Nesli Basgoz

Comprehensive Epitope Analysis of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Responses Directed against the Entire Expressed HIV-1 Genome Demonstrate Broadly Directed Responses, but No Correlation to Viral Load

ABSTRACT Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/106 PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

Metabolic Abnormalities and Cardiovascular Disease Risk Factors in Adults with Human Immunodeficiency Virus Infection and Lipodystrophy

We evaluated metabolic and clinical features of 71 HIV-infected patients with lipodystrophy by comparing them with 213 healthy control subjects, matched for age and body mass index, from the Framingham Offspring Study. Thirty HIV-infected patients without fat redistribution were compared separately with 90 matched control subjects from the Framingham Offspring Study. Fasting glucose, insulin, and lipid levels; glucose and insulin response to standard oral glucose challenge; and anthropometric measurements were determined. HIV-infected patients with lipodystrophy demonstrated significantly increased waist-to-hip ratios, fasting insulin levels, and diastolic blood pressure compared with controls. Patients with lipodystrophy were more likely to have impaired glucose tolerance, diabetes, hypertriglyceridemia, and reduced levels of high-density lipoprotein (HDL) cholesterol than were controls. With the exception of HDL cholesterol level, these risk factors for cardiovascular disease (CVD) were markedly attenuated in patients without lipodystrophy and were not significantly different in comparison with controls. These data demonstrate a metabolic syndrome characterized by profound insulin resistance and hyperlipidemia. CVD risk factors are markedly elevated in HIV-infected patients with fat redistribution.

Nevirapine, Zidovudine, and Didanosine Compared with Zidovudine and Didanosine in Patients with HIV-1 Infection: A Randomized, Double-Blind, Placebo-Controlled Trial

