Screening for pulmonary tuberculosis in HIV-infected individuals: AIDS Clinical Trials Group Protocol A5253.

BACKGROUND: Improved tuberculosis (TB) screening is urgently needed for human immunodeficiency virus (HIV) infected patients. METHODS: An observational, multi-country, cross-sectional study of HIV-infected patients to compare a standardized diagnostic evaluation (SDE) for TB with standard of care (SOC). SOC evaluations included TB symptom review (current cough, fever, night sweats and/or weight loss), sputum Ziehl-Neelsen staining and chest radiography. SDE screening added extended clinical signs and symptoms and fluorescent microscopy (FM). All participants underwent all evaluations. Mycobacterium tuberculosis on sputum culture was the primary outcome. RESULTS: A total of 801 participants were enrolled from Botswana, Malawi, South Africa, Zimbabwe, India, Peru and Brazil. The median age was 33 years; 37% were male, and median CD4 count was 275 cells/mm(3). Thirty-one participants (4%) had a positive culture on Löwenstein-Jensen media and 54 (8%) on MGIT. All but one positive culture came from sub-Saharan Africa, where the prevalence of TB was 54/445 (12%). SOC screening had 54% sensitivity (95%CI 40-67) and 76% specificity (95%CI 72-80). Positive and negative predictive values were respectively 24% and 92%. No elements of the SDE improved the predictive values of SOC. CONCLUSIONS: Symptom-based screening with smear microscopy was insufficiently sensitive. More sensitive diagnostic testing is required for HIV-infected patients.

Impact of the human immunodeficiency virus on early multidrug-resistant tuberculosis treatment outcomes in Botswana.

SETTING: The impact of the human immunodeficiency virus (HIV) on multidrug-resistant tuberculosis (MDR-TB) treatment outcomes in sub-Saharan Africa, where extensive rollout of highly active antiretroviral therapy (HAART) has occurred, remains unclear. OBJECTIVE: To compare the time to initial culture conversion among patients with and those without HIV infection in a setting of individualized MDR-TB care in Botswana. DESIGN: Prospective cohort study of MDR-TB patients receiving ambulatory, integrated TB-HIV care at two public clinics in Botswana. The time to culture conversion was compared by HIV status using Cox proportional hazard ratios (HRs). RESULTS: A total of 40 HIV-infected and 30 non-HIV-infected patients with MDR-TB and follow-up cultures were identified. The median time to initial culture conversion was 78 days (interquartile range [IQR] 42-186) for HIV-infected and 95 days (IQR 70-133) for non-HIV-infected individuals (log rank P > 0.5; unadjusted HR 0.9, 95%CI 0.5-1.5). Adjusting for age, sex, treatment history and number of active anti-tuberculosis drugs did not change this result (adjusted HR 0.8, 95%CI 0.4-1.4). CONCLUSION: We found no difference in the proportion of or time to initial sputum culture conversion between an HIV-infected and a non-infected cohort of MDR-TB patients in Botswana, suggesting that outcomes may be comparable in similar settings with access to individualized anti-tuberculosis treatment and HAART.

Induced sputum improves the diagnosis of pulmonary tuberculosis in hospitalized patients in Gaborone, Botswana.

SETTING: Low sensitivity of acid-fast bacilli (AFB) sputum smears and absence of productive cough are obstacles to the diagnosis of pulmonary tuberculosis (PTB) in hospitals that lack access to bronchoscopy. OBJECTIVES: To evaluate induced sputum, gastric content, blood and urine specimens to improve PTB diagnosis in patients not diagnosed by expectorated sputum AFB smears. DESIGN: Patients admitted to the medical wards of a large public hospital in Gaborone, Botswana, were prospectively enrolled if they had symptoms consistent with PTB, an abnormal chest radiograph, were treated empirically with anti-tuberculosis chemotherapy or had no improvement on antibiotics, and had a non-productive cough or AFB smear-negative sputum. Induced sputum was stained for AFB and Mycobacterium tuberculosis cultures were performed on induced sputum, gastric contents, urine and blood. RESULTS: Of 140 patients meeting the enrollment criteria, 113 (81%) were human immunodeficiency virus (HIV) positive. Fifty-seven (41%) had PTB based on positive cultures from one or more sites, including 48 (84%) from induced sputum, 17 (30%) urine, 13 (23%) gastric contents and 7 (12%) blood. AFB smears were positive in only 18 (32%) culture-proven PTB cases. CONCLUSION: Induced sputum cultures greatly enhanced M. tuberculosis detection in patients with a high prevalence of HIV/AIDS in a hospital without access to bronchoscopy.

