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Bupropion was tested for efficacy in increasing weeks of abstinence in methamphetamine-dependent patients, compared to placebo. This was a double-blind placebo-controlled study, with 12 weeks of treatment and a 30-day follow-up. Five outpatient substance abuse treatment clinics located west of the Mississippi participated in the study. One hundred and fifty-one treatment-seekers with DSM-IV diagnosis of methamphetamine dependence were consented and enrolled. Seventy-two participants were randomized to placebo and 79 to sustained-release bupropion 150 mg twice daily. Patients were asked to come to the clinic three times per week for assessments, urine drug screens, and 90-min group psychotherapy. The primary outcome was the change in proportion of participants having a methamphetamine-free week. Secondary outcomes included: urine for quantitative methamphetamine, self-report of methamphetamine use, subgroup analyses of balancing factors and comorbid conditions, addiction severity, craving, risk behaviors for HIV, and use of other substances. The generalized estimating equation regression analysis showed that, overall, the difference between bupropion and placebo groups in the probability of a non-use week over the 12-week treatment period was not statistically significant ( p =0.09). Mixed model regression was used to allow adjustment for baseline factors in addition to those measured (site, gender, level of baseline use, and level of symptoms of depression). This subgroup analysis showed that bupropion had a significant effect compared to placebo, among male patients who had a lower level of methamphetamine use at baseline ( p <0.0001). Comorbid depression and attention-deficit/hyperactivity disorder did not change the outcome. These data suggest that bupropion, in combination with behavioral group therapy, was effective for increasing the number of weeks of abstinence in participants with low-to-moderate methamphetamine dependence, mainly male patients, regardless of their comorbid condition.

Methamphetamine dependence is an increasing public health problem in the United States. No efficacious medication for methamphetamine dependence has been developed. As ondansetron, a 5-HT3 receptor antagonist and modulator of cortico-mesolimbic dopamine function, has been shown to reduce some of the rewarding effects of d-amphetamine in animal and human laboratory studies, we decided to test whether it would be superior to placebo at reducing methamphetamine use. In a preliminary, multi-site, randomized, double-blind, 8-wk controlled trial, 150 methamphetamine-dependent men and women received ondansetron (0.25 mg, 1 mg, or 4 mg b.i.d.) or placebo. Participants were assessed on several measures of methamphetamine use including urine methamphetamine level up to three times per week. As a psychosocial adjunct to the medication condition, cognitive behavioural therapy also was administered three times per week. Ondansetron was well tolerated and was less likely than placebo to be associated with serious adverse events. Nevertheless, none of the ondansetron doses was superior to placebo at decreasing any of the measures of methamphetamine use, withdrawal, craving, or clinical severity of methamphetamine dependence. Our preliminary results do not support the utility of ondansetron, at the doses tested, as a treatment for methamphetamine dependence. These findings should be viewed in light of the possibility that a less intensive cognitive behavioural therapy regimen might have yielded more positive results in this initial phase II trial exploring for the efficacy of ondansetron.

Background. It is unknown whether sex and race influence clinical outcomes following primary human immunodeficiency virus type 1 (HIV-1) infection. Methods. Data were evaluated from an observational, multicenter, primarily North American cohort of HIV-1 seroconverters. Results. Of 2277 seroconverters, 5.4% were women. At enrollment, women averaged .40 log₁₀ fewer copies/mL of HIV-1 RNA (P < .001) and 66 more CD4⁺ T cells/μL (P = .006) than men, controlling for age and race. Antiretroviral therapy (ART) was less likely to be initiated at any time point by nonwhite women and men compared to white men (P < .005), and by individuals from the southern United States compared to others (P = .047). Sex and race did not affect responses to ART after 6 months (P > .73). Women were 2.17-fold more likely than men to experience >1 HIV/AIDS-related event (P < .001). Nonwhite women were most likely to experience an HIV/AIDS-related event compared to all others (P = .035), after adjusting for intravenous drug use and ART. Eight years after diagnosis, >1 HIV/AIDS-related event had occurred in 78% of nonwhites and 37% of whites from the southern United States, and 24% of whites and 17% of nonwhites from other regions (P < .001). Conclusions. Despite more favorable clinical parameters initially, female HIV-1-seroconverters had worse outcomes than did male seroconverters. Elevated morbidity was associated with being nonwhite and residing in the southern United States.