Strategies to improve the clinical benefit of therapy for human immunodeficiency virus type 1 (HIV-1) infection have included combining antiretroviral drugs to increase antiviral activity. Some combinations of two HIV-1 reverse transcriptase inhibitors synergistically inhibit HIV-1 replication in vitro; these combinations include that of the nucleoside analogues zidovudine and didanosine [1, 2] and that of zidovudine and the non-nucleoside reverse transcriptase inhibitor nevirapine [3-5]. The combination of zidovudine and didanosine also enhances antiviral activity in vivo [6-8] and delays disease progression [9-11] more effectively than zidovudine monotherapy. One way to further suppress HIV-1 replication may be to add a third antiretroviral agent to a combination regimen. Certain three-drug regimens, including the combination of zidovudine, didanosine, and a non-nucleoside reverse transcriptase inhibitor such as nevirapine, inhibit wild-type HIV-1 in vitro more effectively than one or two of these drugs [12-15]. Nevirapine (400 mg/d), as monotherapy or in combination with zidovudine or zidovudine and didanosine, has resulted in sustained antiviral effect in vivo, although nevirapine-resistant virus was isolated from patients in open-label trials [16-18] and from in vitro selection experiments [19, 20]. No adverse drugdrug interactions were noted in patients receiving the triple combination of nevirapine, zidovudine, and didanosine over a short period [18]. Therefore, we tested whether this triple combination would improve immunologic and virologic effects in vivo by comparing it with a combination of zidovudine and didanosine in a 48-week phase II, randomized clinical trial in adults with HIV-1 disease who had previously received prolonged nucleoside therapy. Methods Study Design and Treatment Regimens This multicenter, randomized trial was AIDS (acquired immunodeficiency syndrome) Clinical Trials Group Protocol 241. Patients received either nevirapine with open-label zidovudine and didanosine (the triple combination) or placebo with open-label zidovudine and didanosine (the double combination). The placebo tablets were identical in appearance to the active nevirapine tablets. Patients and investigators were blinded to treatment assignments; a permuted-blocks design within each institution was used for randomization. Patients were stratified according to their screening CD4 cell counts: 50 or fewer cells/mm3, 51 to 200 cells/mm3, and 201 to 350 cells/mm3. Zidovudine (Retrovir, provided by Glaxo Wellcome, Research Triangle Park, North Carolina) was given orally as two 100-mg tablets three times a day. Didanosine (Videx, provided by Bristol-Myers Squibb, Wallingford, Connecticut) was given orally as two 100-mg chewable and dispersable tablets twice daily for patients weighing at least 60 kg and as one 100-mg tablet and one 25-mg tablet twice daily for patients weighing less than 60 kg. Nevirapine (Viramune, provided by Boehringer-Ingelheim Pharmaceuticals, Ridgefield, Connecticut) was given orally as a 200-mg tablet once daily for the first 2 weeks and thereafter as one 200-mg tablet twice daily. This schedule was used because a lead-in period with a lower daily dose of nevirapine had decreased the frequency of nevirapine-related rashes in earlier studies [17]. Data were gathered, analyzed, and interpreted independently of the pharmaceutical companies that provided the drugs, according to the standard operating procedures of the AIDS Clinical Trials Group. Study Sample Adults who had documented HIV-1 infection and CD4 cell counts of 350 cells/mm3 or less within 30 days of randomization and who had had previous nucleoside therapy with zidovudine, didanosine, or zalcitabine for at least 6 months were eligible. Eligibility criteria also included a Karnofsky performance status score of at least 70% within 30 days of randomization, a hemoglobin concentration of at least 91 g/L or more for men and 88 g/L for women, a neutrophil count of 1000 cells/mm3 or more, a platelet count of 75 000 109/L or more, a serum creatinine concentration no more than 1.5 times the upper limit of normal, serum concentrations of alanine aminotransferase and aspartate aminotransferase no more than 3 times the upper limit of normal, and a serum amylase concentration no more than 1.5 times the upper limit of normal (unless the serum lipase concentration was 1.5 times the upper limit of normal or less). Patients were excluded if they were intolerant of zidovudine (at 500 or 600 mg/d) or didanosine (at 400 mg/d for tablets and 500 mg/d for sachets), had moderate peripheral neuropathy ( grade 2 according to the National Institute of Allergy and Infectious Diseases [NIAID] Division of AIDS Table for Grading Adult Adverse Experiences), had pancreatitis, or had previously used non-nucleoside reverse transcriptase inhibitors. The study was approved by the institutional review board at each institution, and all participants gave written informed