Effects of oestrogen on trigeminal ganglia in culture: implications for hormonal effects on migraine.

Although migraine is more common in women than men and often linked to the menstrual cycle, few studies have investigated the biological basis of hormonal influences on the trigeminovascular system. In the present study we investigated the effect of physiological levels (10(-9) m) oestrogen on female rat trigeminal ganglia in vitro. Immunocytochemical analysis demonstrated the presence of oestrogen receptor-alpha in a predominantly cytoplasmic location and in neurites. Microarray analysis demonstrated that oestrogen treatment regulates several genes with potential relevance to menstrual migraine. The genes that were upregulated included synapsin-2, endothelin receptor type B, activity and neurotransmitter-induced early gene 7 (ania-7), phosphoserine aminotransferase, MHC-1b, and ERK-1. Down-regulated genes included IL-R1, bradykinin B2 receptor, N-tropomodulin, CCL20, GABA transporter protein, fetal intestinal lactase-phlorizin hydrolase, carcinoembryonic antigen-related protein, zinc finger protein 36, epsin 1 and cysteine string protein. Protein activity assays demonstrated that exposure of the cultured neurons to oestrogen leads to activation of ERK, which has been linked to inflammatory pain. Immunocytochemistry demonstrated that activated ERK was present in neurons containing peripherin, a marker of nociceptive neurons. Several of the genes in the present study may provide potential targets for understanding the association of oestrogen with migraine and other hormone-related orofacial pain.

Small rat islets are superior to large islets in in vitro function and in transplantation outcomes.

Barriers to the use of islet transplantation as a practical treatment for diabetes include the limited number of available donor pancreata. This project was designed to determine whether the size of the islet could influence the success rate of islet transplantations in rats. Islets from adult rats were divided into two groups containing small (diameter <125 microm) or large (diameter >150 microm) islets. An average pancreas yielded three times more small islets than large. Smaller islets were approximately 20% more viable, with large islets containing a scattered pattern of necrotic and apoptotic cells or central core cell death. Small islets in culture consumed twice as much oxygen as large islets when normalized for the same islet equivalents. In static incubation, small islets released three times more insulin under basal conditions than did large islets. During exposure to high glucose conditions, the small islets released four times more insulin than the same islet equivalencies of large islets, and five times more insulin was released by the small islets in response to glucose and depolarization with K+. Most importantly, the small islets were far superior to large islets when transplanted into diabetic animals. When marginal islet equivalencies were used for renal subcapsular transplantation, large islets failed to produce euglycemia in any recipient rats, whereas small islets were successful 80% of the time. The results indicate that small islets are superior to large islets in in vitro testing and for transplantation into the kidney capsule of diabetic rats.

Clinical protocol. A phase 1 open-label clinical trial of the safety and tolerability of single escalating doses of autologous CD4 T cells transduced with VRX496 in HIV-positive subjects.