HIV-1 infection is complicated by high rates of opportunistic infections against which specific antibodies contribute to immune defense. Antibody function depends on somatic hypermutation (SHM) of variable regions of immunoglobulin heavy chain genes (VH-D-J). We characterized the frequency of SHM in expressed IgG mRNA immunoglobulin transcripts from control and HIV-1-infected patients. We compared utilization of genes in the most prominent VH family (VH3) and mutation frequencies and patterns of cDNA from VH3-IgG genes from 10 seronegative control subjects and 21 patients with HIV-1 infection (6 without and 15 patients with detectable plasma viremia). Unique IgG VH3 family cDNA sequences (n = 1,565) were PCR amplified, cloned, and sequenced from blood. Sequences were analyzed using online (Vbase) and in-house immunoglobulin alignment resources. Mutation frequencies in the antigen-binding hypervariable complementarity determining regions (CDR1/2) of IgG class-switched B cells were lower among viremic HIV-1-infected patients vs. controls for nucleotides (CDR1/2: 10±5% vs. 13.5±6%, p = 0.03) and amino acids (CDR: 20%±10 vs. 25%±12, p = 0.02) and in structural framework regions. Mutation patterns were similar among groups. The most common VH3 gene, VH3-23, was utilized less frequently among viremic HIV-1-infected patients (p = 0.03), and overall, mutation frequencies were decreased in nearly all VH3 genes compared with controls. B cells from HIV-1-infected patients show decreased mutation frequencies, especially in antigen-binding VH3 CDR genes, and selective defects in gene utilization. Similar mutation patterns suggest defects in the quantity, but not quality, of mutator activity. Lower levels of SHM in IgG class-switched B cells from HIV-1-infected patients may contribute to the increased risk of opportunistic infections and impaired humoral responses to preventative vaccines.

The search for effective Hepatitis C antiviral therapies has recently focused on host sterol metabolism and protein prenylation pathways that indirectly affect viral replication. However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. Here, we present a combination chemical genetic study to explore how the sterol and protein prenylation pathways work together to affect hepatitis C viral replication in a replicon assay. In addition to finding novel targets affecting viral replication, our data suggest that the viral replication is strongly affected by sterol pathway regulation. There is a marked transition from antagonistic to synergistic antiviral effects as the combination targets shift downstream along the sterol pathway. We also show how pathway regulation frustrates potential hepatitis C therapies based on the sterol pathway, and reveal novel synergies that selectively inhibit hepatitis C replication over host toxicity. In particular, combinations targeting the downstream sterol pathway enzymes produced robust and selective synergistic inhibition of hepatitis C replication. Our findings show how combination chemical genetics can reveal critical pathway connections relevant to viral replication, and can identify potential treatments with an increased therapeutic window. Synopsis Chemical inhibition of enzymes in either the cholesterol or the fatty acid biosynthetic pathways has been shown to impact viral replication, both positively and negatively (Su et al , 2002 ; Ye et al , 2003 ; Kapadia and Chisari, 2005 ; Sagan et al , 2006 ; Amemiya et al , 2008 ). FBL2 has been identified as a 50 kDa geranylgeranylated host protein that is necessary for localization of the hepatitis C virus (HCV) replication complex to the membranous web through its close association with the HCV protein NS5A and is critical for HCV replication (Wang et al , 2005 ). Inhibition of the protein geranylgeranyl transferase I (PGGT), an enzyme that transfers geranylgeranyl pyrophosphate (GGPP) to cellular proteins such as FBL2 for the purpose of membrane anchoring, negatively impacts HCV replication (Ye et al , 2003 ). Conversely, chemical agents that increase intracellular GGPP concentrations promote viral replication (Kapadia and Chisari, 2005 ). Statin compounds that inhibit 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGCR), the rate‐limiting enzyme in the sterol pathway (Goldstein and Brown, 1990 ), have been suggested to inhibit HCV replication through ultimately reducing the cellular pool of GGPP (Ye et al , 2003 ; Kapadia and Chisari, 2005 ; Ikeda et al , 2006 ). However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. The use of statins for the treatment of HCV is likely to be complicated by the reported compensatory increase in HMGCR expression in vitro and in vivo (Stone et al , 1989 ; Cohen et al , 1993 ) in response to treatment. Enzymes in the sterol pathway are regulated on a transcriptional level by sterol regulatory element‐binding proteins (SREBPs), specifically SREBP‐2 (Hua et al , 1993 ; Brown and Goldstein, 1997 ). When cholesterol stores in cells are depleted, SREBP‐2 activates transcription of genes in the sterol pathway such as HMGCR, HMG‐CoA synthase, farnesyl pyrophosphate (FPP) synthase, squalene synthase (SQLS) and the LDL receptor (Smith et al , 1988 , 1990 ; Sakai et al , 1996 ; Brown and Goldstein, 1999 ; Horton et al , 2002 ). The requirement of additional downstream sterol pathway metabolites for HCV replication has not been completely elucidated. To further understand the impact of the sterol pathway and its regulation on HCV replication, we conducted a high‐throughput combination chemical genetic screen using 16 chemical probes that are known to modulate the activity of target enzymes relating to the sterol biosynthesis pathway (Figure 1 ). Using this approach, we identified several novel antiviral targets including SREBP‐2 as well as targets downstream of HMGCR in the sterol