Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth.

Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.

Differential recognition of HIV-stimulated IL-1ß and IL-18 secretion through NLR and NAIP signalling in monocyte-derived macrophages.

Macrophages are important drivers of pathogenesis and progression to AIDS in HIV infection. The virus in the later phases of the infection is often predominantly macrophage-tropic and this tropism contributes to a chronic inflammatory and immune activation state that is observed in HIV patients. Pattern recognition receptors of the innate immune system are the key molecules that recognise HIV and mount the inflammatory responses in macrophages. The innate immune response against HIV-1 is potent and elicits caspase-1-dependent pro-inflammatory cytokine production of IL-1ß and IL-18. Although, NLRP3 has been reported as an inflammasome sensor dictating this response little is known about the pattern recognition receptors that trigger the "priming" signal for inflammasome activation, the NLRs involved or the HIV components that trigger the response. Using a combination of siRNA knockdowns in monocyte derived macrophages (MDMs) of different TLRs and NLRs as well as chemical inhibition, it was demonstrated that HIV Vpu could trigger inflammasome activation via TLR4/NLRP3 leading to IL-1ß/IL-18 secretion. The priming signal is triggered via TLR4, whereas the activation signal is triggered by direct effects on Kv1.3 channels, causing K+ efflux. In contrast, HIV gp41 could trigger IL-18 production via NAIP/NLRC4, independently of priming, as a one-step inflammasome activation. NAIP binds directly to the cytoplasmic tail of HIV envelope protein gp41 and represents the first non-bacterial ligand for the NAIP/NLRC4 inflammasome. These divergent pathways represent novel targets to resolve specific inflammatory pathologies associated with HIV-1 infection in macrophages.

CTLA-4 and PD-1 dual blockade induces SIV reactivation without control of rebound after antiretroviral therapy interruption.

The primary human immunodeficiency virus (HIV) reservoir is composed of resting memory CD4+ T cells, which often express the immune checkpoint receptors programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which limit T cell activation via synergistic mechanisms. Using simian immunodeficiency virus (SIV)-infected, long-term antiretroviral therapy (ART)-treated rhesus macaques, we demonstrate that PD-1, CTLA-4 and dual CTLA-4/PD-1 immune checkpoint blockade using monoclonal antibodies is well tolerated, with evidence of bioactivity in blood and lymph nodes. Dual blockade was remarkably more effective than PD-1 blockade alone in enhancing T cell cycling and differentiation, expanding effector-memory T cells and inducing robust viral reactivation in plasma and peripheral blood mononuclear cells. In lymph nodes, dual CTLA-4/PD-1 blockade, but not PD-1 alone, decreased the total and intact SIV-DNA in CD4+ T cells, and SIV-DNA and SIV-RNA in B cell follicles, a major site of viral persistence during ART. None of the tested interventions enhanced SIV-specific CD8+ T cell responses during ART or viral control after ART interruption. Thus, despite CTLA-4/PD-1 blockade inducing robust latency reversal and reducing total levels of integrated virus, the degree of reservoir clearance was still insufficient to achieve viral control. These results suggest that immune checkpoint blockade regimens targeting PD-1 and/or CTLA-4, if performed in people living with HIV with sustained aviremia, are unlikely to induce HIV remission in the absence of additional interventions.

Small molecule inhibition of RAS/MAPK signaling ameliorates developmental pathologies of Kabuki Syndrome.