The proposed study is a Phase I trial to evaluate a HIV-based lentiviral vector carrying an antisense sequence targeted to HIV in the treatment of HIV infection. The primary objective of this Phase I study is to determine the safety and tolerability of treatment with autologous CD4+ T cells modified (transduced) ex vivo with VRX496 when administered to HIV-infected patients. VRX496 is a completely gutted lentiviral vector and does not code for any viral proteins. The viral vector contains an antisense sequence targeted to the HIV envelope (env) gene. VRX496 directly interferes with wild-type HIV (wt-HIV) expression via anti-env antisense expression in vector transduced CD4 cells that become infected with wt-HIV. Expression of the anti-HIV antisense env from a HIV vector transcript would target wt-HIV RNA and destroy it, and hence, decrease productive HIV replication from CD4 T cells. The clinical goal for this treatment approach is to decrease viral loads and promote CD4 T cell survival in vivo. Data from in vitro studies suggest that HIV vectors such as VRX496 could potentially reduce viral loads in HIV-infected individuals and thus could delay the onset to AIDS while promoting CD4 T cell survival and providing the immune system with a better chance to control the infection. Additionally, preliminary results from experiments in SCID mice (mice with transplanted human immune cells) indicate that the human cells transduced with VRX496 and implanted into the SCID mice do not elicit any overt adverse effects. HIV-infected patients (CD4 T cell count of >200/mm3, discontinued from HAART therapy) will undergo leukoapheresis with subsequent CD4 T cell isolation. Patient CD4 T cells will be transduced ex vivo with the vector, expanded for 8-11 days, and then the modified cells will be reintroduced into the patient. Each subject will receive a single intravenous injection infused over 30 minutes; subjects will be examined 24, 48, and 72 hours post-injection and weekly for 4 weeks. Patients will receive one of four different ascending doses (1 x 10(9), 3 x 10(9), 1 x 10(10), and 3 X 10(10) cells/patient). Doses will be administered to four independent, sequential subject cohorts of 3 patients. Groups will be administered escalating doses at 6-week intervals after safety has been demonstrated in the previous group. Follow-up examinations will be conducted 1, 3, and 6 months post-injection. Long term follow-up including RCR testing will be performed.

Species differences in [11C]clorgyline binding in brain.

[11C]Clorgyline selectively binds to MAO A in the human brain. This contrasts with a recent report that [11C]clorgyline (in contrast to other labeled MAO A inhibitors) is not retained in the rhesus monkey brain [4]. To explore this difference, we compared [11C]clorgyline in the baboon brain before and after clorgyline pretreatment and we also synthesized deuterium substituted [11C]clorgyline (and its nor-precursor) for comparison. [11C]Clorgyline was not retained in the baboon brain nor was it influenced by clorgyline pretreatment or by deuterium substitution, contrasting to results in humans. This suggests a species difference in the susceptibility of MAO A to inhibition by clorgyline and represents an unusual example of where the behavior of a radiotracer in the baboon brain does not predict its behavior in the human brain.

Increased plasma tryptophan in HIV-infected patients treated with pharmacologic doses of nicotinamide.

OBJECTIVE: Decreased plasma tryptophan in persons infected with human immunodeficiency virus (HIV) was first reported over a decade ago, and this observation has since been confirmed by many groups. Before this study, only zidovudine (an antiviral medication) had been reported to reverse plasma tryptophan depletion in HIV-infected persons. Starting with the hypothesis that HIV induces a pellagra-like state and that plasma tryptophan in HIV-infected patients is decreased as a known biochemical correlate of pellagra, we predicted that niacin therapy would reverse plasma tryptophan depletion as it does in pellagra. METHODS: After receiving approval from the institutional review board, we treated HIV-infected patients for 2 mo with high-dose niacin in the form of oral nicotinamide. RESULTS: There was an average 40% increase in plasma tryptophan (P = 0.01) in the four HIV-infected individuals who completed the 2-mo protocol. This finding was specific in that four other amino acids, which have been shown to have significant plasma concentration alterations during HIV infection (i.e., cystine, methionine, taurine, and lysine), showed no significant change with nicotinamide therapy. CONCLUSIONS: There were no adverse side effects attributable to this treatment. The effects of high-dose nicotinamide treatment on morbidity or mortality in HIV-infected persons are yet to be determined. This report marks the first successful use of a vitamin to reverse this HIV-induced metabolic abnormality.

Maintenance of brain monoamine oxidase B inhibition in smokers after overnight cigarette abstinence.

OBJECTIVE: The authors' goal was to replicate a previous finding that smokers have lower brain monoamine oxidase B (MAO-B) levels than comparison nonsmoking subjects and to determine if levels recover after overnight cigarette abstinence. METHOD: Brain MAO-B levels were measured by means of positron emission tomography in six smokers who were scanned twice: 11.3 hours (baseline) and 10 minutes after smoking one cigarette. RESULTS: Average MAO-B levels in smokers in the present study were similar to those found in the previous study and averaged 39% (SD=17) lower than those found in a comparison group of nonsmokers. Brain MAO-B levels did not differ between baseline levels and 10 minutes after smoking. CONCLUSIONS: This study reinforces the need to investigate whether MAO-B inhibition may account for some of the behavioral and epidemiological features of smoking.