pathway such as oxidosqualene cyclase (OSC) and lanosterol demethylase. Many of our chemical probes, specifically SR‐12813, farnesol and squalestatin, strongly promoted replicon replication. The actions of both farnesol and squalestatin ultimately result in an increase in the cellular pool of GGPP, which is known to increase HCV replication (Ye et al , 2003 ; Kapadia and Chisari, 2005 ; Wang et al , 2005 ). Chemical combinations targeting enzymes upstream of squalene epoxidase (SQLE) at the top of the sterol pathway (Figure 4A ) elicited Bateson‐type epistatic responses (Boone et al , 2007 ), where the upstream agent's response predominates over the effects of inhibiting all downstream targets. This was especially notable for combinations including simvastatin and either U18666A or squalestatin, and for squalestatin in combination with Ro48‐8071. Treatment with squalestatin prevents the SQLS substrate, farnesyl pyrophosphate (FPP) from being further metabolized by the sterol pathway. As FPP concentrations increase, the metabolite can be shunted away from the sterol pathway toward farnesylation and GGPP synthetic pathways, resulting in an increase in host protein geranylgeranylation, including FBL2, and consequently replicon replication. This increase in replicon replication explains the source of the observed epistasis over Ro48‐8071 treatment. Combinations between probes targeting enzymes downstream of and including OSC produced robust synergies with each other or with a PGGT inhibitor. Figure 4B highlights examples of antiviral synergy resulting from treatment of cells with an OSC inhibitor in combination with an inhibitor of either an enzyme upstream or downstream of OSC. A combination of terconazole and U18666A is synergistic without similar combination effects in the host proliferation screen. Likewise, clomiphene was also synergistic when added to replicon cells in combination with U18666A. One of the greatest synergies observed downstream in the sterol pathway is a combination of amorolfine and AY 9944, suggesting that there is value in developing combinations of drugs that target enzymes in the sterol pathway, which are downstream of HMGCR. Interactions with the protein prenylation pathway also showed strong mechanistic patterns (Figure 4C ). GGTI‐286 is a peptidomimetic compound resembling the CAAX domain of a protein to be geranylgeranylated and is a competitive inhibitor of protein geranylgeranylation. Simvastatin impedes the antiviral effect of GGTI‐286 at low concentrations but that antagonism is balanced by comparable synergy at higher concentrations. At the low simvastatin concentrations, a compensatory increase in HMGCR expression leads to increased cellular levels of GGPP, which are likely to result in an increase in PGGT enzymatic turnover and decreased GGTI‐286 efficacy. The antiviral synergy observed at the higher inhibitor concentrations is likely nonspecific as synergy was also observed in a host viability assay. Further downstream, however, a competitive interaction was observed between GGTI‐286 and squalestatin, where the opposing effect of one compound obscures the other compound's effect. This competitive relationship between GGTI and SQLE explains the epistatic response observed between those two agents. For inhibitors of targets downstream of OSC, such as amorolfine, there are strong antiviral synergies with GGTI‐286. Notably, combinations with OSC inhibitors and GGTI‐286 were selective, in that comparable synergy was not found in a host viability assay. This selectivity suggests that jointly targeting OSC and PGGT is a promising avenue for future HCV therapy development. This study provides a comprehensive and unique perspective into the impact of sterol pathway regulation on HCV replication and provides compelling insight into the use of chemical combinations to maximize antiviral effects while minimizing proviral consequences. Our results suggest that HCV therapeutics developed against sterol pathway targets must consider the impact on underlying sterol pathway regulation. We found combinations of inhibitors of the lower part of the sterol pathway that are effective and synergistic with each other when tested in combination. Furthermore, the combination effects observed with simvastatin suggest that, though statins inhibit HMGCR activity, the resulting regulatory consequences of such inhibition ultimately lead to undesirable epistatic effects. Inhibitors that prevent SREBP‐2 activation, inhibit PGGT or encourage the production of polar sterols have great potential as HCV therapeutics if associated toxicities can be reduced. SREBP‐2, oxidosqualene cyclase (OSC) or lanosterol demethylase were identified as novel sterol pathway‐associated targets that, when probed with chemical agents, can inhibit hepatitis C virus (HCV) replication. Using a combination chemical genetics approach, combinations of chemicals targeting sterol pathway enzymes downstream of and including OSC or protein geranylgeranyl transferase I (PGGT) produce robust and selective synergistic inhibition of HCV replication. Inhibition of enzymes upstream of OSC elicit proviral responses that are dominant to the effects of inhibiting all downstream targets. Inhibition of the sterol pathway without inhibition of regulatory feedback mechanisms ultimately results in an increase in HCV replication because of a compensatory upregulation of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGCR) expression. Increases in HMGCR expression without inhibition of HMGCR enzymatic activity ultimately stimulate HCV replication through increasing the cellular pool of geranylgeranyl pyrophosphate (GGPP). Chemical inhibitors that ultimately prevent SREBP‐2 activation, inhibit PGGT or encourage the production of polar sterols have great potential as HCV therapeutics if associated toxicities can be reduced.