Kabuki Syndrome (KS) is a rare disorder characterized by distinctive facial features, short stature, skeletal abnormalities, and neurodevelopmental deficits. Previously, we showed that loss of function of RAP1A, a RAF1 regulator, can activate the RAS/MAPK pathway and cause KS, an observation recapitulated in other genetic models of the disorder. These data suggested that suppression of this signaling cascade might be of therapeutic benefit for some features of KS. To pursue this possibility, we performed a focused small molecule screen of a series of RAS/MAPK pathway inhibitors, where we tested their ability to rescue disease-relevant phenotypes in a zebrafish model of the most common KS locus, kmt2d. Consistent with a pathway-driven screening paradigm, two of 27 compounds showed reproducible rescue of early developmental pathologies. Further analyses showed that one compound, desmethyl-Dabrafenib (dmDf), induced no overt pathologies in zebrafish embryos but could rescue MEK hyperactivation in vivo and, concomitantly, structural KS-relevant phenotypes in all KS zebrafish models (kmt2d, kmd6a and rap1). Mass spectrometry quantitation suggested that a 100 nM dose resulted in sub-nanomolar exposure of this inhibitor and was sufficient to rescue both mandibular and neurodevelopmental defects. Crucially, germline kmt2d mutants recapitulated the gastrulation movement defects, micrognathia and neurogenesis phenotypes of transient models; treatment with dmDf ameliorated all of them significantly. Taken together, our data reinforce a causal link between MEK hyperactivation and KS and suggest that chemical suppression of BRAF might be of potential clinical utility for some features of this disorder.

Single- and Multiple-Day Dosing Studies to Investigate High-Dose Pharmacokinetics of Epelsiban and Its Metabolite, GSK2395448, in Healthy Female Volunteers.

Open-label single- and double-blind repeat-dose studies in healthy female volunteers were conducted to investigate the pharmacokinetics (PK) and safety/tolerability of epelsiban total daily doses ranging from 600 to 900 mg. In 1 study (n = 12), epelsiban was dosed at 300 or 450 mg twice daily (every 12 hours) for a single day. In the repeat-dose double-blind study, epelsiban and placebo were administered to 31 subjects as 200 mg 3 times daily, 300 mg 3 times daily (TID), or 450 mg twice daily (BID) for 14 days. After both single and 14 daily repeat doses, the PK profiles for epelsiban and its metabolite, GSK2395448, remained linear at all administered doses. The exposures at a given total daily dose were also similar between BID and TID dosing regimens. Exposure (AUC0-τ ), based on dosing intervals, for both epelsiban and GSK2395448 was similar. However, compared with morning dosing, Cmax was lower after evening dosing, possibly because of a food effect. The highest accumulation of epelsiban and GSK2395448 exposures (AUC0-τ ) was approximately 34% for each after repeat dosing, consistent with the short half-life. At total daily doses of 600 and 900 mg, epelsiban was generally well tolerated, and there were no significant safety concerns identified.

spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States.

A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants.

Incidence and cost of hospitalizations associated with Staphylococcus aureus skin and soft tissue infections in the United States from 2001 through 2009.

BACKGROUND: The emergence of community-associated methicillin-resistant Staphylococcus aureus (SA) and its role in skin and soft tissue infections (SSTIs) accentuated the role of SA-SSTIs in hospitalizations. METHODS: We used the Nationwide Inpatient Sample and Census Bureau data to quantify population-based incidence and associated cost for SA-SSTI hospitalizations. RESULTS: SA-SSTI associated hospitalizations increased 123% from 160,811 to 358,212 between 2001 and 2009, and they represented an increasing share of SA- hospitalizations (39% to 51%). SA-SSTI incidence (per 100,000 people) doubled from 57 in 2001 to 117 in 2009 (p<0.01). A significant increase was observed in all age groups. Adults aged 75+ years and children 0-17 years experienced the lowest (27%) and highest (305%) incidence increase, respectively. However, the oldest age group still had the highest SA-SSTI hospitalization incidence across all study years. Total annual cost of SA-SSTI hospitalizations also increased and peaked in 2008 at $4.84 billion, a 44% increase from 2001. In 2009, the average associated cost of a SA-SSTI hospitalization was $11,622 (SE=$200). CONCLUSION: There has been an increase in the incidence and associated cost of SA-SSTI hospitalizations in U.S.A. between 2001 and 2009, with the highest incidence increase seen in children 0-17 years. However, the greatest burden was still seen in the population over 75 years. By 2009, SSTI diagnoses were present in about half of all SA-hospitalizations.

Molecular characterization of Staphylococcus aureus in the United States 2004-2008 reveals the rapid expansion of USA300 among inpatients and outpatients.