Reproducibility of repeated measures of deuterium substituted [11C]L-deprenyl ([11C]L-deprenyl-D2) binding in the human brain.

The purpose of this study was to assess the reproducibility of repeated positron emission tomography (PET) measures of brain monoamine oxidase B (MAO B) using deuterium-substituted [11C]L-deprenyl ([11C]L-deprenyl-D2) in normal subjects and to validate the method used for estimating the kinetic constants from the irreversible 3-compartment model applied to the tracer binding. Five normal healthy subjects (age range 23-73 years) each received two PET scans with [11C]L-deprenyl-D2. The time interval between scans was 7-27 days. Time-activity data from eight regions of interest and an arterial plasma input function was used to calculate lambda k3, a model term proportional to MAO B, and K1, the plasma to brain transfer constant that is related to blood flow. Linear (LIN) and nonlinear least-squares (NLLSQ) estimation methods were used to calculate the optimum model constants. A comparison of time-activity curves for scan 1 and scan 2 showed that the percent of change for peak uptake varied from -18.5 to 15.0% and that increases and decreases in uptake on scan 2 were associated with increases and decreases in the value of the arterial input of the tracer. Calculation of lambda k3 showed a difference between scan 1 and scan 2 in the global value ranging between -6.97 and 4.5% (average -2.1 +/- 4.7%). The average percent change for eight brain regions for the five subjects was -2.84 +/- 7.07%. Values of lambda k3 for scan 1 and scan 2 were highly correlated (r2 = 0.98; p < 0.0001; slope 0.955). Similarly, values of K1 showed a significant correlation between scan 1 and scan 2 (r2 = 0.61; p < 0.0001; slope 0.638) though the values for scan 2 were generally lower than those of scan 1. There was essentially no difference between the values of model constants calculated using the NLLSQ or LIN methods. Regional brain uptake of [11C]L-deprenyl-D2 varied between scan 1 and scan 2, driven by the differences in arterial tracer input. Application of a 3-compartment model to regional time-activity data and arterial input function yielded lambda k3 values for scan 1 and scan 2 with an average difference of -2.84 +/- 7.07%. Linear regression applied to values of lambda k3 from the LIN and NLLSQ methods validated the use of the linear method for calculating lambda k3.

Effects of Mycobacterium avium complex-infection treatment on cytokine expression in human immunodeficiency virus-infected persons: results of AIDS clinical trials group protocol 853.

Human immunodeficiency virus (HIV) type 1-infected persons with newly diagnosed Mycobacterium avium complex (MAC) bacteremia were enrolled in an 8-week study to determine whether treatment of MAC infection is associated with decreases in plasma tumor necrosis factor (TNF)-alpha levels. Blood specimens were obtained for quantitative MAC cultures and to determine plasma levels of HIV RNA, TNF-alpha, and other proinflammatory cytokines. MAC levels decreased by 1.75 log at week 4 (P=.008) and by 2.48 log at week 8 (P=.001). Plasma TNF-alpha decreased by 0.15 log at week 4 (P=.042) and by 0. 40 log at week 8 (P=.027). Plasma interleukin (IL)-6 decreased by 0. 56 log at week 8 (P=.039). There were nonsignificant trends (P<.10) for plasma levels of IL-1beta and HIV RNA to decrease at week 8. Nonsignificant decreases in plasma levels of TNF-alpha, IL-1beta, IL-6, and HIV RNA were also seen in those individuals who remained on stable antiretroviral therapy throughout the 8 weeks of the study.

Vaccination of seronegative volunteers with a human immunodeficiency virus type 1 env/rev DNA vaccine induces antigen-specific proliferation and lymphocyte production of beta-chemokines.

