Publications

The HIV-1 protein Nef enhances viral pathogenicity and accelerates disease progression in vivo. Nef potentiates T cell activation by an unknown mechanism, probably by optimizing the intracellular environment for HIV replication. Using a new T cell reporter system, we have found that Nef more than doubles the number of cells expressing the transcription factors NF-kappaB and NFAT after TCR stimulation. This Nef-induced priming of TCR signaling pathways occurred independently of calcium signaling and involved a very proximal step before protein kinase C activation. Engagement of the TCR by MHC-bound Ag triggers the formation of the immunological synapse by recruiting detergent-resistant membrane microdomains, termed lipid rafts. Approximately 5-10% of the total cellular pool of Nef is localized within lipid rafts. Using confocal and real-time microscopy, we found that Nef in lipid rafts was recruited into the immunological synapse within minutes after Ab engagement of the TCR/CD3 and CD28 receptors. This recruitment was dependent on the N-terminal domain of Nef encompassing its myristoylation. Nef did not increase the number of cell surface lipid rafts or immunological synapses. Recently, studies have shown a specific interaction of Nef with an active subpopulation of p21-activated kinase-2 found only in the lipid rafts. Thus, the corecruitment of Nef and key cellular partners (e.g., activated p21-activated kinase-2) into the immunological synapse may underlie the increased frequency of cells expressing transcriptionally active forms of NF-kappaB and NFAT and the resultant changes in T cell activation.

Increasing resistance to chloroquine necessitates the evaluation of other antimalarial therapies in Africa. We compared the efficacies of amodiaquine (AQ), sulfadoxine-pyrimethamine (SP), and AQ + SP for the treatment of uncomplicated falciparum malaria in a randomized trial of patients 6 months of age or older in Bobo-Dioulasso, Burkina Faso. Of the 944 patients enrolled, 829 (88%; 53% under 5 years of age) were assigned 28-day efficacy outcomes. For all regimens, early treatment failures were uncommon (< 2%). Considering all treatment failures based on WHO criteria, AQ + SP was most efficacious (failures in 4.2%), followed by SP (9.1%) and AQ (17.9%; P < 0.02 for all pairwise comparisons). Considering only clinical failures, relative efficacies were similar (failures in 2.1% with AQ + SP, 6.5% with SP, and 13.2% with AQ; P < 0.02 for all pairwise comparisons). The risk of recrudescence was lower with AQ + SP (2.1%) compared with SP (6.1%, P = 0.02) and AQ (8.1%, P = 0.001). Risks of new infection were lower with AQ + SP (2.1%) and SP (2.4%) compared with AQ (9.1%, P < 0.001 for both comparisons). No serious adverse events were seen. AQ + SP appears to offer a highly effective, inexpensive, and available therapy for the treatment of uncomplicated malaria in Burkina Faso.

