Differences in the Selection Bottleneck between Modes of Sexual Transmission Influence the Genetic Composition of the HIV-1 Founder Virus.

Due to the stringent population bottleneck that occurs during sexual HIV-1 transmission, systemic infection is typically established by a limited number of founder viruses. Elucidation of the precise forces influencing the selection of founder viruses may reveal key vulnerabilities that could aid in the development of a vaccine or other clinical interventions. Here, we utilize deep sequencing data and apply a genetic distance-based method to investigate whether the mode of sexual transmission shapes the nascent founder viral genome. Analysis of 74 acute and early HIV-1 infected subjects revealed that 83% of men who have sex with men (MSM) exhibit a single founder virus, levels similar to those previously observed in heterosexual (HSX) transmission. In a metadata analysis of a total of 354 subjects, including HSX, MSM and injecting drug users (IDU), we also observed no significant differences in the frequency of single founder virus infections between HSX and MSM transmissions. However, comparison of HIV-1 envelope sequences revealed that HSX founder viruses exhibited a greater number of codon sites under positive selection, as well as stronger transmission indices possibly reflective of higher fitness variants. Moreover, specific genetic "signatures" within MSM and HSX founder viruses were identified, with single polymorphisms within gp41 enriched among HSX viruses while more complex patterns, including clustered polymorphisms surrounding the CD4 binding site, were enriched in MSM viruses. While our findings do not support an influence of the mode of sexual transmission on the number of founder viruses, they do demonstrate that there are marked differences in the selection bottleneck that can significantly shape their genetic composition. This study illustrates the complex dynamics of the transmission bottleneck and reveals that distinct genetic bottleneck processes exist dependent upon the mode of HIV-1 transmission.

Frequent and strong antibody-mediated natural killer cell activation in response to HIV-1 Env in individuals with chronic HIV-1 infection.

Natural killer (NK) cells play a critical role in the control of HIV-1 infection, and NK cells that respond to HIV-1 peptides have been recently described. However, the mechanisms by which NK cells recognize HIV-1 antigens are not fully understood. We investigated NK cell activation in response to HIV-1 peptides during early and chronic HIV-1 clade B infection using a whole-blood assay and multiparameter flow cytometry. Antibody-mediated NK cell activation in response to HIV-1 peptides was not detected in HIV-1-uninfected individuals. In contrast, 79% of individuals with chronic infection and 22% of individuals with early infection had detectable gamma interferon (IFN-γ) NK cell responses to HIV-1 antigens (P < 0.00001). IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing NK cells most frequently targeted Env gp120 (median of 4% and range of 0 to 31% of all NK cells). NK cells rarely targeted other HIV-1 proteins such as Gag, Pol, and Nef. Antibody-mediated NK cell responses to peptides mapped predominantly to Env protein, required the presence of plasma or plasma IgG, and resulted in lower CD16 expression on NK cells, suggesting an antibody-mediated activation of NK cells. Further studies are needed to assess the consequences of these antibody-mediated NK cell responses for HIV-1 disease progression and vaccine-induced protection from infection.

Medication Exposure in Pregnancy Risk Evaluation Program.

To describe a program to study medication safety in pregnancy, the Medication Exposure in Pregnancy Risk Evaluation Program (MEPREP). MEPREP is a multi-site collaborative research program developed to enable the conduct of studies of medication use and outcomes in pregnancy. Collaborators include the U.S. Food and Drug Administration and researchers at the HMO Research Network, Kaiser Permanente Northern and Southern California, and Vanderbilt University. Datasets have been created at each site linking healthcare data for women delivering an infant between January 1, 2001 and December 31, 2008 and infants born to these women. Standardized data files include maternal and infant characteristics, medication use, and medical care at 11 health plans within 9 states; birth certificate data were obtained from the state departments of public health. MEPREP currently involves more than 20 medication safety researchers and includes data for 1,221,156 children delivered to 933,917 mothers. Current studies include evaluations of the prevalence and patterns of use of specific medications and a validation study of data elements in the administrative and birth certificate data files. MEPREP can support multiple studies by providing information on a large, ethnically and geographically diverse population. This partnership combines clinical and research expertise and data resources to enable the evaluation of outcomes associated with medication use during pregnancy.

Challenges and Errors in Genetic Testing: The Fifth Case Series.