There is a pressing need to test novel vaccine concepts in an effort to develop an effective vaccine for human immunodeficiency virus (HIV) type 1. A phase I clinical study was done to test the immunogenicity of an HIV env/rev DNA vaccine, which was administered intramuscularly to HIV-1-seronegative persons. Subjects received 3 doses of vaccine at a single concentration (100 or 300 microgram) at 0, 4, 8, and 24 weeks. In at least 1 of multiple assays, the 6 subjects who received the 300-microgram dose had DNA vaccine-induced antigen-specific lymphocyte proliferative responses and antigen-specific production of both interferon-gamma and beta-chemokine. Furthermore, 4 of 5 subjects in the 300 microgram-dose group responded to both the rev and env components of the vaccine. The responses did not persist within inoculated individuals and scored in different individuals at different times in the trial. This study supports that HIV-1 DNA vaccine antigens can stimulate multiple immune responses in vaccine-naive individuals, and it warrants additional studies designed to enhance DNA vaccine immunogenicity.

Induction of tissue plasminogen activator secretion from rat heart microvascular cells by fM 1,25(OH)(2)D(3).

We investigated the effects of 1,25-dihydroxyvitamin D(3) [25(OH)(2)D(3)] on tissue plasminogen activator (tPA) secretion from primary cultures of rat heart microvascular cells. After an initial 5-day culture period, cells were treated for 24 h with 1,25(OH)(2)D(3) and several of its analogs. The results showed that 1,25(OH)(2)D(3) induced tPA secretion at 10(-10) to 10(-16) M. A less calcemic analog, Ro-25-8272, and an analog that binds the vitamin D receptor but is ineffective at perturbing Ca(2+) channels, Ro-24-5531, were approximately 10% as active as 1,25(OH)(2)D(3). An analog that binds the vitamin D receptor poorly but is an effective Ca(2+) channel agonist, Ro-24-2287, required approximately 10(-13) M to induce tPA secretion. Combinations of Ro-24-5531 and Ro-24-2287 were approximately as potent as 1,25(OH)(2)D(3). Treatment of the cells with BAY K 8644 or thapsigargin also increased tPA secretion, suggesting that increased cytosolic calcium concentration ([Ca(2+)]) induces tPA secretion. The results suggested that the sensitivity of the tPA secretory response of microvascular cells to 1,25(OH)(2)D(3) was due in part to generation of a vitamin D-depleted state in vitro and in part to synergistic effects of 1,25(OH)(2)D(3) on two different induction pathways of tPA release.

Azithromycin as treatment for disseminated Mycobacterium avium complex in AIDS patients.

This multicenter, randomized, dose-ranging study was performed to determine the safety and efficacy of two different doses of azithromycin for treating disseminated Mycobacterium avium complex (MAC) in patients with AIDS. Eighty-eight AIDS patients with symptoms and blood cultures consistent with disseminated MAC were treated with 600 or 1,200 mg of azithromycin daily for 6 weeks; 62 patients completed the entire 6 weeks of study. Of note, this study was done prior to the time when combination antiretroviral or anti-MAC regimens were the standard of care. Over the 6-week study period, symptomatic improvement was noted in both dose groups. Microbiological responses were comparable, with mean decreases of 1. 5 and 2.0 log CFU/ml in the high- and low-dose groups, respectively. Sterilization of blood cultures occurred in 54% of samples; patients with lower baseline colony counts were more likely to achieve culture negativity. Resistance developed in one patient. Gastrointestinal symptoms were the most common side effects and were more frequent in patients receiving 1,200 mg. Azithromycin is a useful alternative treatment for disseminated MAC infection in AIDS patients. Symptomatic improvement correlates with measurable decreases in mycobacterial load.

HIV-1 DNA vaccines and chemokines.

DNA vaccines have a demonstrated ability to induce humoral and cellular immune responses in animal models and humans. The technology, although it dates back to the 1950's, has had an insurgence of interest within the past few years following concurrent research papers. The basic technology is being applied broadly to viral, bacterial and parasitic infections. It has also been demonstrated that genes delivered via plasmid expression vectors result in expression of functional proteins in the inoculated host. Further, injection of plasmids encoding cytokine, chemokine or co-stimulatory molecules, also referred to as immunomodulatory plasmids can lead to the further expansion of this technology to include directed immunology. We have been developing DNA technology specifically with a focus as a vaccine against HIV-1 infection. We report that such vaccines can stimulate immune responses in a variety of relevant animal systems including humoral and cellular responses as well as the production of beta-chemokines. We describe that the beta-chemokines can both modulate the immune response induced by DNA vaccines and be modulated by the DNA vaccines in the murine and chimpanzee models as well as in humans.

