify numbers of specific leukocyte subsets, NK-cell adhesion molecule expression, and NK cytotoxicity. Couples responded to the conflict task with cardiovascular reactivity; increases in the percentages of circulating NK cells and CD8(+) T cells and decreases in the percentage of circulating CD4(+) T cells; decreases in the percentage of NK cells that express L-selectin; and increases in NK-cell cytotoxicity without a commensurate increase in per-cell cytotoxicity. Rapid downregulation or shedding of L-selectin (CD62L) from NK cells did not contribute to their mobilization during conflict. Instead, CD62L(-) NK cells were mobilized while CD62L(+) NK cells were selectively retained in the vascular marginating pool and/or in extravascular tissue. From a broader perspective, the data support the hypothesis that altered trafficking of specific leukocyte subsets is an integral component of the fight-or-flight response to an acute stressor.

Thalidomide for the treatment of esophageal aphthous ulcers in patients with human immunodeficiency virus infection. National Institute of Allergy and Infectious Disease AIDS Clinical Trials Group.

A multicenter, double-blind, randomized, placebo-controlled clinical trial was conducted to determine the safety and efficacy of thalidomide for treating esophageal aphthous ulceration in persons infected with human immunodeficiency virus (HIV). Twenty-four HIV-infected patients with biopsy-confirmed aphthous ulceration of the esophagus were randomly assigned to receive either oral thalidomide, 200 mg/day, or oral placebo daily for 4 weeks. Eight (73%) of 11 patients randomized to receive thalidomide had complete healing of aphthous ulcers at the 4-week endoscopic evaluation, compared with 3 (23%) of 13 placebo-randomized patients (odds ratio, 13.82; 95% confidence interval, 1.16-823.75; P=.033). Odynophagia and impaired eating ability caused by esophageal aphthae were improved markedly by thalidomide treatment. Adverse events among patients receiving thalidomide included somnolence (4 patients), rash (2 patients), and peripheral sensory neuropathy (3 patients). Thalidomide is effective in healing aphthous ulceration of the esophagus in patients infected with HIV.

Psychosocial predictors of natural killer cell mobilization during marital conflict.

This study examined how specific emotions relate to autonomic nervous and immune system parameters and whether cynical hostility moderates this relationship. Forty-one married couples participated in a 15-min discussion about a marital problem. Observers recorded spouses' emotional expressions during the discussion, and cardiovascular, neuroendocrine, and immunologic parameters were assessed throughout the laboratory session. Among men high in cynical hostility, anger displayed during the conflict was associated with greater elevations in systolic and diastolic blood pressure, cortisol, and increases in natural killer cell numbers and cytotoxicity. Among men low in cynical hostility, anger was associated with smaller increases in heart rate and natural killer cell cytotoxicity. These findings suggest that models describing the impact of stress on physiology should be refined to reflect the joint contribution of situational and dispositional variables.

Worry affects the immune response to phobic fear.

Worry, the cognitive enumeration and anticipation of potential future negative events, is associated with autonomic dysregulation, which may in turn have implications for the immune system. People endorsing high (n = 7) and normal levels of trait worry (n = 8) were briefly exposed to a phobic stimulus and the autonomic and immune responses and recovery were assessed. A time-matched control group (n = 6) was not exposed to any stimulus. Both worry groups showed increased heart rate and skin conductance in response to phobic fear. However, only the normal worry group showed a concomitant increase in natural killer cells in peripheral blood. Patterns of change during the follow-up period suggested that phobic fear had disrupted a normal circadian increase in natural killer cells. Adrenergic and hypothalamus-pituitary-adrenal mechanisms may be responsible for the differences between high and normal worry groups in their natural killer cell response to and recovery from phobic fear.

Stability of plasma levels of cytokines and soluble activation markers in patients with human immunodeficiency virus infection.