The etiology of atherosclerosis is complex and multifactorial but there is extensive evidence indicating that oxidized lipoproteins may play a key role. At present, the site and mechanism by which lipoproteins are oxidized are not resolved, and it is not clear if oxidized lipoproteins form locally in the artery wall and/or are sequestered in atherosclerotic lesions following the uptake of circulating oxidized lipoproteins. We have been focusing our studies on demonstrating that such potentially atherogenic oxidized lipoproteins in the circulation are at least partially derived from oxidized lipids in the diet. Thus, the purpose of our work has been to determine in humans whether oxidized dietary oxidized fats such as oxidized fatty acids and oxidized cholesterol are absorbed and contribute to the pool of oxidized lipids in circulating lipoproteins. When a meal containing oxidized linoleic acid was fed to normal subjects, oxidized fatty acids were found only in the postprandial chylomicron/chylomicron remnants (CM/RM) which were cleared from circulation within 8 h. No oxidized fatty acids were detected in low density lipoprotein (LDL) or high density lipoprotein (HDL) fractions at any time. However, when alpha-epoxy cholesterol was fed to human subjects, alpha-epoxy cholesterol in serum was found in CM/RM and also in endogenous very low density lipoprotein, LDL, and HDL and remained in the circulation for 72 h. In vitro incubation of the CM/RM fraction containing alpha-epoxy cholesterol with human LDL and HDL that did not contain alpha-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. We have suggested that cholesteryl ester transfer protein is mediating the transfer. Thus, alpha-epoxy cholesterol in the diet is incorporated into CM/RM fraction and then transferred to LDL and HDL contributing to lipoprotein oxidation. We hypothesize that diet-derived oxidized fatty acids in chylomicron remnants and oxidized cholesterol in remnants and LDL accelerate atherosclerosis by increasing oxidized lipid levels in circulating LDL and chylomicron remnants. This hypothesis is supported by our feeding experiments in animals. When rabbits were fed oxidized fatty acids or oxidized cholesterol, the fatty streak lesions in the aorta were increased by 100%. Moreover, dietary oxidized cholesterol significantly increased aortic lesions in apo-E and LDL receptor-deficient mice. A typical Western diet is rich in oxidized fats and therefore could contribute to the increased arterial atherosclerosis in our population.

BACKGROUND

Tropospheric ozone (O3) is an oxidant, outdoor air pollutant. Chronic exposure has been associated with decreased lung function in children and adolescents. This study investigated the effects of long-term exposure to O3 on lung function in college freshmen.

METHODS

We recruited University of California, Berkeley students (n=255) who were lifelong residents of the Los Angeles and San Francisco Bay areas and who never smoked. Lifetime exposures to O3, small particulate matter (PM10), and nitrogen dioxide (NO2) were based on spatial interpolation of compliance monitor measurements to all residences at which students lived. Spirometry was performed between February and May, times when students would not have had recent exposure to increased levels of O3.

RESULTS

Lifetime exposure to O3 was associated with decreased levels of measures of small airways (<2 mm) function (FEF75 and FEF25-75). There was an interaction with the FEF25-75/FVC ratio, a measure of intrinsic airway size. Subjects with a large ratio were less likely to have decreases in FEF75 and FEF25-75 for a given estimated lifetime exposure to O3. This association was not altered by history of chronic respiratory disease, allergy, second-hand exposure to environmental tobacco smoke, exposure to PM10 and NO2, or measurement errors in exposure assessment.

CONCLUSIONS

A history of increased level of lifetime exposure to ambient O3 is associated with decreased function of airways in which O3 deposition in the lungs is the greatest. Adolescents with intrinsically smaller airways appear to be at greatest risk. Any environmental or genetic factors that lead to reduced airway size may lead to increased susceptibility to the adverse effects of ambient ozone.

BACKGROUND

Arsenic is a unique human carcinogen in that it causes lung cancer by exposure through ingestion (in drinking water) as well as through inhalation. Less is known about nonmalignant pulmonary disease after exposure to arsenic in drinking water.

METHODS

We recruited 108 subjects with arsenic-caused skin lesions and 150 subjects without lesions from a population survey of over 7000 people in an arsenic-exposed region in West Bengal, India. Thirty-eight study participants who reported at least 2 years of chronic cough underwent high-resolution computed tomography (CT); these scans were read by investigators in India and the United States without knowledge of the presence or absence of skin lesions.

RESULTS

The mean (+/-standard deviation) bronchiectasis severity score was 3.4 (+/-3.6) in the 27 participants with skin lesions and 0.9 (+/-1.6) in the 11 participants without these lesions. In subjects who reported chronic cough, CT evidence of bronchiectasis was found in 18 (67%) participants with skin lesions and 3 (27%) subjects without skin lesions. Overall, subjects with arsenic-caused skin lesions had a 10-fold increased prevalence of bronchiectasis compared with subjects who did not have skin lesions (adjusted odds ratio=10; 95% confidence interval=2.7-37).