PURPOSE: In this ongoing case series, 33 genetic testing cases are documented in which tests were recommended, ordered, interpreted, or used incorrectly and/or in which clinicians faced challenges related to history/reports provided by patients or laboratories. METHODS: An invitation to submit cases of challenges or errors in genetic testing was issued to the general National Society of Genetic Counselors Listserv, the National Society of Genetic Counselors Cancer Special Interest Group members, as part of a case series with Precision Oncology News, and via social media (i.e., Facebook, Twitter, LinkedIn). Deidentified clinical documentation was requested and reviewed when available. Thirty-three cases were submitted, reviewed, and accepted. A thematic analysis was performed. Submitters were asked to approve cases before submission. RESULTS: All cases took place in the United States, involved hereditary cancer testing and/or findings in cancer predisposition genes, and involved medical-grade genetic testing, direct-to-consumer testing, or research genetic testing. In 9 cases, test results were misinterpreted, leading to incorrect screening or risk-reducing procedures being performed/recommended. In 5 cases, incorrect or unnecessary testing was ordered/recommended. In 3 cases, incorrect clinical diagnoses were made, or opportunities for diagnoses were delayed. In 3 cases, errors or challenges arose related to medical intervention after testing or reported genetic diagnosis. In 2 cases, physicians provided incorrect information related to the inheritance pattern of a syndrome. In 2 cases, there were challenges related to the interpretation of genetic variants. In 2 cases, challenges arose after direct-to-consumer testing. One case involved test results that should never have been reported based on sample quality. In 1 case, a patient presented a falsified test result. In 5 cases, multiple errors were made. DISCUSSION: As genetic testing continues to become more complicated and common, it is critical that patients and nongenetics providers have access to accurate and timely genetic counseling information. Even as multiple medical bodies highlight the value of genetic counselors (GCs), tension exists in the genomics community as GCs work toward licensure and Medicare provider status. It is critical that health care communities leverage, rather than restrict, the expertise and experience of GCs so that patients can benefit from, and not be harmed by, genetic testing. In order to responsibly democratize genomics, it will be important for genetics and nongenetic health care providers to collaborate and use alternative service delivery models and technology solutions at point of care. To deliver on the promise of precision medicine, accurate resources and tools must be utilized.

Antidepressant medication use and risk of persistent pulmonary hypertension of the newborn.

PURPOSE: To determine the prevalence of persistent pulmonary hypertension of the newborn (PPHN) among infants whose mothers were exposed to antidepressants in the third trimester of pregnancy compared to the prevalence among infants whose mothers were not exposed to antidepressants in the third trimester. METHODS: A retrospective study was conducted using the automated databases of four health plans participating in the HMO Research Network Center for Education and Research on Therapeutics. Women who delivered an infant in a hospital from 1 January 1996 through 31 December 2000 were identified. The administrative databases were used to identify full-term infants whose mothers received an antidepressant during the third trimester of pregnancy and unexposed infants whose mothers did not receive an antidepressant during the third trimester. Hospitalization data were used to identify diagnoses or procedure codes potentially indicative of PPHN. RESULTS: Among 1104 infants exposed to antidepressants in the third trimester and a matched sample of 1104 unexposed infants, five infants were classified by the expert reviewers as having PPHN. Among those infants whose mothers were exposed to selective serotonin reuptake inhibitors (SSRIs) in the third trimester, the prevalence of PPHN was 2.14 per 1000 (95% confidence interval (CI) 0.26, 7.74), while the prevalence among infants whose mothers were not exposed was 2.72 per 1000 (95%CI 0.56, 7.93). CONCLUSIONS: We did not find an association between SSRI use in late pregnancy and PPHN. Limitations of the present study, including the small number of confirmed cases, suggest further research in this area may be warranted.

A randomized trial of direct-to-patient communication to enhance adherence to beta-blocker therapy following myocardial infarction.

BACKGROUND: Although beta-blockers are routinely prescribed at hospital discharge after myocardial infarction (MI), patients' adherence has been shown to decline substantially over time. We sought to test the hypothesis that a simple, direct-to-patient intervention can improve adherence to beta-blocker therapy following MI. METHODS: We conducted a cluster randomized controlled trial in 4 geographically dispersed health maintenance organizations testing the hypothesis that a simple direct-to-patient intervention could improve adherence. The study was carried out from June 2004 to March 2005. The primary analyses were based on 836 post-MI patients who were dispensed a beta-blocker prescription after discharge. The intervention consisted of 2 mailings 2 months apart describing the importance of beta-blocker use. The main outcomes were proportion of days covered with beta-blocker therapy and percentage of patients with at least 80% of days covered in the 9 months after the first mailing. Analyses were adjusted for age, sex, total medications dispensed, days between MI and intervention, and intervention site. RESULTS: Over the entire follow-up period, patients in the treatment arm had a mean absolute increase of 4.3% of days covered per month compared with patients in the control arm (a 5.7% relative change from baseline), representing 1.3 extra days (P = .04). Treatment patients were 17% more likely (relative risk, 1.17; 95% confidence interval, 1.02-1.29) to have 80% of days covered. For every 16 patients receiving the intervention, 1 additional patient would become adherent (80% or more days covered per month). CONCLUSION: A low-cost, easily replicable effort to increase adherence can have a demonstrable impact on beta-blocker adherence following MI. Trial Registration clinicaltrials.gov Identifier: NCT00211172.