To assess the clonal structure of Staphylococcus aureus in the United States, we performed a molecular epidemiological study of 1,055 S. aureus isolates from a nationally representative clinical isolate collection from 2004-2008. Resistant and susceptible isolates were typed with multilocus sequence typing, tested for the presence of Panton-Valentine leukocidin (PVL), and serotyped. USA300 (multilocus sequence typing clonal complex 8, PVL positive, and methicillin-resistant) was the most frequently isolated clone, expanding from 12% of all isolates in 2004 to 38% in 2006. The USA300 clone increased significantly in frequency among both outpatients and inpatients. USA300 increased in both skin and soft-tissue and invasive infection isolates. The second most frequently observed clone was clonal complex 5, PVL-negative, and methicillin-resistant, and its frequency was stable from 2004-2008. The methicillin-susceptible S. aureus in the study was polyclonal, and decreased in frequency as it was replaced by USA300.

Prevalence and sequence variation of panton-valentine leukocidin in methicillin-resistant and methicillin-susceptible staphylococcus aureus strains in the United States.

Panton-Valentine leukocidin (PVL), encoded by the lukSF-PV genes, is a putative virulence factor and marker for community-associated methicillin-resistant Staphylococcus aureus. Here we report the prevalence of PVL among a representative sample of 1,055 S. aureus infection isolates from the United States and describe the sequence variation of the lukSF-PV genes. We performed multilocus sequence typing (MLST) on all isolates and sequenced fragments of the lukSF-PV genes from a sample of 86 isolates. We assigned isolates to a PVL R or H sequence type based on a polymorphism that results in an amino acid change from arginine (R) to histidine (H). Overall, we found that 36% of S. aureus isolates were positive for lukSF-PV. Among the 86 we typed, we identified 72 R variants and 14 H variants. Among the 47 methicillin-resistance S. aureus (MRSA) isolates, 43 harbored the R variant, and among the 39 methicillin-susceptible S. aureus (MSSA) isolates, 29 harbored the R variant. Almost all (97%) of the R variants were found in MLST clonal complex 8 (CC8), while the H variant was broadly distributed among 6 CCs. Within CC8, all 38 MRSA (USA300) and all 28 MSSA isolates harbored the R variant. Of the 20 isolates from blood and the lower respiratory tract, 19 (95%) harbored the R variant. While the R variant had been linked primarily to USA300 MRSA, we found that all CC8 MSSA isolates also contained the R variant, suggesting that some strains of USA300 may have lost methicillin resistance as an adaptation in the community.

Increasing role of Staphylococcus aureus and community-acquired methicillin-resistant Staphylococcus aureus infections in the United States: a 10-year trend of replacement and expansion.

BACKGROUND: There is the need to properly characterize the temporal trend of U.S. Staphylococcus aureus infections, including methicillin-resistant S. aureus (MRSA) and community-acquired (CA) MRSA in inpatient and outpatient settings. METHODS: The study used the Surveillance Network(®) surveillance database (Eurofins Medinet) and the National Hospitalization Discharge Survey for the period 1998-2007. CA-MRSA phenotype was defined by a resistance profile that includes susceptibility to gentamicin and cotrimoxazole, and coresistance to ciprofloxacin/clindamycin. Adjusted rates, rate ratios, and 95% confidence intervals (CIs) were computed using multivariate logistic regression. RESULTS: The study consisted of 1,761,991 S. aureus isolates. Annual MRSA prevalence continuously increased over the 10-year period from 32.7% in 1998 to 53.8% in 2007 (odds ratio 2.4, 95% CI 2.3-2.5). CA-MRSA replaced competing strains by increasing its share of MRSA from 22.3% in 1998 to 66.1% in 2007 (odds ratio 6.7, 95% CI 6.5-6.9). MRSA-related hospitalization rate per 1,000 discharges doubled from 3.5 ± 0.9 in 1998 to 7.6 ± 1.5 in 2007 (RR 2.2, 95% CI 1.8-3.1), whereas CA-MRSA increased from 0.4 ± 0.14 hospitalizations per 1,000 discharges in 1998 to 3.1 ± 0.5 in 2007 (RR 8.1, 95% CI 5.2-14.1), By 2007, 81.5% of all MRSA isolates were categorized as CA-MRSA among children, whereas CA-MRSA represented 48.9% of MRSA isolates from the elderly. CONCLUSION: MRSA not only replaced methicillin susceptible S. aureus (MSSA) isolates as a percentage of all S. aureus isolates, but its hospitalization rates increased over and above the replacement process. This trend also applies to CA-MRSA over hospital-acquired (HA) MRSA.