consent. Concomitant therapy with other antiretroviral agents, foscarnet, biological response modifiers, erythromycin, clavulanate-containing antibacterial agents, warfarin, phenytoin, or phenobarbital was not permitted. Prophylaxis for Pneumocystis carinii pneumonia was required for all patients with a CD4 cell count of 200 cells/mm3 or less or a history of P. carinii pneumonia. Maintenance therapies for other opportunistic infections were permitted. Patients could continue their prestudy antiretroviral nucleoside therapy until the day on which therapy with the study medications was started. Management of Toxicities Toxicity was graded according to the NIAID Division of AIDS Table for Grading Adult Adverse Experiences. Modification, interruption, or discontinuation of therapy with the study medications was done for many adverse effects one drug at a time, starting with the drug most likely to have caused the toxicity. Zidovudine was the first drug to be modified for severe anemia, myositis, neutropenia, fatigue, and headache; didanosine was the first to be modified for severe nausea, vomiting, diarrhea, constipation, hyperamylasemia, fasting hypertriglyceridemia, hyperuricemia, and peripheral neuropathy; and nevirapine or placebo was the first to be modified for rash, severe thrombocytopenia, and adverse neuropsychological effects. Therapy with all study medications was interrupted for other severe adverse experiences, including hepatotoxicity. Therapy with a medication or medications was interrupted until the toxicity resolved and was then resumed at half the original dose, except that didanosine therapy was permanently discontinued for pancreatitis and nevirapine or placebo use was permanently discontinued for severe rash. Recurrent or persistent adverse effects led to the permanent discontinuation of either zidovudine or didanosine therapy. All study treatment was stopped if therapy with nevirapine or placebo or any two study medications was permanently discontinued. Patient Evaluation The schedule of patient evaluations is shown in Table 1. Standardized assays were used for CD4 cell counts [21, 22] and serum p24 antigen levels (AIDS Clinical Trials Group Virus Quality Assurance Laboratory reference standards were used with enzyme-linked immunosorbent assay kits from either Coulter [Hialeah, Florida], Abbott Laboratories [North Chicago, Illinois], or DuPont [Wilmington, Delaware]). Plasma nevirapine concentrations were measured at Boehringer-Ingelheim Pharmaceuticals by a validated high-performance liquid chromatography assay [23]. Additional virologic and pharmacologic assessments were done on specimens from all patients enrolled at 8 of the 16 study sites (n = 198). The HIV-1 infectivity titers in peripheral blood mononuclear cells were calculated by using maximum likelihood estimation from a standardized HIV-1 quantitative microculture assay in quality-assured AIDS Clinical Trials Group virology laboratories [24]. Plasma HIV-1 RNA levels were measured by quantitative reverse transcriptase polymerase chain reaction (Roche Molecular Systems, Alameda, California, and Branchburg, New Jersey) done in batch at Roche Biomedical Laboratories (Research Triangle Park, North Carolina) [25]. Plasma zidovudine and didanosine concentrations were measured at the University of Alabama at Birmingham by validated radioimmunoassays [26, 27]. Viruses isolated at entry were classified as either syncytium-inducing or non-syncytium-inducing according to the results of an MT-2 cell assay [28]. Table 1. Schedule of Patient Evaluations Primary outcome measures were differences between treatment groups at the end of the study in mean absolute CD4 cell count, HIV-1 infectivity titer in peripheral blood mononuclear cells, serum p24 antigen level, and time to HIV-1 disease progression or death. Secondary outcome measures were differences between treatment groups at the end of the study in the mean percentage of T lymphocytes that were CD4 cells and in plasma HIV-1 RNA levels. These measurements at the end of the study were the geometric means of measurements obtained between weeks 40 and 48, expressed as the change from the geometric mean of the two measurements made before treatment. Standardized area-under-the-curve analyses of immunologic and virologic measures over time were additional secondary outcome measures used to assess differences between treatments that included short-term effects. Progression of HIV-1 disease was defined as the development of a new AIDS-defining clinical event according to the Centers for Disease Control and Prevention case definition (except that we excluded CD4 cell count as a disease progression criterion) [29], a newly diagnosed deep-seated bacterial infection or bacteremia unrelated to the use of injection drugs or an intravascular catheter, 1 month or more of symptomatic microsporidiosis, or the recurrence of either P. carinii pneumonia or central nervous system toxoplasmosis. All disease prog

Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response

The duration of disease-free survival after infection with human immunodeficiency virus type 1 (HIV-1) varies considerably during antiretroviral therapy. Patients with similar CD4+ lymphocyte counts progress at different rates when they are given the same antiretroviral therapy. Better prediction of risk for progression and its association with viral suppression may help improve antiretroviral management for individual patients and speed the development of new drugs. Higher plasma HIV-1 RNA levels are associated with poorer clinical status and lower CD4+ lymphocyte counts [1-3] and predict subsequent outcome [4-11]. The biological variability of plasma HIV-1 RNA levels in patients receiving stable therapeutic regimens must be quantified to define the magnitude of an antiviral effect that can be reliably detected after antiretroviral treatment is initiated. Determination of infectious HIV-1 titers in mononuclear cells of peripheral blood by quantitative microculture [12, 13] or syncytium-inducing phenotype of an HIV-1 isolate may provide information that is different from or complementary to the information gleaned from measuring plasma HIV-1 RNA levels [14-16]. However, studies have not yet conclusively determined whether measurements of CD4+ lymphocytes in conjunction with any or all of these virological variables should be recommended to optimize prediction or guide antiretroviral treatment more effectively. In this report, we quantify the relative roles of CD4+ lymphocyte counts, plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and the syncytium-inducing viral phenotype as predictors of disease progression during a clinical trial of combination therapy [17]. Our approach was to assess the value of plasma HIV-1 RNA levels and CD4+ lymphocyte count, both of which are readily available to clinicians, and then to assess the additional value of the infectious HIV-1 titer in mononuclear cells of peripheral blood and the syncytium-inducing viral phenotype. We also quantify the variability of plasma HIV-1 RNA levels. Our results suggest guidelines for using these measures in clinical practice for predicting the effectiveness of antiretroviral therapy over 1 year. Methods Study Design We prospectively evaluated virological, immunologic, and clinical data from patients who participated in the intensive virology substudy of ACTG (AIDS Clinical Trials Group) Protocol 241; ACTG Protocol 241 was a multicenter, randomized, double-blind, placebo-controlled trial of 398 patients receiving nevirapine, zidovudine, and didanosine compared with zidovudine and didanosine [17]. All patients at 8 of the 16 AIDS Clinical Trials Units who participated in the main study were enrolled in the substudy (n = 198). For 48 weeks, all 198 patients received open-label zidovudine (600 mg/d) and didanosine (400 mg/d for patients weighing 60 kg and 250 mg/d for patients weighing <60 kg). One hundred of the substudy patients were randomly assigned to receive nevirapine (200 mg/d for the first 2 weeks and 400 mg/d thereafter), and 98 were assigned to receive matching placebo. Participants gave written informed consent, and the protocol was approved by the institutional review board at each participating AIDS Clinical Trials Unit. The study was funded by the ACTG of the National Institute of Allergy and Infectious Diseases; supplemental funding for virology was provided by Boehringer Ingelheim Pharmaceuticals (Ridgefield, Connecticut). Study drugs were provided by Glaxo Wellcome (Research Triangle Park, North Carolina), Bristol-Myers Squibb (Princeton, New Jersey), and Boehringer Ingelheim Pharmaceuticals. However, all data were gathered by members of the ACTG and were analyzed and interpreted by the authors, who had sole responsibility for the decision to submit the manuscript for publication. Evaluation of Patients Stable therapy at baseline was defined as the absence of reported change in antiretroviral therapy from 30 days before the preentry visit until the entry visit. All patients were followed prospectively for progression of HIV-related disease. Progression was defined as the development of a new acquired immunodeficiency syndrome (AIDS)-defining event [18]; a newly diagnosed, deep-seated bacterial infection or bacteremia that was not related to injection drug use or an intravascular catheter; pulmonary or extrapulmonary tuberculosis; recurrent Pneumocystis carinii pneumonia; recurrent toxoplasmosis of the central nervous system; or death. Reports of disease progression were reviewed by the study chair; only events that could be confirmed were used in the analysis. We measured CD4+ lymphocyte counts, plasma HIV-1 RNA levels, and infectious HIV-1 titers in mononuclear cells of peripheral blood at the preentry visit (within 14 days of starting study treatment), at the entry visit (before starting study treatment and at least 72 hours after the preentry visit), and at the visits 8 and 48 weeks after the start of study treatment. Specimens could be obtained at any time of day. We used the geometric mean of preentry and entry measurements as the baseline value for each variable. The presence of the syncytium-inducing viral phenotype was determined at the entry visit. Standardized assays were used to determine CD4+ lymphocyte counts [19, 20], infectious HIV-1 titer in mononuclear cells of peripheral blood (in infectious units per million cells) using real-time testing [13, 21], and syncytium-inducing viral phenotype of a virus isolated from mononuclear cells of peripheral blood using MT-2 cells [22]. Plasma samples were frozen at 70C; HIV-1 RNA levels were measured by quantitative reverse transcription polymerase chain reaction assay (Roche Molecular Systems, Alameda, California, and Branchburg, New Jersey) [23]. The lower limit of detection for this assay was 200 HIV-1 RNA copies/mL. Levels of HIV-1 RNA in plasma samples collected from the same patient at the preentry, entry, week 8, and week 48 visits were determined in a single laboratory assay. Statistical Analysis Analysis of plasma HIV-1 RNA levels and infectious HIV-1 titers in mononuclear cells of peripheral blood was done after log10 transformation. Plasma levels of HIV-1 RNA that were below the detectable limit were assigned the value of 200 copies/mL. Infectious HIV-1 titers in mononuclear cells of peripheral blood outside the measurable range (0.22 to 7493 infectious units per million cells) were assigned the value of 0.22 infectious units per million cells if they were below the range and 7493 infectious units per million cells if they were above the range. Linear regression analysis [24] was used to compare the mean plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and CD4+ lymphocyte counts according to patient characteristics at baseline and to assess factors associated with the long-term change (from baseline to week 48) in CD4+ lymphocyte counts. Logistic regression analysis [25] was used to assess the association at baseline of the percentage of patients who had AIDS with virological measures and CD4+ lymphocyte counts. The intrapatient SD of plasma HIV-1 RNA levels was estimated using the method of moments for variance components [26]. Spearman correlation coefficients were used to assess the association between preentry and entry measurements. Proportional hazards models [27] were used to assess the association between the risk for disease progression or death and baseline levels and early changes (from baseline to week 8) in plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and log-transformed CD4+ lymphocyte counts as well as baseline syncytium-inducing viral phenotype. These models were stratified by study treatment to control for any differential effects of the two study regimens. Results Patient Characteristics at Study Entry The mean CD4+ lymphocyte count of the 198 patients before treatment was 145 cells/mm3 (range, 1 to 443 cells/mm3). Patients were a median of 39 years of age, predominantly male (81%), predominantly white (76%), and predominantly free of a previous AIDS-defining diagnosis (86%). All but 3 patients had taken zidovudine before study entry, 44% had taken didanosine, and 35% had taken zalcitabine. The median duration of cumulative previous nucleoside therapy was 25 months, and 34% of patients had received therapy for longer than 36 months. Virological Measures at Baseline by Patient Characteristics Table 1 shows the mean plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and CD4+ lymphocyte counts at baseline for patients stratified by characteristics that were significantly associated with viral load. We also assessed the associations with age, sex, racial or ethnic group, self-reported homosexuality, and duration of previous nucleoside therapy, but these associations were not significant. Table 1. Plasma HIV-1 RNA Level, Infectious HIV-1 Titer in Mononuclear Cells of Peripheral Blood, and CD4+ Lymphocyte Count at Baseline* Patients with a history of AIDS had a significantly higher mean baseline level of HIV-1 RNA in plasma and a significantly lower mean CD4+ lymphocyte count than did those without such a history (Table 1). More patients with a history of AIDS than those without had baseline HIV-1 isolates with the syncytium-inducing viral phenotype (58% compared with 36%; P = 0.015). However, in a multivariate analysis, only the CD4+ lymphocyte count at baseline was significantly associated with a history of AIDS. Thus, disease status at baseline was explained by CD4+ lymphocyte counts and not by any of the virological measures that were considered. Variability of Virological Measures in Patients Receiving Stable Treatment Variation in plasma HIV-1 RNA levels was evaluated by comparing the preentry and entry measures from the 167 patients who reported no changes in treatment from 30 days