Use of antidepressant medications during pregnancy: a multisite study.

OBJECTIVE: This study was undertaken to provide information on the prevalence of use of antidepressant drugs among pregnant women in the United States. STUDY DESIGN: A retrospective study was conducted using the automated databases of 7 health plans. Women who delivered an infant in a hospital were identified. Antidepressant drug use was evaluated assuming a gestational duration of 270 days. RESULTS: Among the 118,935 deliveries occurring from 2001-2005, 6.6% of women were dispensed an antidepressant during pregnancy. Antidepressant drug use increased from 2.0% in 1996 to 7.6% of deliveries in 2004 and 2005. Selective serotonin reuptake inhibitor use increased from 1.5% in 1996 to 6.4% in 2004 and 6.2% in 2005. CONCLUSION: Our finding that nearly 8% of pregnant women were prescribed antidepressants drugs during the years 2004 and 2005 highlights the importance of understanding the effects of these medications on the developing fetus and on the pregnant woman.

Outpatient use of cardiovascular drugs during pregnancy.

PURPOSE: To provide information on the prevalence of use of cardiovascular drugs, some of which may have fetotoxic or teratogenic effects, in the outpatient setting among pregnant women in the United States. METHODS: A retrospective study was conducted using the automated databases of seven health plans participating in the HMO Research Network Center for Education and Research on Therapeutics (CERT). Women who delivered an infant from 1 January 2001 to 31 December 2005 were identified. Cardiovascular drug use was evaluated assuming a gestational duration of 270 days. RESULTS: During the period 2001 through 2005, 118,935 deliveries were identified that met the criteria for study; 3.1% of women (N = 3672) were dispensed an antihypertensive medication and 0.12% of women (N = 146) were dispensed an antihyperlipidemic medication at any time during pregnancy. The most common antihypertensive drugs dispensed during pregnancy were nifedipine (1219 deliveries; 1.0%), methyldopa (961 deliveries; 0.8%), atenolol (593 deliveries; 0.5%), and labetalol (576 deliveries; 0.5%). Overall, 134 women (0.11%) received an angiotensin converting enzyme (ACE) inhibitor and 7 women (0.006%) received an angiotensin II receptor blocker (ARB) during pregnancy. Statins were the most commonly dispensed antihyperlipidemic drugs (71 deliveries; 0.06%). CONCLUSIONS: The prevalence of use of cardiovascular drugs that are suspected to be fetotoxic or teratogenic (ACE inhibitors, ARBs, and statins) was low in this cohort of pregnant women. Differing patterns of use across health plans suggests that further research is needed to evaluate the potential differential effects of cardiovascular drugs to assist prescribers and patients in making informed treatment decisions.

Diabetes and lipid screening among patients in primary care: a cohort study.

BACKGROUND: Obesity is associated with increased cardiovascular diseases and diabetes mellitus. Guidelines call for intensified glucose and lipid screening among overweight and obese patients. Data on compliance with these guidelines are scarce. The purpose of this study was to assess rates of diabetes and lipid screening in primary care according to demographic variables and weight status. METHODS: Over a 3-year follow-up period, we assessed screening rates for blood glucose, triglycerides, and HDL- and LDL-cholesterol among 5025 patients in primary care. From proportional hazards models we estimated screening rates among low, moderate, high, and very-high risk patients and compared them with recommendations of the American Diabetes Association (ADA), National Cholesterol Education Program (ATP III) and U.S. Preventive Services Task Force (USPSTF). RESULTS: Mean (SD) age was 47.4 (15.6); 69% were female, 21% were non-white, and 30% of males and 25% of females were obese (BMI > or = 30 kg/m2). For both diabetes and lipid screening, the adjusted hazard was 260-330% higher among > or = 65 than < 35 year-olds, 50-90% higher in persons with BMI > or = 35 than < 25 kg/m2, 10-30% lower for females than males, and not lower among racial/ethnic minorities. Screening rates were at least 80% among very-high risk persons, which we defined as 55-64 years old, BMI > or = 35 kg/m2, non-white, with baseline hypertension. In contrast, high-risk persons who were younger (35-44 years old) and less obese (BMI 30-<35 kg/m2) were screened less often (43% for LDL-cholesterol among females to 83% for diabetes among males) even though ADA, ATP III and USPSTF recommend diabetes and lipid screening among them. CONCLUSION: Patients with higher BMI or age were more likely to be screened for cardiometabolic risk factors. Women were screened at lower rates than men. Even in a highly structured medical group practice, some obese patients were under-screened for diabetes and dyslipidemia.