Impact of new Clinical Laboratory Standards Institute Streptococcus pneumoniae penicillin susceptibility testing breakpoints on reported resistance changes over time.

The analysis comprised a total of 97,843 U.S. isolates from the Surveillance Network(®) database for the period 1996-2008. Penicillin resistance, when defined using the old Clinical Laboratory Standards Institute breakpoint (≥2 µg/ml), had an initial rise that started in 1996, peaked in 2000, declined until 2003, and rebounded through 2008 (15.6%, 23.2%, 15.4%, and 16.9%, respectively). Using the new Clinical Laboratory Standards Institute criteria and applying a breakpoint of ≥8 µg/ml to blood and bronchial isolates, resistance was unchanged (0.24% in 2003) but rose to 1.52% in 2008. Using the new meningitis criteria (≥0.12 µg/ml), resistance prevalence was 34.8% in 2008, whereas it was 12.3% using the old criteria (≥2 µg/ml) for cerebrospinal fluid isolates. The rise, fall, and subsequent rebound of penicillin resistance in the United States, presumably influenced by the introduction of the conjugate pneumococcal vaccine, is clearly seen with the old definition, but only the rebound is seen when the new criteria are applied. In the postvaccine period, isolates with minimum inhibitory concentrations of 1 and 2 µg/ml decline, whereas those with minimum inhibitory concentrations of 0.12-0.5 increase, which may signal the loss of resistant vaccine serotypes and the acquisition of resistance by nonvaccine serotypes.

Acinetobacter baumannii 2002-2008: increase of carbapenem-associated multiclass resistance in the United States.

The study consisted of data for 55,330 U.S. Acinetobacter baumannii isolates from The Surveillance Network(®) database for the period 2002-2008. Risk factors were time, age, sex, census region, location (Ward or ICU), and isolate source. Antimicrobial susceptibility data were available for carbapenems, cephalosporins, aminoglycosides, fluoroquinolones, and ß-lactam/ß-lactamase inhibitor combinations. Multiclass resistance was defined as nonsusceptibility to carbapenems and two or more additional classes. Odds of resistance were obtained using a logistic regression model with cubic splines. Carbapenem-associated multiclass resistance has had a 3.7-fold (95% confidence interval [CI] 3.4-4.3) increase from 20.6% in 2002 to 49.2% in 2008. Among blood isolates the increase was by 2.2 times (95% CI 1.7-2.9). Subjects <18 years old had significantly (p < 0.001) lower rates in 2002 (6.9%) than those 65 years or older (21.5%), but by 2008 this difference diminished as rates increased to 44.2% and 54.2%, respectively. A similar divergence was also observed between ICU and Ward, with no differences in 2002, whereas in 2008 ICU isolates had significantly higher rates (55.2%, 95% CI 53.6%-56.9%) than Ward isolates (45.6%, 95% CI 44.2%-47.0%). Over half of all A. baumannii-resistant isolates were carbapenem and multiclass resistant in 2008. Rates among subjects <18 years old have increased faster than those of the elderly, and in the ICU as compared to Ward.

HIV type 1 from a patient with baseline resistance to CCR5 antagonists uses drug-bound receptor for entry.

