Household aggregation of Staphylococcus aureus by clonal complex and methicillin resistance profiles in Starr County, Texas.

Staphylococcus aureus is one of the most common causes of skin and soft tissue infections in health-care and community settings, but transmission of S. aureus in community-based populations is incompletely understood. S. aureus carriage phenotypes (persistent, intermittent, and non-carriers) were determined for households from Starr County, TX. Nasal swabs were collected from a cohort of 901 residents and screened for the presence of S. aureus. Isolated strains were spa-typed and assigned to clonal complexes. Of the 901 participants there were 134 pairs, 28 trios, 11 quartets, 3 quintets and 1 septet residing in the same household. There was a significant increase in "ever" carriers (persistent and intermittent carriers combined) in these households over that expected based on population frequencies (p = 0.029). There were 42 ever carrier pairs of individuals with 21 concordant for clonal complex type whereas only 4.7 were expected to be so (p = 6.9E-11). These results demonstrated clear aggregation of S. aureus carriage and concordance for strain types within households. As antibiotic-resistant S. aureus strains increase in community settings, it is important to better understand risk factors for colonization, mechanisms of transmission, clonal complexes present, and the role of household concordance/transmission.

Sylvatic Transmission of Trypanosoma cruzi Among Domestic and Wildlife Reservoirs in Texas, USA: A Review of the Historical Literature.

Chagas disease (Trypanosoma cruzi infection) is one of the most important neglected tropical diseases affecting the Americas. The transmission dynamic of this parasite is a complicated process that involves three genera of Triatominae subfamily and over 100 known mammalian reservoirs composed of domestic, peridomestic and wildlife species. Understanding the complex relationship between vector species and mammalian hosts is important for preventing transmission to humans. We performed a historical literature review to assess the disease burden in the Texas wildlife and domestic animal population. Reports of sylvatic transmission in Texas date back to the 1940s. We found that up to 23 species can serve as reservoirs for T. cruzi in the state with wood rats, raccoons, and wild and domestic canine species most frequently reported as positive for the parasite. We finish with a discussion of the current research gaps, implications for high-risk populations and future directions for research.

Prevalence and behavioural risk factors of Staphylococcus aureus nasal colonization in community-based injection drug users.

The aims of this study were to identify Staphylococcus aureus nasal colonization prevalence, behavioural risk factors, and to determine staphylococcal protein A (spa) types in community-based injection drug users (IDUs). Nasal swabs were collected and methicillin susceptibility testing and spa/SCCmec typing were performed on S. aureus isolates. Generalized estimating equations were used to report adjusted odds ratios and 95% confidence intervals. Of the 440 participants, 24·1% were colonized and 5·7% had methicillin-resistant S. aureus (MRSA). Colonization was associated with age, employment/marital status, and the presence of scabs but not with sexually transmitted disease co-infection, HIV status, antibiotic use, hospitalization, or drug treatment programme participation. The USA300 MRSA clone spa types were most common, but 15/49 spa types were new to one of the international databases. Community-based IDUs appear to have different risk factors compared to IDUs from clinical studies. In addition, the number of newly identified spa types indicates a diverse, understudied population.

The Panton-Valentine leukocidin vaccine protects mice against lung and skin infections caused by Staphylococcus aureus USA300.

Methicillin-resistant Staphylococcus aureus is increasingly responsible for staphylococcal infections in the community. A large percentage of the community-acquired methicillin-resistant (CA-MRSA) strains in the USA produce Panton-Valentine leukocidin (PVL), which is associated with severe infections. The virulence of the clinical CA-MRSA strain USA300 was compared to that of its isogenic pvl-deleted mutant, and it was shown that PVL contributes to lung and muscle tissue destruction, respectively, in murine necrotizing pneumonia and skin infection models. Mice infected with the USA300 strain developed a dominant anti-PVL response. The PVL subunits were therefore tested as vaccinogens against this isolate, and their vaccine efficacy correlated with both the route of vaccination and infection. These data suggest that PVL is a virulence factor in murine CA-MRSA infections.