Background/objective HIV-1 infection is complicated by high rates of opportunistic infections against which specific antibodies contribute to immune defense. Antibody function depends on somatic hypermutation (SHM) of variable regions of immunoglobulin heavy chain genes (VH-D-J). We characterized the frequency of SHM in expressed IgG mRNA immunoglobulin transcripts from control and HIV-1-infected patients. Design We compared utilization of genes in the most prominent VH family (VH3) and mutation frequencies and patterns of cDNA from VH3-IgG genes from 10 seronegative control subjects and 21 patients with HIV-1 infection (6 without and 15 patients with detectable plasma viremia). Methods Unique IgG VH3 family cDNA sequences (n = 1,565) were PCR amplified, cloned, and sequenced from blood. Sequences were analyzed using online (Vbase) and in-house immunoglobulin alignment resources. Results Mutation frequencies in the antigen-binding hypervariable complementarity determining regions (CDR1/2) of IgG class-switched B cells were lower among viremic HIV-1-infected patients vs. controls for nucleotides (CDR1/2: 10 plus or minus 5% vs. 13.5 plus or minus 6%, p = 0.03) and amino acids (CDR: 20% plus or minus 10 vs. 25% plus or minus 12, p = 0.02) and in structural framework regions. Mutation patterns were similar among groups. The most common VH3 gene, VH3-23, was utilized less frequently among viremic HIV-1-infected patients (p = 0.03), and overall, mutation frequencies were decreased in nearly all VH3 genes compared with controls. Conclusions B cells from HIV-1-infected patients show decreased mutation frequencies, especially in antigen-binding VH3 CDR genes, and selective defects in gene utilization. Similar mutation patterns suggest defects in the quantity, but not quality, of mutator activity. Lower levels of SHM in IgG class-switched B cells from HIV-1-infected patients may contribute to the increased risk of opportunistic infections and impaired humoral responses to preventative vaccines.

Although prostate cancer is the leading cause of cancer mortality for African men, the vast majority of known disease associations have been detected in European study cohorts. Furthermore, most genome-wide association studies have used genotyping arrays that are hindered by SNP ascertainment bias. To overcome these disparities in genomic medicine, the Men of African Descent and Carcinoma of the Prostate (MADCaP) Network has developed a genotyping array that is optimized for African populations. The MADCaP Array contains more than 1.5 million markers and an imputation backbone that successfully tags over 94% of common genetic variants in African populations. This array also has a high density of markers in genomic regions associated with cancer susceptibility, including 8q24. We assessed the effectiveness of the MADCaP Array by genotyping 399 prostate cancer cases and 403 controls from seven urban study sites in sub-Saharan Africa. We find that samples from Ghana and Nigeria cluster together, while samples from Senegal and South Africa yield distinct ancestry clusters. Using the MADCaP array, we identified cancer-associated loci that have large allele frequency differences across African populations. Polygenic risk scores were also generated for each genome in the MADCaP pilot dataset, and we found that predicted risks of CaP are lower in Senegal and higher in Nigeria. Significance: We have developed an Africa-specific genotyping array which enables investigators to identify novel disease associations and to fine-map genetic loci that are associated with prostate and other cancers.