CCR5 antagonists are a new class of antiretroviral drugs that block viral entry by disrupting interactions between the viral envelope (Env) glycoprotein and coreceptor. During the CCR100136 (EPIC) Phase IIb study of the CCR5 antagonist aplaviroc (APL) in treatment-naive individuals, a patient was identified who harbored virus strains that exhibited partial resistance to APL at the time of virologic failure. Retrospectively, it was found that APL resistance was present at baseline as well. To investigate the mechanism of APL resistance in this patient, we cloned HIV-1 env genes from plasma obtained at baseline and after virologic failure. Approximately 85% of cloned Envs were functional, and all exhibited partial resistance to APL. All Envs were R5-tropic, were partially resistant to other CCR5 antagonists including maraviroc on cells with high CCR5 expression, but remained sensitive to the fusion inhibitor enfuvirtide. Competition studies with natural CCR5 ligands revealed that the mechanism of drug resistance entailed the use of the drug-bound conformation of CCR5 by the Env proteins obtained from this individual. The degree of drug resistance varied between Env clones, and also varied depending on the cell line used or the donor from whom the primary T cells were obtained. Thus, both virus and host factors contribute to CCR5 antagonist resistance. This study shows that R5 HIV-1 strains resistant to CCR5 inhibitors can arise in patients, confirming a mechanism of resistance previously characterized in vitro. In addition, some patients can harbor CCR5 antagonist-resistant viruses prior to treatment, which may have implications for the clinical use of this new class of antiretrovirals.

The impact of the pneumococcal conjugate vaccine on antimicrobial resistance in the United States since 1996: evidence for a significant rebound by 2007 in many classes of antibiotics.

BACKGROUND: The impact of the introduction of the pneumococcal conjugate vaccine over antimicrobial resistance has not been well established. The present study models the changes in resistance over time for all major classes of antibiotics. METHODS: Susceptibility data on a total of 129,652 isolates from The Surveillance Network surveillance database during the period 1996-2007 were available for analysis, as well as age, specimen source, inpatient or outpatient location, and census region. Cubic splines in a logistic regression mixed model were used to model changes of the resistance rates over time in the United States, taking into account risk factors, so that separate adjusted curves were modeled for each antibiotic. RESULTS: Yearly resistance prevalence to most antibiotics had been increasing in the period 1996-2001. Adjusted prevalence rates in a multivariate model declined in the period 2001-2004 for penicillin, erythromycin, amoxicillin/clavulanate, trimethoprim/sulfamethoxazole, tetracycline, ceftriaxone, and multidrug. These same antibiotics showed a significant rebound for the period 2004-2007, with the largest overall increase for erythromycin, followed by amoxicillin/clavulanate, tetracycline, multidrug, penicillin, trimethoprim/sulfamethoxazole, and ceftriaxone. Changes in both decline and rebound were more marked for children <5 years old and for otitis media isolates. CONCLUSION: The indirect effect of the pneumococcal conjugate vaccine introduction on yearly resistance prevalence for several antibacterials as well as for multidrug resistance is one of blunting of a prior sustained increase, with a significant but short-lived decrease in resistance rates, and a significant rebound in adjusted rates for the period 2004-2007.

Virologic failure in first-line human immunodeficiency virus therapy with a CCR5 entry inhibitor, aplaviroc, plus a fixed-dose combination of lamivudine-zidovudine: nucleoside reverse transcriptase inhibitor resistance regardless of envelope tropism.

The CCR102881 (ASCENT) study evaluated the antiviral activity of the novel CCR5 entry inhibitor aplaviroc plus a fixed-dose combination of lamivudine-zidovudine (Combivir) in drug-naïve human immunodeficiency virus type 1-infected subjects with only CCR5-tropic virus detected in plasma. Although the trial was stopped prematurely due to idiosyncratic hepatotoxicity, eight subjects met protocol-defined virologic failure criteria. Clonal analyses of the viral envelope tropism, aplaviroc susceptibility, and env sequencing were performed on plasma at baseline and at the time of virologic failure. Molecular evolutionary analyses were also performed. The majority of the subjects with virologic failure (six of eight) acquired the lamivudine resistance-associated mutation M184V, and none had evidence of reduced susceptibility to aplaviroc at the time of virologic failure, even at the clonal level. Six subjects with virologic failure maintained CCR5 tropism, while two exhibited a change in population tropism readout to dual/mixed-tropic with R5X4-tropic clones detected prior to therapy. Two evolutionary patterns were observed: five subjects had no evidence of population turnover, while three subjects had multiple lines of evidence for env population turnover. The acquisition of the M184V mutation is the primary characteristic of virologic failure in first-line therapy with aplaviroc plus lamivudine-zidovudine, regardless of the envelope tropism.