High risk of human immunodeficiency virus in men who have sex with men with herpes simplex virus type 2 in the EXPLORE study.

The relation between herpes simplex virus type 2 (HSV-2) and human immunodeficiency virus (HIV) acquisition was evaluated among 4,295 high-risk, HIV-negative men who have sex with men in an intensive behavioral intervention (colloquially referred to as "EXPLORE") study in the United States from 1999 to 2003. Sexual behavior data were obtained by computer-assisted self-interview, and sera were collected semiannually for HIV and HSV-2 serology. HSV-2 infection was classified as "recent incident" (at the first HSV-2 seropositive visit), "remote incident" (within 24 months of the first positive visit), and "prevalent" (for visits >24 months after the first HSV-2 positive visit). Baseline HSV-2 prevalence was 20.3%. HSV-2 incidence was 1.9 (95% confidence interval (CI): 1.6, 2.2) per 100 person-years; significant risk factors were African-American race, unprotected receptive anal intercourse, an HIV-positive male sex partner, and six or more male partners in the prior 6 months. The behavioral intervention did not reduce HSV-2 acquisition (adjusted hazard ratio (HR) = 1.2, 95% CI: 0.9, 1.6). Overall HIV incidence was 1.9 (95% CI: 1.7, 2.2) per 100 person-years. HIV risk was elevated among men who have sex with men with recent incident HSV-2 (adjusted HR = 3.6, 95% CI: 1.7, 7.8), remote incident HSV-2 (adjusted HR = 1.7, 95% CI: 0.8, 3.3), and prevalent HSV-2 (adjusted HR = 1.5, 95% CI: 1.1, 2.1) infection compared with HSV-2 seronegative participants. HIV intervention strategies targeting HSV-2 prevention and suppression among men who have sex with men should be evaluated.

Approaches to the release of a master cell bank of PER.C6 cells; a novel cell substrate for the manufacture of human vaccines.

At Merck and Co. we have developed a recombinant E1 deficient adenovirus type 5 vaccine vector for HIV-1 and have adopted the PER.C6 cell line as a cell substrate for the manufacture of this vector for Phase I and II clinical trials. The PER.C6 cell line was developed at Crucell by the transfection of human primary embryonic retinoblasts with a transgene of E1 constructed with a minimum of E1 coding sequences to preclude homologous recombination generating replication-competent adenovirus, between E1 sequences in PER.C6 and adenovirus vectors with E1 deletions of the same molecular coordinates. We have developed a Master Cell Bank (MCB) of PER.C6 cells under serum-free conditions of suspension culture from a vial of PER.C6 cells obtained from Crucell. This MCB has been released according to an extensive panel of testing for the detection of adventitious viral agents, including assays for sterility and mycoplasma, in vivo and in vitro assays for the detection of viruses of human, bovine and porcine origin, replication competent adenoviruses, sensitive PERT assays for the detection of RT in supernatants of co-cultivations, electron microscopy and a panel of PCR-based assays for specific human and animal viruses. This MCB has been used for the manufacture of vaccine vector supporting a number of IND submissions for Phase I clinical trials over a three-year period during which the panel of PCR testing applied to the MCB has been judiciously expanded. Advances in QPCR technology, liquid handling systems, and more recently mass spectrometry offer the possibility that very broad panels of primers and probes capable of the detection of all known human viruses can be applied routinely to support the release of biologicals for human clinical trials. The impact of this breadth of testing on the continued reliance of classical in vivo and in vitro assays for adventitious viruses is clearly an emerging issue worthy of serious debate.

Selective and opposing actions of progesterone receptor isoforms in human endometrial stromal cells.