Effects of Androgen Administration in Men with the AIDS Wasting Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial

The AIDS wasting syndrome is characterized by loss of lean body mass out of proportion to weight [1, 2]. The few effective treatments that have been identified are short-term pharmacologic agonists. Because loss of lean body mass is associated with decreased survival in men with the AIDS wasting syndrome [3], development of therapeutic strategies to increase lean body mass is of critical importance. Half of all men with AIDS are hypogonadal [4], and serum androgen levels correlate with lean body mass among hypogonadal men with the AIDS wasting syndrome [5]. Previous studies in non-HIV-infected hypogonadal men show that androgen administration has a significant anabolic effect on body composition [6-10]. We hypothesized that loss of the potent anabolic hormone testosterone in men with the AIDS wasting syndrome may contribute to the critical loss of lean body mass. Therefore, we investigated the effects of physiologic testosterone administration in men with the AIDS wasting syndrome. Methods Patients In 1995 and 1996, 51 HIV-positive men (42 8 years of age) were recruited from the multidisciplinary HIV practice at the Massachusetts General Hospital and from newspaper, television, and radio advertisements. Weight, testosterone levels, and medication history were determined at a screening assessment. To be included in the study, patients had to have decreased free testosterone levels, defined as less than 42 pmol/L at screening (normal range for men 18 to 49 years of age, 42 to 121 pmol/L [12.0 to 35.0 pg/mL]), and wasting, defined as weight less than 90% of ideal body weight or involuntary weight loss greater than 10% of baseline weight [11]. The CD4 count was not an inclusion criterion. We excluded patients with severe diarrhea (>6 stools/d); hemoglobin value less than 5.0 mmol/L (<8 g/dL); platelet count less than 50 000 cells/mm3; creatinine concentration greater than 177 mol/L (>2 mg/dL); new opportunistic infection within 6 weeks of screening; use of testosterone, anabolic steroids, growth hormone, ketoconazole, or systemic steroid therapy within 3 months before screening; or history of prostate cancer. In addition, patients receiving antiretroviral agents, including protease inhibitors, were required to be receiving a stable regimen for at least 6 weeks before study entry. Ten patients were receiving long-term, stable therapy with megestrol acetate for at least 8 weeks before study entry and were equally distributed between the two treatment groups (5 in the testosterone group and 5 in the placebo group). All patients gave written consent, and the study was approved by the Human Studies Committee of the Massachusetts General Hospital. Protocol Patients were randomly assigned to receive testosterone enanthate, 300 mg (Bio-Technology General Corp., Iselin, New Jersey), or placebo intramuscularly every 3 weeks by self-injection. Participants were stratified for weight less than or greater than 90% of ideal body weight and megestrol acetate use before randomization. Randomization was performed by the Massachusetts General Hospital Pharmacy by using a permuted block algorithm. The correspondence between patient code number and drug was generated by the study statistician; this list was available to the hospital pharmacist but not to the investigators or patients. The placebo contained sesame oil with chlorobutanol as a preservative and matched testosterone enanthate in color and consistency. The study drug was bottled by the Massachusetts General Hospital Pharmacy in containers labeled with the study name, expiration date, and patient code. Before the first injection, participants returned within approximately 2 weeks of the screening visit for a 3-day baseline inpatient visit to the General Clinical Research Center at the Massachusetts General Hospital for hormonal, nutritional, immune function, and body composition analysis, which included assessment by dual-energy x-ray absorptiometry, bioimpedance analysis, potassium-4040 K) isotope analysis, and measurement of urinary creatinine excretion. No patient experienced the onset of a new opportunistic infection, other complication, or substantial weight change between the screening and baseline visits. Patients were instructed on the proper technique for intramuscular injection; those who were unable to self-administer the study drug received injections every 3 weeks from the nursing staff of the General Clinical Research Center. Patients returned for an outpatient visit at 3 months for assessment of weight and determination of total-body potassium content and for a 3-day inpatient visit at 6 months; this visit was identical to the baseline evaluation. Patients also reported on response to therapy at the 6-month visit. Baseline data from 26 patients have been reported elsewhere [5]. Subsequent study visits were timed to correspond to the midpoint between study drug injections. Study drug compliance was