CONCLUSIONS

These results suggest that, in addition to being a cause of lung cancer, ingestion of high concentrations of arsenic in drinking water may be a cause of bronchiectasis.

The human immunodeficiency virus (HIV)-mediated immune response may be beneficial or harmful, depending on the balance between expansion of HIV-specific T cells and the level of generalized immune activation. The current study utilizes multicolor cytokine flow cytometry to study HIV-specific T cells and T-cell activation in 179 chronically infected individuals at various stages of HIV disease, including those with low-level viremia in the absence of therapy ("controllers"), low-level drug-resistant viremia in the presence of therapy (partial controllers on antiretroviral therapy [PCAT]), and high-level viremia ("noncontrollers"). Compared to noncontrollers, controllers exhibited higher frequencies of HIV-specific interleukin-2-positive gamma interferon-positive (IL-2(+) IFN-gamma(+)) CD4(+) T cells. The presence of HIV-specific CD4(+) IL-2(+) T cells was associated with low levels of proliferating T cells within the less-differentiated T-cell subpopulations (defined by CD45RA, CCR7, CD27, and CD28). Despite prior history of progressive disease, PCAT patients exhibited many immunologic characteristics seen in controllers, including high frequencies of IL-2(+) IFN-gamma(+) CD4(+) T cells. Measures of immune activation were lower in all CD8(+) T-cell subsets in controllers and PCAT compared to noncontrollers. Thus, control of HIV replication is associated with high levels of HIV-specific IL-2(+) and IFN-gamma(+) CD4(+) T cells and low levels of T-cell activation. This immunologic state is one where the host responds to HIV by expanding but not exhausting HIV-specific T cells while maintaining a relatively quiescent immune system. Despite a history of advanced HIV disease, a subset of individuals with multidrug-resistant HIV exhibit an immunologic profile comparable to that of controllers, suggesting that functional immunity can be reconstituted with partially suppressive highly active antiretroviral therapy.

The sequence diversity of human immunodeficiency virus type 1 (HIV-1) represents a major obstacle to the development of an effective vaccine, yet the forces impacting the evolution of this pathogen remain unclear. To address this issue we assessed the relationship between genome-wide viral evolution and adaptive CD8+ T-cell responses in four clade B virus-infected patients studied longitudinally for as long as 5 years after acute infection. Of the 98 amino acid mutations identified in nonenvelope antigens, 53% were associated with detectable CD8+ T-cell responses, indicative of positive selective immune pressures. An additional 18% of amino acid mutations represented substitutions toward common clade B consensus sequence residues, nine of which were strongly associated with HLA class I alleles not expressed by the subjects and thus indicative of reversions of transmitted CD8 escape mutations. Thus, nearly two-thirds of all mutations were attributable to CD8+ T-cell selective pressures. A closer examination of CD8 escape mutations in additional persons with chronic disease indicated that not only did immune pressures frequently result in selection of identical amino acid substitutions in mutating epitopes, but mutating residues also correlated with highly polymorphic sites in both clade B and C viruses. These data indicate a dominant role for cellular immune selective pressures in driving both individual and global HIV-1 evolution. The stereotypic nature of acquired mutations provides support for biochemical constraints limiting HIV-1 evolution and for the impact of CD8 escape mutations on viral fitness.

BACKGROUND

Estimating an individual woman's absolute risk for breast cancer is essential for decision making about screening and preventive recommendations. Although the current standard, the Gail model, is well calibrated in populations, it performs poorly for individuals. Mammographic breast density (BD) may improve the predictive accuracy of the Gail model.

METHODS

Prospective observational cohort of 81,777 women in the San Francisco Mammography Registry presenting for mammography during 1993 through 2002 who had no prior diagnosis of breast cancer. Breast density was rated by clinical radiologists using the Breast Imaging Reporting and Data System classification (almost entirely fat; scattered fibroglandular densities; heterogeneously dense; extremely dense). Breast cancer cases were identified through linkage to Northern California Surveillance Epidemiology End Results (SEER) program. We compared the predictive accuracy of models with Gail risk, breast density, and the combination. All models were adjusted for age and ethnicity.