Transcriptional regulation by progesterone is mediated primarily through the two progesterone receptor (PR) isoforms, PR-A and PR-B. Primary human endometrial stromal cell cultures, in which endogenous PR expression was lost, were infected with adenovirus expressing PR-A, PR-B, or both. Global gene expression analysis was conducted on vehicle and 30 nM progesterone (P4) treated cells following 12 h treatment. Interestingly, many genes regulated by PR-B alone or upon PR-A and PR-B co-expression, did not overlap with each other or with the PR-A expression group. Although many genes known to be progestin regulated in the uterus in vivo were also regulated in this study, markedly little overlap with published P4 regulated genes from human breast cancer cells was observed. Progesterone dose response curves were generated for several genes demonstrating gene selective potency and efficacy for each PR isoform. Furthermore, the PR isoforms opposed each other in regulation of tissue factor, with PR-B increasing and PR-A decreasing both mRNA and protein levels. Our data provide a view of global gene expression by PR isoforms in human endometrial cells and a comparison with other cell types. The specific genes and regulation patterns found provide groundwork to revealing the mechanism of PR isoform selectivity, and perhaps ultimately to the tissue selective properties these receptors appear to exhibit.

Transcriptional profiling of human osteoblast differentiation.

Osteoblast differentiation is a key aspect of bone formation and remodeling. To further our understanding of the differentiation process, we have developed a collection of conditionally immortalized adult human osteoblast cell lines representing discrete stages of differentiation. To evaluate changes in gene expression associated with differentiation, polyA((+)) RNA from pre-osteoblasts, early and late osteoblasts, and pre-osteocytes was subjected to gene chip analysis using the Affymetrix Hu6800 chip in conjunction with an Affymetrix custom chip enriched in bone and cartilage cDNAs. Overall, the expression of 47 genes was found to change threefold or more on both chips between the pre-osteoblastic and pre-osteocytic stages of differentiation. Many of the observed differences, including down-regulation of collagen type I and collagen-processing enzymes, reflect expected patterns and support the relevance of our results. Other changes have not been reported and offer new insight into the osteoblast differentiation process. Thus, we observed regulation of factors controlling cell cycle and proliferation, reflecting decreased proliferation, and increased apoptosis in pre-osteocytic cells. Elements maintaining the cytoskeleton, extracellular matrix, and cell-cell adhesion also changed with differentiation reflecting profound alterations in cell architecture associated with the differentiation process. We also saw dramatic down-regulation of several components of complement and other immune response factors that may be involved in recruitment and differentiation of osteoclasts. The decrease in this group of genes may provide a mechanism for controlling bone remodeling of newly formed bone. Our screen also identified several signaling proteins that may control osteoblast differentiation. These include an orphan nuclear receptor DAX1 and a small ras-related GTPase associated with diabetes, both of which increased with increasing differentiation, as well as a high mobility group-box transcription factor, SOX4, that was down-regulated during differentiation. In summary, our study provides a comprehensive transcriptional profile of human osteoblast differentiation and identifies several genes of potential importance in controlling differentiation of osteoblasts.

Primary care physicians attitude toward diagnosis and treatment of late-life depression.

Underdiagnosis and undertreatment of late-life depression is common, especially in primary care settings. To help assess whether physicians attitude and confidence in diagnosing and managing depression serve as barriers to care, a total of 176 physicians employed in 18 primary care groups were administered surveys to assess attitudes towards diagnosis, treatment, and management of depression in elderly patients, (individuals over 65 years of age). Logistic regression was performed to assess the association of physician characteristics on attitudes. Nearly all of the physicians surveyed felt that depression in the elderly was a primary care problem, and 41% reported late-life depression as the most common problem seen in older patients. Physicians were confident in their ability to diagnose and manage depression, yet 45% had no medical education on depression in the previous three years. Physicians confidence in their ability to diagnose, treat, and manage depression, and their reported adequacy of training, do not appear to correspond to the amount of continuing medical education in depression, suggesting that physician overconfidence may potentially be serving as a barrier to care.

Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci.

The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the Staphylococcus aureus genome, was used to identify open reading frames that are regulated by agr and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and agr, sarA, and agr sarA mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up- or downregulated in an agr- and/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including hla (alpha-toxin) and spa (protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and protein A expression. Several putative virulence factors were also identified as regulated by agr and/or SarA. In addition, genes that are involved in several biological processes but which are difficult to reconcile as playing a direct role in the organism's pathogenesis also appeared to be regulated by each effector, suggesting that products of both the agr and the sarA locus are more-global transcription regulators than previously realized.