confirmed by history, medication diaries, outpatient injection records, empty vial counts, and serum testosterone levels. History of medication use was assessed at each visit. The change in fat-free mass assessed by dual-energy x-ray absorptiometry was the primary clinical end point; changes in weight, muscle mass, total body potassium content, and quality of life were secondary end points. Body Composition Analysis Body composition was determined by four methods: 1) dual-energy x-ray absorptiometry to assess fat and fat-free mass (Hologic-2000 densitometer, Hologic, Inc., Waltham, Massachusetts; precision error, 3% for fat and 1.5% for fat-free mass [12], 2) 40K isotope analysis to assess total-body potassium content in a whole-body counter with sodium iodide detectors fixed above and below the patient at the xiphoid level (Canberra Nuclear, Meriden, Connecticut; precision error < 2.5% on the basis of repeated calibration with a known potassium chloride source [Appendix]), 3) urinary creatinine excretion averaged over 3 days (during which the patient received a meat-free diet) multiplied by a constant of 18 kg of muscle per gram of urinary creatinine and indexed for height to determine the percentage of predicted muscle mass [13, 14], and 4) bioimpedance analysis to determine total-body water content (Bioelectrical Impedance Analyzer Model BIA-101, RJL Systems, Clinton Turnpike, Michigan; correlation with deuterium oxide equivalent to R = 0.99 [15]). Lean body mass was derived from total-body potassium content by using the Equation of Forbes and Lewis of 68.1 mEq of potassium per kg of lean body mass [16]. Nutritional Assessment Weight was measured on the first day of each visit after an overnight fast. The percentage of ideal body weight was calculated on the basis of standard height and weight tables [17]. Patients were instructed on completion of a 4-day food record, which was analyzed for total calorie, fat, protein, and carbohydrate content (Minnesota Nutrition Data Systems, version 8A/2.6, Minneapolis, Minnesota) by the Clinical Research Center dietitian. Patients received an isocaloric, meat-free, protein-substituted diet 3 days before and during the inpatient assessments at baseline and at 6 months, during which creatinine excretion and nitrogen balance were measured. Total urinary nitrogen excretion was measured by the Kjeldal technique from consecutive 24-hour collections averaged over 3 days. Nonurinary nitrogen losses were assumed to be constant at 4 g/d [13, 18, 19]. Nitrogen intake was derived from total protein intake divided by a constant of 6.25 g of protein per g of nitrogen [19]. Calorie and protein intake were monitored on a daily basis and were modified to match the reports in the outpatient food records immediately before these visits. Resting energy expenditure was measured by indirect calorimetry with a metabolic cart. Energy requirements were calculated by using the Harris-Benedict equation [20]. Patient Reports of Response to Therapy Each patients perceived well-being was assessed at the end of the study by using nine linear analogue-scale questions on the overall treatment effect, change in quality of life, personal appearance, weight, and appetite (Table 1) [21]. A Karnofsky score was also determined at each visit. Table 1. Assessment of Patient Response to Therapy* Exercise Functional Testing Exercise history was assessed by a standardized questionnaire adapted from the study by Kohl and coworkers [22]. Exercise functional status was determined at the baseline and final visits by the physical therapy department of the Massachusetts General Hospital by using the 6-minute walk test, the timed sit-to-stand test, and the timed get-up-and-go test [23-25]. The distance covered in 6 minutes, the number of times the patient was able to move from a sitting to standing position in 10 seconds, and the time to cover a distance of 3 meters after standing from a seated position was recorded for each patient. Biochemical and Immunologic Assays Hematocrit and serum levels of follicle-stimulating hormone, luteinizing hormone, sex hormone-binding globulin, and prolactin were measured at the baseline and final visits by using published methods [26]. Serum levels of total and free testosterone were measured by radioimmunoassay kit (Diagnostics Products Corp., Los Angeles, California) with intra-assay coefficients of variation of 5% to 12% for total testosterone and 3.2% to 4.3% for free testosterone. CD4 cell counts were measured by flow cytometry (Becton Dickinson Immunocytochemistry Systems, San Jose, California). Viral burden was determined by using the Amplicor HIV-1 monitor test (Roche Molecular Systems, Branchburg, New Jersey). Statistical Analysis Sample size was based on the change in lean body mass in response to testosterone administration among adult men with acquired hypogonadism [8]. A change of 3.2% 4.0% was expected over 6 months. With 20 patients in each group, the study had an 80% chance of seeing an effect of testosterone at a two-sided