Cytokine and immune activation marker levels in plasma are valuable measurements of immune status and treatment effects in human immunodeficiency virus (HIV) infection and AIDS. Five populations representing various stages of disease were studied: controls, 2 AIDS groups with <50/mm3 CD4 cells, and 2 groups of HIV-positive subjects-1 with stable CD4 T cells (median, 545/mm3) and 1 with >100/mm3 CD4 cell decline in 1 year. Relatively stable levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptor (R)II, soluble interleukin-2R, neopterin, and beta2-microglobulin (beta2M) were documented over 5-8 weeks in patients with AIDS and for 1-4 years in the other groups. beta2M was generally the most stable marker. Interferon-gamma levels, however, fluctuated substantially. Individuals, whether normal or HIV-positive, maintain characteristic plasma levels of cytokines and immune activation markers. Thus, documented changes, in excess of the variability observed in this study, are likely to be significant indicators of change in disease status or effects of therapy.

The prognostic significance in HIV infection of immune activation represented by cell surface antigen and plasma activation marker changes.

One hundred and eighteen HIV-infected homosexual men without AIDS and 40 control seronegative homosexual men were assessed for 23 parameters reflecting immune activation to determine prognostic significance for occurrence of AIDS. Samples cryopreserved in 1987-1989 were analyzed, with AIDS occurrence determined by mid-1992. Cell surface antigens assessed on the major lymphocyte subsets were HLA-DR, CD38, CD71, and CD25. Soluble serum molecules assessed were tumor necrosis factor alpha, soluble TNFalpha receptor II, soluble IL-2 receptor alpha, neopterin, and beta2-microglobulin. Using a proportional hazards model, prognostic markers included decreased CD4 number and percentage; increased sIL-2R, neopterin, and beta2M; increased percentage HLA-DR+ total lymphocytes and CD4+ cells; increased CD38+ total lymphocytes and CD8+ cells; increased CD71+ total lymphocytes and CD4+ cells; and decreased CD25+ total lymphocytes and CD19+ cells. After adjustment for CD4 cell levels, sIL-2R, neopterin, beta2M, and CD25+ CD19 cells remained significant, indicating that additional information about AIDS risk was provided by these markers.

Variables that affect assays for plasma cytokines and soluble activation markers.

Cytokines and soluble immune activation markers that reflect cytokine activities in vivo are increasingly being measured in plasma, serum, and other body fluids. They provide useful diagnostic and prognostic information as well as insight into disease pathogenesis. Assays of neopterin, beta2-microglobulin, soluble interleukin-2 receptor, and soluble tumor necrosis factor receptor type II as well as of the cytokines tumor necrosis factor alpha and gamma interferon (IFN-gamma) were evaluated by using serum and plasma samples of human immunodeficiency virus (HIV)-positive and HIV-negative subjects. Many factors were found to influence the outcomes of these assays. Substantial differences in apparent levels of analytes were frequently found when enzyme-linked immunosorbent assay (ELISA) kits from different manufacturers were used. In some cases, differences were found in the standards provided by separate manufacturers. Furthermore, the analytic results from different lots of ELISA kits supplied by single manufacturers differed by as much as 50%. The need for uniformity in the standards for quantitative assays was clearly illustrated. International reference standards are available for cytokines but not for soluble cytokine receptors or soluble activation markers. Marker levels in serum or in plasma were similar except those for IFN-gamma. Most of the analytes were stable under several storage conditions. Thus, batch testing of frozen stored samples is feasible. The findings indicate that for longitudinal studies, the levels of cytokines and immune activation markers in plasma or serum should be measured by using preverified reagents from one manufacturer. The quality of laboratory performance can have an impact on clinical relevance. Proficiency testing and external quality assurance programs can help to develop the needed consensus.

Phenotypic and functional characteristics of circulating monocytes of elderly persons.