Comparative effectiveness of beta-adrenergic antagonists (atenolol, metoprolol tartrate, carvedilol) on the risk of rehospitalization in adults with heart failure.

Placebo-controlled randomized trials have demonstrated the efficacy of selected beta blockers on outcomes in chronic heart failure (HF), but the relative effectiveness of different beta blockers in usual clinical care is poorly understood. We compared 12-month risk of rehospitalization for HF associated with receipt of different beta blockers in 7,883 adults hospitalized for HF within 2 large health plans between January 1, 2001 and December 31, 2002. Beta-blocker use was ascertained from electronic pharmacy databases and readmissions within 12 months were identified from hospital discharge databases. Extended Cox regression was used to examine the association between receipt of different beta blockers and risk of readmission for HF after adjustment for potential confounders. During follow-up, there were 3,234 person-years of exposure to beta blockers (39.3% atenolol, 42.0% metoprolol tartrate, 12.3% carvedilol, and 6.4% other). Crude 12-month rates of readmissions for HF were high overall (42.6 per 100 person-years). After adjustment for potential confounders, cumulative exposure to each beta blocker, and propensity to receive carvedilol compared with atenolol, adjusted risks of readmission were not significantly different for metoprolol tartrate (adjusted hazard ratio 0.95, 95% confidence interval 0.85 to 1.05) or for carvedilol (adjusted hazard ratio 0.92, 95% confidence interval 0.74 to 1.14). In conclusion, in a contemporary cohort of high-risk patients hospitalized with HF, we found that adjusted risks of rehospitalization for HF within 12 months were not significantly different in patients receiving atenolol, shorter-acting metoprolol tartrate, or carvedilol.

The United States pediatric vaccine stockpile program.

The initial goal of the national vaccine stockpile program was to establish a 6-month supply of all recommended childhood vaccines, to meet national demands if a manufacturing process was interrupted. When the first vaccine stockpiles were created in 1983, the childhood immunization schedule was much less complicated than it is today, and the first stockpiles included only measles-mumps-rubella, poliovirus, and pertussis vaccines, as well as diphtheria and tetanus toxoids. However, today's vaccine needs are much greater, and current stockpiles do not include all recommended childhood vaccines, partially because inclusion of vaccines that are universally recommended, fully implemented, and produced by a single manufacturer has been made a priority. Future planning must also consider substantially higher vaccine costs, the development of new combination vaccines, a wide range of production times, and changes in immunization recommendations. Expansion and strengthening of the national vaccine stockpile program are critical to protect against future disruptions in vaccine supply.

Laminin-311 (Laminin-6) fiber assembly by type I-like alveolar cells.

Two epithelial cell types cover the alveolar surface of the lung. Type II alveolar epithelial cells produce surfactant and, during development or following wounding, give rise to type I cells that are involved in gas exchange and alveolar fluid homeostasis. In culture, freshly isolated alveolar type II cells assume a more squamous (type I-like) appearance within 4 days after plating. They assemble numerous focal adhesions that associate with the actin cytoskeleton at the cell margins. These alveolar epithelial cells lose expression of type II cell markers including SP-C and after 4 days in culture express the type I cell marker T1alpha. Those cells that express T1alpha also deposit fibers of laminin-311 in their matrix. The latter appears to be related to their development of a type I phenotype because freshly isolated, primary type I cells also assemble laminin-311-rich fibers in vitro. A beta1 integrin antibody antagonist inhibits the assembly of laminin-311 matrix fibers. Moreover, the formation of laminin fibers is dependent on the activity of the small GTPases and is perturbed by ML-7, a myosin light chain kinase inhibitor. In summary, our data indicate that assembly of laminin-311 fibers by lung epithelial cells is integrin and actin cytoskeleton dependent, and that these fibers are characteristic of type I alveolar cells.