Effects of Testosterone and Progressive Resistance Training in Eugonadal Men with AIDS Wasting: A Randomized, Controlled Trial

Substantial loss of lean body and muscle mass occur among HIV-infected patients with relatively preserved body weight (1); these changes are associated with reduced functional status and strength (2). Protease inhibitor therapy has not been shown to increase muscle mass in patients with AIDS wasting (3), suggesting the need for successful anabolic strategies in these patients. Testosterone therapy and progressive resistance training increase lean body mass in hypogonadal men with AIDS wasting (4-6). However, most men with AIDS wasting have normal testosterone levels (7). We assessed the independent effects of progressive resistance training and testosterone in eugonadal men with AIDS wasting. Baseline (2) and screening data (7) from a subset of participants were previously reported. Methods Patients From 1997 to 1999, 54 HIV-infected men with AIDS-related wasting (weight<90% ideal body weight or self-reported weight loss>10%) and a normal serum level of free testosterone (>42 pmol/L) were recruited through community advertisements and contact with physicians in the multidisciplinary HIV practice at the Massachusetts General Hospital, Boston, Massachusetts, and other community clinics. Exclusion criteria were new opportunistic infection diagnosed within 6 weeks of the study; other contraindication to exercise; use of a new antiretroviral agent within 8 weeks of the study; abnormal prostate-specific antigen level; symptomatic prostatism; prostate malignancy; bipolar disorder; use of parenteral nutrition, megestrol acetate, glucocorticoids, androgen, estrogen, growth hormone or other anabolic agent within 3 months of the study; hemoglobin value less than 90 g/L or greater than 170 g/L; platelet count less than 50 000 cells/mm3; or serum creatinine concentration greater than 177 mol/L (2.0 mg/dL). All patients gave written consent, and the study was approved by the Human Studies Committee of the Massachusetts General Hospital. Protocol Eligible patients were stratified for weight less than 90% of ideal body weight or 90% or greater than ideal body weight. Using a 2 2 factorial design, we randomly assigned patients to receive intramuscular injections of testosterone enanthate (200 mg/wk; Bio-Technology General Corp., Iselin, New Jersey) or placebo and to progressive resistance training (three times per week) or no training for 12 weeks. The study statistician used a permuted-block algorithm with blocks of 8 to perform randomization; the code was available only to the hospital pharmacy that bottled the study drug. Placebo contained sesame oil with chlorobutanol as a preservative and matched testosterone enanthate in color and consistency. Compliance with drug therapy was confirmed by history, outpatient injection records, and vial counts. Patients assigned to training participated in supervised progressive strength training and aerobic conditioning three times per week for 12 weeks. During each session, patients began by performing 20 minutes of aerobic exercise on a stationary bicycle at a target heart rate of 60% to 70% of their age-predicted maximum, in accordance with American College of Sports Medicine recommendations (8). A cool-down period of 15 minutes and normalization of heart rate preceded resistance training. Training was performed isotonically on the following computerized equipment (Life Fitness, Franklin Park, Illinois): leg extension, leg curl, leg press, latissimus dorsi pull-down, arm curl, and triceps extension. A one-repetition maximum weight was established at baseline for each patient on each machine in the best of three efforts. Patients increased resistance as follows: weeks 1 and 2, 2 sets, 8 repetitions/set, 60% one-repetition maximum; weeks 3 through 6, 2 sets, 8 repetitions/set, 70% one-repetition maximum; weeks 7 through 12, 3 sets, 8 repetitions/set, 80% one-repetition maximum. Patients were asked to refrain from exercise for 2 weeks before the baseline visit and to refrain from any exercise or activity beyond normal daily activity during the study. Food intake was ad libitum; caloric intake was determined by using a 4-day food record (Nutrition Data System for Research, version 12A/2.91, Nutrition Coordinating Center, University of Minnesota, Minneapolis, Minnesota). Resting and predicted energy expenditure were calculated (VMAX 29N, SensorMedics, Inc., Loma Linda, California). Clinical End Points Clinical end points were assessed at baseline and 12 weeks. Lean body mass and fat mass were measured by using dual-energy x-ray absorptiometry (QDR-4500 Densitometer, Hologic, Inc., Waltham, Massachusetts) with a precision error of 1.5% for fat-free mass (9). Cross-sectional muscle areas of the leg and arm were assessed by performing computed tomography of the midfemur and humerus (General Electric High Speed Helical CAT Scanner, Milwaukee, Wisconsin; SE 3% for arm muscle area and 1% for leg muscle area). The location of the midfemur and humerus were determined from the scout image. Upper- and lower-extremity muscle strength were measured by using the quantitative muscle function test (10, 11). Peak isometric force of shoulder flexion, shoulder extension, elbow flexion, elbow extension, knee flexion, knee extension, dorsiflexion, and grip were measured on the best of two repetitions (10, 12). Z scores were calculated for upper- and lower-extremity strength (MVCT Computer Analysis Software, Boston, Massachusetts) by standardizing to a group of healthy male controls (11, 12). Serum levels of total and free testosterone were measured by using a radioimmunoassay kit (Diagnostics Products Corp., Los Angeles, California) (4). CD4 cell counts were measured by using flow cytometry (Becton-Dickinson Immunocytochemistry Systems, San Jose, California); viral load was measured by using the Amplicor HIV-1 Monitor (Roche Molecular Systems, Branchburg, New Jersey). Other tests were done according to published methods (13). A digital prostate examination was performed at each visit. Statistical Analysis The effects of training and testosterone were simultaneously assessed in the same factorial model. In the primary analysis, we used analysis of covariance to assess change from baseline at 3 months simultaneously in the testosterone arm (testosterone recipients vs. placebo recipients) and the training arm (trained patients vs. nontrained patients), controlling for baseline values. To test for an interaction between testosterone and training, we used analysis of covariance with an interaction term. Change in lean body mass was the primary clinical end point for the effect of testosterone, and change in cross-sectional muscle area was the primary end point for the effect of resistance training. Change from baseline was also determined within each individual treatment group and was compared with zero change by using analysis of covariance. The t- test was used to compare treatment groups at baseline. All available data are included in the analysis. Results are reported as the mean SD. Results No patient withdrew from the study because of an adverse event or side effect; dropout rates did not differ by group (Appendix Figure). Patients had lost significant weight but were not severely ill or low weight at study entry (Table 1). Seventy-six percent of patients were receiving antiretroviral therapy and 72% were receiving highly active antiretroviral therapy. Seventy-six percent of patients had previously had an opportunistic infection. Appendix Figure. Flow of participants through the study. Table 1. Results of Factorial Analysis Changes in response to testosterone therapy and training are shown in Table 1. Lean body mass and muscle area increased significantly in response to training and testosterone therapy. Muscle strength on elbow flexion and shoulder extension and overall upper-extremity Z score increased in response to testosterone therapy. The change in muscle area correlated with the change in muscle strength (R =0.48; P =0.001 for mid-thigh muscle area and strength on knee extension). No interaction was found between testosterone therapy and training. Levels of high-density lipoprotein (HDL) cholesterol increased in response to training but decreased in response to testosterone therapy. Levels of total and free testosterone increased in response to testosterone therapy, and levels of gonadotropin and sex hormonebinding globulin decreased. Caloric intake did not change significantly between the groups. The CD4 count did not change significantly in response to training or testosterone therapy (P >0.2). Viral load decreased in testosterone-treated patients. Use of antiretroviral therapy did not change in any study group. Levels of aspartate aminotransferase or prostate-specific antigen did not change significantly (P >0.2). No patient developed new prostate nodules. Three patients developed breast tenderness or gynecomastia (two were receiving testosterone and one was receiving placebo). Compliance with the training program was 78% among patients who completed the study; compliance with testosterone therapy was 98%. Discussion Previous studies suggest that testosterone therapy, alone (4, 5) and in combination with resistance training, increases lean body mass in hypogonadal men with AIDS wasting (6). However, recent data indicate that androgen levels are normal in most HIV-infected men (7), and the independent effects of testosterone and supervised exercise in eugonadal men with AIDS wasting are not known. The patients in our study generally had normal body weight and a normal Karnofsky score but had lost substantial weight. Most patients had a history of opportunistic infection, and although they were not cachectic or malnourished, they had reduced muscle mass (2). Previous studies have shown that resistance training in combination with testosterone or anabolic steroid therapy increases lean body mass (6, 14, 15). In contrast, we found that training had a significant effect (increase of 2.3 kg) on lean bod