Virologic failure in therapy-naive subjects on aplaviroc plus lopinavir-ritonavir: detection of aplaviroc resistance requires clonal analysis of envelope.

The CCR100136 (EPIC) study evaluated the antiviral activity of the novel CCR5 entry inhibitor aplaviroc in combination with lopinavir-ritonavir in drug-naïve human immunodeficiency virus type 1-infected subjects. Although the trial was stopped prematurely due to idiosyncratic hepatotoxicity, 11 subjects met the protocol-defined virologic failure criteria. Clonal analyses of the viral envelope tropism, aplaviroc susceptibility, and env sequencing were performed on plasma at day 1 and at the time of virologic failure. Molecular evolutionary analyses were also performed. Treatment-emergent resistance to aplaviroc or lopinavir-ritonavir was not observed at the population level. However, aplaviroc resistance was detected prior to therapy at both the clonal and population levels in one subject with virologic failure and in six subjects in a minority (<50%) of clones at day 1 or at the time of virologic failure. Reduced aplaviroc susceptibility manifested as a 50% inhibitory concentration curve shift and/or a plateau. Sequence changes in the clones with aplaviroc resistance were unique to each subject and scattered across the envelope coding region. Clones at day 1 and at the time of virologic failure were not phylogenetically distinct. Two subjects with virologic failure had a population tropism change from CCR5- to dual/mixed-tropic during treatment. Virologic failure during a regimen of aplaviroc and lopinavir-ritonavir may be associated with aplaviroc resistance, only at the clonal level, and/or, infrequently, tropism changes.

The role of positive selection in determining the molecular cause of species differences in disease.

BACKGROUND: Related species, such as humans and chimpanzees, often experience the same disease with varying degrees of pathology, as seen in the cases of Alzheimer's disease, or differing symptomatology as in AIDS. Furthermore, certain diseases such as schizophrenia, epithelial cancers and autoimmune disorders are far more frequent in humans than in other species for reasons not associated with lifestyle. Genes that have undergone positive selection during species evolution are indicative of functional adaptations that drive species differences. Thus we investigate whether biomedical disease differences between species can be attributed to positively selected genes. RESULTS: We identified genes that putatively underwent positive selection during the evolution of humans and four mammals which are often used to model human diseases (mouse, rat, chimpanzee and dog). We show that genes predicted to have been subject to positive selection pressure during human evolution are implicated in diseases such as epithelial cancers, schizophrenia, autoimmune diseases and Alzheimer's disease, all of which differ in prevalence and symptomatology between humans and their mammalian relatives. In agreement with previous studies, the chimpanzee lineage was found to have more genes under positive selection than any of the other lineages. In addition, we found new evidence to support the hypothesis that genes that have undergone positive selection tend to interact with each other. This is the first such evidence to be detected widely among mammalian genes and may be important in identifying molecular pathways causative of species differences. CONCLUSION: Our dataset of genes predicted to have been subject to positive selection in five species serves as an informative resource that can be consulted prior to selecting appropriate animal models during drug target validation. We conclude that studying the evolution of functional and biomedical disease differences between species is an important way to gain insight into their molecular causes and may provide a method to predict when animal models do not mirror human biology.

Molecular epidemiology of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from global clinical trials.