Multistate outbreak of Listeria monocytogenes infection linked to delicatessen turkey meat.

BACKGROUND: Despite a decreasing incidence of listeriosis in the United States, molecular subtyping has increased the number of recognized outbreaks. In September 2000, the New York City Department of Health identified a cluster of infections caused by Listeria monocytogenes isolates with identical molecular subtypes by pulsed-field gel electrophoresis (PFGE) and ribotyping. METHODS: To determine the magnitude of the outbreak and identify risk factors for infection, we notified state health departments and conducted a case-control study. A case was defined as a patient or mother-infant pair infected with Listeria monocytogenes whose isolate yielded the outbreak PFGE pattern. Controls were patients infected with Listeria monocytogenes whose isolate yielded a different PFGE pattern. Patients were asked about food and drink consumed during the 30 days before the onset of illness. RESULTS: Between May and December 2000, there were 30 clinical isolates of Listeria monocytogenes with identical PFGE patterns identified in 11 US states. Cases of infection caused by these isolates were associated with 4 deaths and 3 miscarriages. A case-control study implicated sliced processed turkey from a delicatessen (Mantel-Haenszel odds ratio, 8.0; 95% confidence interval, 1.2-43.3). A traceback investigation identified a single processing plant as the likely source of the outbreak, and the company voluntarily recalled 16 million pounds of processed meat. The same plant had been identified in a Listeria contamination event that had occurred more than a decade previously. CONCLUSIONS: Prevention of persistent L. monocytogenes contamination in food processing plants presents a critical challenge to food safety professionals.

Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli ß-Glucuronidase.

ß-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While ß-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins.

Complexities in understanding attentional functioning among children with fetal alcohol spectrum disorder.

Parental reports of attention problems and clinical symptomatology of ADHD among children with fetal alcohol syndrome disorder (FASD) were assessed in relation to performance on standardized subtests of attentional control/shifting and selective attention from the Test of Everyday Attention for Children (TEA-Ch; Manly etal., 1998). The participants included 14 children with FASD with a mean chronological age (CA) of 11.7 years and a mean mental age (MA) of 9.7 years, and 14 typically developing (TD) children with no reported history of prenatal exposure to alcohol or attention problems with a mean CA of 8.4 years and a mean MA of 9.6 years. The children with FASD were rated by their caregivers as having clinically significant attention difficulties for their developmental age. The reported symptomatology for the majority of the children with FASD were consistent with a diagnosis of ADHD, combined type, and only one child had a score within the average range. These reports are consistent with the finding that the children with FASD demonstrated difficulties with attentional control/shifting, but inconsistent with the finding that they outperformed the TD children on a test assessing selective attention. These findings are considered within the context of the complexity in understanding attentional functioning among children with FASD and discrepancies across sources of information and components of attention.

Why is school closed today? Unplanned K-12 school closures in the United States, 2011-2013.

INTRODUCTION: We describe characteristics of unplanned school closures (USCs) in the United States over two consecutive academic years during a non-pandemic period to provide context for implementation of school closures during a pandemic. METHODS: From August 1, 2011 through June 30, 2013, daily systematic internet searches were conducted for publicly announced USCs lasting ≥ 1 day. The reason for closure and the closure dates were recorded. Information on school characteristics was obtained from the National Center for Education Statistics. RESULTS: During the two-year study period, 20,723 USCs were identified affecting 27,066,426 students. Common causes of closure included weather (79%), natural disasters (14%), and problems with school buildings or utilities (4%). Only 771 (4%) USCs lasted ≥ 4 school days. Illness was the cause of 212 (1%) USCs; of these, 126 (59%) were related to respiratory illnesses and showed seasonal variation with peaks in February 2012 and January 2013. CONCLUSIONS: USCs are common events resulting in missed school days for millions of students. Illness causes few USCs compared with weather and natural disasters. Few communities have experience with prolonged closures for illness.

Weekly doxorubicin increases coronary arteriolar wall and adventitial thickness.