Aging is associated with impairment of immune functions. Age-dependent alterations in T-cells are well known. Although the pivotal role of monocytes in immune regulation by their production of proinflammatory and inhibitory cytokines is acknowledged, limited information is available on monocyte changes in aging. The present study focused on phenotypic changes in circulating monocytes in elderly subjects and in the level of cytokines they produce. The results demonstrated a significant expansion of CD14dim/CD16bright circulating monocytes in elderly. In contrast, the majority of circulating monocytes of healthy young individuals were CD14bright/CD16dim. The CD14dim/CD16bright monocytes are considered to have phenotypic evidence for activation. Furthermore, significant increases of constitutive production of monocytic cytokines including interleukin (IL)-1beta. IL-1 receptor antagonist, and IL-6 by nonstimulated monocytes from elderly was also indicative of activation. This was also observed when monocytes from elderly were cultured with autologous lymphocytes. However, after stimulation, significantly lowered IL-1beta production was observed and IL-6 and IL-10 tended to be higher in the elderly. Collectively, these results indicate that monocytes of aged individuals, in contrast to a younger population exhibit in vivo activation as well as imbalanced production of cytokines. Such age-related alterations in monocytes may contribute to impaired immune competence of aging.

Relationship of worry to immune sequelae of the Northridge earthquake.

Worry is a cognitive activity in which potential problems are anticipated and enumerated in an attempt to control the future. Worry has been associated with dysregulation of the autonomic nervous system, which may extend to the immune system. The relationship between trait worry and immune parameters was investigated at three follow-up points after the Northridge earthquake in a sample of 47 hospital employees. Participants with scores above the median on a trait worry measure had fewer natural killer cells than participants with worry scores below the median and controls. This effect was not mediated by intrusive thoughts, avoidance, anxious mood, or health behavior. These results suggest that worry may have a detrimental effect on the regulation of natural killer cells during stress. This effect may be due to differences in autonomic responsiveness associated with worry.

Levels of cytokines and immune activation markers in plasma in human immunodeficiency virus infection: quality control procedures.

Procedures for quality control (QC) in a laboratory that concentrates on cytokine and soluble marker measurements in biological fluids are outlined. Intra-assay, interassay, and interlaboratory experiences are presented. Plasma and serum beta2-microglobulin (beta2M) and neopterin test data are presented in greatest detail, along with substantial tumor necrosis factor alpha (TNF-alpha), gamma interferon, soluble interleukin-2 receptor-alpha (sIL-2Ralpha), sTNF-RII, IL-4, and IL-6 data. Recommended QC procedures for cytokine and soluble-marker testing include replicate testing of two or more reference samples provided by the kit manufacturer, replicate testing of in-house frozen reference QC samples that represent normal and abnormal analyte contents, retesting 15 to 20% of randomly selected samples, and comparing normal reference ranges each year. Also, eight cytokines and soluble markers were evaluated in human immunodeficiency virus (HIV)-seronegative and HIV-seropositive individuals stratified on the basis of CD4 T-cell numbers. Levels of some but not all cytokines in serum increased in HIV infection. There was a tendency for cytokines to increase with more advanced disease, defined by reduced CD4 T-cell numbers. Cytokine changes did not relate closely to CD4 level, indicating that separate information was provided by the measurements of TNF-alpha, sTNF-RII, sIL-2Ralpha, beta2M, and neopterin. Serum IL-4 and TNF-alpha levels were not increased. The quality of laboratory data can impact on clinical relevance. Interlaboratory comparisons revealed substantial differences at some sites and documented the need for external proficiency-testing quality assurance programs.

Pentoxifylline is not a promising treatment for multiple sclerosis in progression phase.

Fourteen MS patients took pentoxifylline at varying doses for up to 24 months. In vitro production of tumor necrosis factor alpha was reduced in patients taking 2,400 to 3,200 mg/day of pentoxifylline for 12 weeks or more. Twelve of the 14 patients experienced worsening of the disease during the study according to clinical, MRI, or visual evoked potential criteria. These results provide no hint of efficacy for pentoxifylline as a treatment for MS in progression phase.