Pseudo-Cushing's Syndrome in Human Immunodeficiency Virus—Infected Patients

To our knowledge, an association between human immunodeficiency virus infection and pseudo-Cushings syndrome has not previously been described. We describe four HIV-infected patients with pseudo-Cushings syndrome, characterized by striking dorsocervical and submandibular fat accumulation and central obesity. In each case, cortisol levels were either normal or suppressed adequately with administration of dexamethasone, excluding the diagnosis of true Cushings syndrome. Immune function and weight improved significantly preceding the development of pseudo-Cushings syndrome. Three of the four patients were taking a common protease inhibitor at the onset of symptoms, and the fourth reported the exacerbation of his symptoms with the addition of a protease inhibitor. The observed characteristic pattern of fat deposition may be attributable to a specific effect of new antiretroviral therapies or may relate to recovery independent of medication usage. Distinguishing between pseudo-Cushings syndrome and true Cushings syndrome is critical for preventing the unnecessary and potentially harmful treatment of such patients. Further research into the mechanisms of this novel phenomenon is needed.

Comparison of overlapping peptide sets for detection of antiviral CD8 and CD4 T cell responses

Increasing efforts are directed towards the development of effective vaccines through induction of virus-specific T cell responses. Although emerging data indicate a significant role of these cells in determining viral set point in infections such as HIV, there is as yet no consensus as to the best methods for assaying the breadth of these responses. In this study, we used sensitive interferon gamma-based intracellular cytokine staining (ICS) and Elispot assays to determine the optimal overlapping peptide set to screen for these responses. Twenty persons with established HIV infection were studied, focusing on responses to the highly immunogenic Nef protein. Six different HIV-1 Nef peptide sets were used, ranging in length from 15 to 20 amino acids (aa), in overlap from 10 to 11 amino acids, and derived from two different B clade sequences. A total of 54 CD8 T cell responses to Nef peptides were found in this cohort, of which only 12 were detected using previously defined Nef optimal epitopes. No single peptide set detected all responses. Though there was a trend of the shorter peptides detecting more CD8 T cell responses than the 20 amino acid long peptides and longer peptides detecting more CD4 T cell responses, neither was statistically significant. There was no difference between an overlap of 10 or 11 amino acids. All responses detected with the six different sets of overlapping peptides were towards the more highly conserved regions of Nef. We conclude that peptides ranging from 15 to 20 amino acids yield similar results in IFN-gamma-based Elispot and ICS assays, and that all are likely to underestimate the true breadth of responses to a given reference strain of virus.

Prevalence of Hypogonadism among Men with Weight Loss Related to Human Immunodeficiency Virus Infection Who Were Receiving Highly Active Antiretroviral Therapy

Previous studies have indicated that there is a significant prevalence (50%) of hypogonadism among men with acquired immunodeficiency syndrome (AIDS)-associated wasting, and for these patients testosterone administration has been shown to increase lean body mass and improve quality of life. However, the prevalence of hypogonadism is not known among men with weight loss related to human immunodeficiency virus (HIV) infection who are receiving highly active antiretroviral therapy (HAART). From 1997 through 1999, we investigated total and free testosterone levels in 90 men who were <90% of ideal body weight or had weight loss of >10% from preillness weight; 71% of these subjects were receiving HAART. Twenty-one percent of the subjects receiving HAART had low free testosterone levels. No correlation was seen between weight, CD4 cell count, medication status, and other clinical factors. These data suggest that hypogonadism remains relatively common in men with AIDS wasting, despite treatment with HAART. HIV-infected men with wasting syndrome should be screened for hypogonadism and receive physiological androgen replacement therapy if they are hypogonadal.

Prediction of Coronary Heart Disease Risk in HIV-Infected Patients with Fat Redistribution

A metabolic syndrome has been described among human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy; the syndrome is characterized by fat redistribution, insulin resistance, and dyslipidemia. We compared the 10-year coronary heart disease (CHD) risk estimates for 91 HIV-infected men and women with fat redistribution with the risk estimates for 273 age-, sex-, and body mass index (BMI)-matched subjects enrolled in the Framingham Offspring Study. Thirty HIV-infected patients without fat redistribution were also compared with 90 age- and BMI-matched control subjects. The 10-year CHD risk estimate was significantly elevated among HIV-infected patients with fat redistribution, particularly among men; however, when they were matched with control subjects by waist-to-hip ratio, the 10-year CHD risk estimate did not significantly differ between groups. HIV-infected patients without fat redistribution did not have a greater CHD risk estimate than did control subjects. In addition, the CHD risk estimate was greatest in HIV-infected patients who had primary lipoatrophy, compared with those who had either lipohypertrophy or mixed fat redistribution. Therefore, although CHD risk is increased in HIV-infected patients with fat redistribution, the pattern of fat distribution and sex are potential important components in determining the risk in this population.