RESULTS

During 5.1 years of follow-up, 955 women were diagnosed with invasive breast cancer. The Gail model had modest predictive accuracy (concordance index (c-index) 0.67; 95% CI 0.65-0.68). Adding breast density to the model increased the predictive accuracy to 0.68 (95% CI .66-.70, p < 0.01 compared with the Gail model alone). The model containing only breast density adjusted for age and ethnicity had predictive accuracy equivalent to the Gail model (c-index 0.67, 95% CI 0.65-0.68).

CONCLUSION

The addition of breast density measured by BI-RADS categories minimally improved the predictive accuracy of the Gail model. A model based on breast density alone adjusted for age and ethnicity was as accurate as the Gail model.

Zomepirac [ZP, 5-(chlorobenzoyl)-1,4-dimethylpyrrole-2-acetic acid] was withdrawn from the market because of unpredictable allergic reactions that may have been caused by ZP-protein adducts formed by reaction of the reactive acyl glucuronide of ZP (ZP-O-G) with endogenous proteins. To test the hypothesis that the reactive ZP acyl coenzyme A thioester (ZP-CoA) was formed and potentially could contribute to formation of ZP-protein adducts, we investigated the acyl CoA-dependent metabolism of ZP in freshly isolated rat hepatocytes (1 mM) and in vivo (100 mg ZP/kg, ip) in rat livers (2 h after dose administration), rat bile (0-4 h), and rat urine (0-24 h). ZP-CoA was detected in freshly isolated hepatocytes and in vivo in rat livers by LC/MS/MS. In addition, the ZP glycine conjugate (ZP-Gly) and ZP taurine conjugates (ZP-Tau) were identified by LC/MS/MS in rat hepatocytes and in vivo in rat livers, rat urine, and rat bile. The identities of ZP-CoA, ZP-Gly, and ZP-Tau were confirmed by comparison of retention times and MS/MS spectra with those of authentic standards. Moreover, the ZP acyl carnitine ester was detected in rat urine and rat bile based upon (i) the chlorine isotope pattern, (ii) MS/MS spectra showing significant ions characteristic for carnitine (m/z 60, 144 and loss of m/z 59) and ZP (m/z 139), and (iii) accurate mass measurements with a mass accuracy of 0.2 ppm. ZP-CoA serves as an obligatory intermediate in the formation of ZP-Gly, ZP-Tau, and ZP carnitine ester, and it is therefore of mechanistic significance that these conjugates were identified. Finally, time-dependent concentration profiles obtained in experiments with rat hepatocytes and in vivo from quantitative analysis of rat livers indicate that ZP-CoA, in addition to ZP-O-G, may contribute to formation of the potentially toxic covalent ZP-protein adducts.

BACKGROUND

Undernutrition in homebound older adults is a significant problem. The purpose of this study was to investigate the effect of the presence of others, both within the household and during meals, on caloric intake in homebound older adults.

METHODS

In-depth interviews and three 24-hour dietary recalls were obtained from 50 older adults who were receiving home health services. Descriptive statistics were used to characterize participants, and hierarchical linear modeling was performed to evaluate predictors of caloric intake per meal.

RESULTS

Participants' mean age was 77. Females composed 65% and African Americans composed 42% of the sample. Analyses are based on 553 meal observations. The majority (84%) of participants consumed all meals for each of the 3 days of data collection; however, they consumed an average of only 1305 calories per day. Hierarchical linear modeling analysis indicated that persons who had others present during meals consumed an average of 114.0 calories more per meal than those who ate alone (p =.009) and that women consumed 76.7 fewer calories per meal than did men (p =.045). The presence of others within the household had no effect on caloric intake.

CONCLUSION

This research suggests that a simple and inexpensive way to increase caloric intake in homebound older adults is to make arrangements for family members or caregivers to eat with them.