C1.7 antigen expression on CD8+ T cells is activation dependent: increased proportion of C1.7+CD8+ T cells in HIV-1-infected patients with progressing disease.

The C1.7 Ag is a surface marker previously shown to be expressed on all NK cells and on a subset of CD8+ T cells. We report in this study that C1.7 Ag expression on peripheral blood-derived CD8+ T cells overlaps with activation markers S6F1high and CD29high and is reciprocally expressed with CD62L. C1.7 Ag expression can be induced in vitro on CD8+ T cells by anti-CD3 cross-linking, suggesting that C1.7 Ag is activation dependent. In contrast to NK cells, C1.7 Ag does not signal on CD8+ T cells, nor does it induce redirected lysis upon ligation. The proportion of C1.7 Ag+CD8+ T cells is increased in HIV-infected patients compared with healthy donors. In 69 HIV-infected patients, we observed a significant inverse correlation between the percentage of C1.7 Ag-expressing CD8+ T cells and the absolute CD4+ T cell count. Two-year clinical follow-up of patients with initial CD4+ T cell count of >400 cells/mm3 and a normal proportion of C1.7 Ag+CD8+ T cells revealed that these patients were clinically stable with minimal HIV-associated symptoms. In contrast, 10 of 12 patients with CD4+ T cell counts of >400 cells/mm3 and an elevated proportion of C1.7 Ag+CD8+ T cells were symptomatic. ANOVA analysis of patients indicates that C1.7 Ag is a better predictor of disease progression than CD4 count. Overall, our findings indicate that C1.7 Ag is the first described marker for activated/memory CD8+ T cells and a useful parameter for evaluating the level of CD8+ T cell activation in vivo.

Enhancement of cellular immune response in HIV-1 seropositive individuals: A DNA-based trial.

A DNA-based vaccine containing HIV-1 Env and Rev genes was tested for safety and host immune response in 15 HIV-infected asymptomatic patients with CD4-positive lymphocyte counts >/=500/microl of blood and receiving no antiviral therapy. Successive groups of patients received three doses of vaccine at 30, 100, or 300 microg at 10-week intervals in a dose-escalation trial. Some changes were noted in cytotoxic T-lymphocyte activity against gp160-bearing targets. Importantly, enhanced specific lymphocyte proliferative activity against HIV-1 envelope was observed in multiple patients. Three of three patients in the 300-microg dose group also developed increased MIP-1alpha levels which were detectable in their serum. Interestingly patients in the lowest dose group showed no overall changes in the immune parameters measured. The majority of patients who exhibited increases in any immune parameters were contained within the 300 microg, which was the highest dose group. These studies support further investigation of this technology for the production of antigen-specific immune responses in humans.

Use of PCR in detection of Mycobacterium avium complex (MAC) bacteremia: sensitivity of the assay and effect of treatment for MAC infection on concentrations of human immunodeficiency virus in plasma.

We evaluated the sensitivity and specificity of a PCR-based qualitative test for the rapid diagnosis of Mycobacterium avium-M. intracellulare complex (MAC) bacteremia in patients with AIDS disease. Eleven subjects with newly culture-proven MAC bacteremia had the following tests performed at biweekly intervals during the first 8 weeks of therapy: blood culture, Mycobacterium-specific PCR, and quantitative human immunodeficiency virus (HIV) viral-load testing. Mycobacterium genus-specific biotinylated primers were used to amplify a sequence of approximately 582 nucleotides within the 16S rRNA genes of M. avium and M. intracellulare. Detection of the amplified product was performed with an oligonucleotide probe-coated microwell plate combined with an avidin-horseradish peroxidase-tetramethylbenzidine conjugate-substrate system. While not as sensitive as BACTEC culture, PCR detected 17 of 18 specimens which grew >/=40 organisms/ml (94.4% sensitivity) and 9 of 16 specimens which grew