Determining the genetic characteristics of Staphylococcus aureus is important for better understanding of the global and dynamic epidemiology of this organism as we witness the emergence and spread of virulent and antibiotic-resistant clones. We genotyped 292 S. aureus isolates (105 methicillin resistant and 187 methicillin susceptible) using a combination of pulsed-field gel electrophoresis, multilocus sequence typing, and SCCmec typing. In addition, S. aureus isolates were tested for the presence of the Panton-Valentine leukocidin (PVL) genes. Isolates were recovered from patients with uncomplicated skin infections in 10 different countries during five phase III global clinical trials of retapamulin, a new topical antibiotic agent. The most common methicillin-resistant clone had multilocus sequence type 8, pulsed-field type USA300, and SCCmec type IV and possessed the PVL genes. This clone was isolated exclusively in the United States. The most common PVL-positive, methicillin-susceptible clone had multilocus sequence type 121 and pulsed-field type USA1200. This clone was found primarily in South Africa and the Russian Federation. Other clones were found at lower frequencies and were limited in their geographic distribution. Overall, considerable genetic diversity was observed within multilocus sequence type clonal complexes and pulsed-field types.

Chemokine (C-C motif) receptor 5-using envelopes predominate in dual/mixed-tropic HIV from the plasma of drug-naive individuals.

OBJECTIVE: HIV-1 utilizes CD4 and either chemokine (C-C motif) receptor 5 (CCR5) or chemokine (C-X-C motif) receptor 4 (CXCR4) to gain entry into host cells. Small molecule CCR5 antagonists are currently being developed for the treatment of HIV-1 infection. Because HIV-1 may also use CXCR4 for entry, the use of CCR5 entry inhibitors is controversial for patients harboring CCR5-using and CXCR4-using (dual/mixed-tropic) viruses. The goal of the present study was to determine the proportion of CCR5-tropic and CXCR4-tropic viruses in dual/mixed-tropic virus isolates from drug-naïve patients and the phenotypic and genotypic relationships of viruses that use CCR5 or CXCR4 or both. DESIGN: Fourteen antiretroviral-naive HIV-1-infected patients were identified as having population coreceptor tropism readout of dual/mixed-tropic viruses. Intrapatient comparisons of coreceptor tropism and genotype of env clones were conducted on plasma virus from each patient. METHODS: Population HIV-1 envelope tropism and susceptibility to the CCR5 entry inhibitor, aplaviroc, were performed using the Monogram Biosciences Trofile Assay. Twelve env clones from each patient were analyzed for coreceptor tropism, aplaviroc sensitivity, genotype, and intrapatient phylogenetic relationships. RESULTS: Viral populations from antiretroviral-naive patients with dual/mixed-tropic virus are composed primarily of CCR5-tropic env clones mixed with those that use both coreceptors (R5X4-tropic) and, occasionally, CXCR4-tropic env clones. Interestingly, the efficiency of CXCR4 use by R5X4-tropic env clones varied with their genetic relationships to CCR5-tropic env clones from the same patient. CONCLUSION: These data show that the majority of viruses in these dual/mixed-tropic populations use CCR5 and suggest that antiretroviral-naive patients may benefit from combination therapy that includes CCR5 entry inhibitors.

Temporal and spatial distribution of clonal complexes of Streptococcus pneumoniae isolates resistant to multiple classes of antibiotics in Belgium, 1997 to 2004.

We performed multilocus sequence typing on 203 invasive disease isolates of Streptococcus pneumoniae to assess the clonal compositions of isolates from two provinces in Belgium and to determine the relationship between clones and antibiotic nonsusceptibility, particularly nonsusceptibility to two or more classes of antibiotics. The frequency of multiclass nonsusceptibility (MCNS) was higher in the province of West Flanders (38%) than in Limburg (21%). This difference was largely attributable to five clonal complexes (CC156, CC81, CC143, CC193, and CC1848), which contained high proportions of isolates with MCNS (>47%) and which were circulating at higher frequencies in West Flanders. The S. pneumoniae population changed over time, as CC156 and CC81 declined in frequency from 1997 to 1999 to 2001 to 2004. Over the same time period, the frequency of pneumococcal conjugate vaccine 7 (PCV7) serotypes dropped from 69% to 41%. In contrast, the nonvaccine serotype 19A increased in frequency from 2.1% to 6.6%. None of these changes can be attributed to PCV7 vaccine, as it was not in use in Belgium during the time period studied. There was evidence that MCNS clones flowed from West Flanders to Limburg.