BACKGROUND: Doxorubicin (DOX) is associated with premature cardiovascular events including myocardial infarction. This study was performed to determine if the weekly administration of DOX influenced coronary arteriolar medial and/or adventitial wall thickening. METHODS: Thirty-two male Sprague-Dawley rats aged 25.1± 2.4 weeks were randomly divided into three groups and received weekly intraperitoneal injections of normal saline (saline, n = 7), or low (1.5 mg/kg to 1.75 mg/kg, n = 14) or high (2.5 mg/kg, n = 11) doses of DOX. The animals were treated for 2-12 weeks, and euthanized at pre-specified intervals (2, 4, 7, or 10+ weeks) to obtain histopathologic assessments of coronary arteriolar lumen diameter, medial wall thickness, adventitial wall thickness, and total wall thickness (medial thickness + adventitial thickness). RESULTS: Lumen diameter was similar across all groups (saline: 315±34 µm, low DOX: 286±24 µm, high DOX: 242±27 µm; p = 0.22). In comparison to animals receiving weekly saline, animals receiving weekly injections of 2.5 mg/kg of DOX experienced an increase in medial (23±2 µm vs. 13±3 µm; p = 0.005), and total wall thickness (51±4 µm vs. 36±5 µm; p = 0.022), respectively. These increases, as well as adventitial thickening became more prominent after normalizing for lumen diameter (p<0.05 to p<0.001) and after adjusting for age, weight, and total cumulative DOX dose (p = 0.02 to p = 0.01). Animals receiving low dose DOX trended toward increases in adventitial and total wall thickness after normalization to lumen diameter and accounting for age, weight, and total cumulative DOX dose (p = 0.06 and 0.09, respectively). CONCLUSION: In conclusion, these data demonstrate that weekly treatment of rats with higher doses of DOX increases coronary arteriolar medial, adventitial, and total wall thickness. Future studies are warranted to determine if DOX related coronary arteriolar effects are reversible or preventable, exacerbate the known cardiomyopathic effects of DOX, influence altered resting or stress-induced myocardial perfusion, or contribute to the occurrence of myocardial infarction.

HIV-specific cytolytic CD4 T cell responses during acute HIV infection predict disease outcome.

Early immunological events during acute HIV infection are thought to fundamentally influence long-term disease outcome. Whereas the contribution of HIV-specific CD8 T cell responses to early viral control is well established, the role of HIV-specific CD4 T cell responses in the control of viral replication after acute infection is unknown. A growing body of evidence suggests that CD4 T cells-besides their helper function-have the capacity to directly recognize and kill virally infected cells. In a longitudinal study of a cohort of individuals acutely infected with HIV, we observed that subjects able to spontaneously control HIV replication in the absence of antiretroviral therapy showed a significant expansion of HIV-specific CD4 T cell responses-but not CD8 T cell responses-compared to subjects who progressed to a high viral set point (P = 0.038). Markedly, this expansion occurred before differences in viral load or CD4 T cell count and was characterized by robust cytolytic activity and expression of a distinct profile of perforin and granzymes at the earliest time point. Kaplan-Meier analysis revealed that the emergence of granzyme A(+) HIV-specific CD4 T cell responses at baseline was highly predictive of slower disease progression and clinical outcome (average days to CD4 T cell count <350/µl was 575 versus 306, P = 0.001). These data demonstrate that HIV-specific CD4 T cell responses can be used during the earliest phase of HIV infection as an immunological predictor of subsequent viral set point and disease outcome. Moreover, these data suggest that expansion of granzyme A(+) HIV-specific cytolytic CD4 T cell responses early during acute HIV infection contributes substantially to the control of viral replication.

A High Throughput Assay for Discovery of Bacterial ß-Glucuronidase Inhibitors.

CPT-11 is a widely-used anti-cancer drug that is converted in vivo to its active metabolite, SN-38. In the liver, enzymes detoxify SN-38 by coupling it to a glucuronidate moiety and this inactive compound (SN-38G) is excreted into the gastrointestinal tract. In the intestine, commensal bacteria convert the SN-38G back to the active and toxic SN-38 using bacterial ß-glucuronidase enzyme (GUS). This intestinal SN-38 causes debilitating diarrhea that prevents dose-intensification and efficacy in a significant fraction of patients undergoing CPT-11 treatment for cancer. This CPT-11 metabolic pathway suggests that small molecule inhibitors of GUS may have utility as novel therapeutics for prevention of dose-limiting diarrhea resulting from CPT-11 therapy. To identify chemical inhibitors of GUS activity, we employed and validated a high throughput, fluorescence-based biochemical assay and used this assay to screen a compound library. Novel inhibitors of GUS were identified with IC(50) values ranging from 50 nM to 4.8 µM. These compounds may be useful as chemical probes for use in proof-of-concept experiments designed to determine the efficacy of GUS inhibitors in altering the intestinal metabolism of drugs. Our results demonstrate that this high throughput assay can be used to identify small molecule inhibitors of GUS.