Suppression of antigen-specific lymphocyte activation in modeled microgravity.

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

The effect of UV irradiation on infection of mice with Borrelia burgdorferi.

These studies addressed the hypothesis that UV radiation (UVR) could affect immune responses in mice infected with Borrelia burgdorferi. Immunity against the Lyme spirochete B. burgdorferi was studied in a murine model of UV-induced immune suppression. Borrelia-specific cellular and humoral responses were examined following immunosuppressive doses of UVR. Low-passage Borrelia were injected intradermally at the base of the tail following irradiation. At various time points after infection the blood was cultured for the presence of Borrelia and the serum analyzed for Borrelia-specific antibodies. Two weeks after infection one hind-limb joint was cultured for the presence of spirochetes and the contralateral joint was examined histologically for arthritis formation. The results demonstrated that UV irradiation, administered at the site of infection or at a distant site, suppressed Borrelia-specific cellular and humoral responses in infected mice. Suppression of delayed-type hypersensitivity and antibody responses to UV was abrogated by administration of anti-interleukin (IL)-10 after UV irradiation. In addition, UV irradiation altered the dissemination pattern of the bacteria from the skin into the blood and exacerbated arthritis when compared with unirradiated controls. From these studies we concluded that UV irradiation can modulate the immune response to Borrelia and exacerbate the subsequent arthritic component of Lyme disease in mice. Furthermore, our studies suggest that IL-10 is in part responsible for the suppression of both cellular and humoral responses in addition to playing a role in the development of Lyme arthritis.

Resistance to Lyme disease in decorin-deficient mice.

Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn(-/-) mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn(-/-) mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn(+/+) or Dcn(+/-) mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borreila-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn(-/-) mice had diminished Borrelia numbers compared with issues harvested from Dcn(+/+) controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn(-/-) mice. Conversely, no differences in the numbers of Borreila-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.

Quantitative analysis of mRNA amplification by in vitro transcription.

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

Evaluation of normalization procedures for oligonucleotide array data based on spiked cRNA controls.

BACKGROUND: Affymetrix oligonucleotide arrays simultaneously measure the abundances of thousands of mRNAs in biological samples. Comparability of array results is necessary for the creation of large-scale gene expression databases. The standard strategy for normalizing oligonucleotide array readouts has practical drawbacks. We describe alternative normalization procedures for oligonucleotide arrays based on a common pool of known biotin-labeled cRNAs spiked into each hybridization. RESULTS: We first explore the conditions for validity of the 'constant mean assumption', the key assumption underlying current normalization methods. We introduce 'frequency normalization', a 'spike-in'-based normalization method which estimates array sensitivity, reduces background noise and allows comparison between array designs. This approach does not rely on the constant mean assumption and so can be effective in conditions where standard procedures fail. We also define 'scaled frequency', a hybrid normalization method relying on both spiked transcripts and the constant mean assumption while maintaining all other advantages of frequency normalization. We compare these two procedures to a standard global normalization method using experimental data. We also use simulated data to estimate accuracy and investigate the effects of noise. We find that scaled frequency is as reproducible and accurate as global normalization while offering several practical advantages. CONCLUSIONS: Scaled frequency quantitation is a convenient, reproducible technique that performs as well as global normalization on serial experiments with the same array design, while offering several additional features. Specifically, the scaled-frequency method enables the comparison of expression measurements across different array designs, yields estimates of absolute message abundance in cRNA and determines the sensitivity of individual arrays.

Genomic analysis of gene expression in C. elegans.

Until now, genome-wide transcriptional profiling has been limited to single-cell organisms. The nematode Caenorhabditis elegans is a well-characterized metazoan in which the expression of all genes can be monitored by oligonucleotide arrays. We used such arrays to quantitate the expression of C. elegans genes throughout the development of this organism. The results provide an estimate of the number of expressed genes in the nematode, reveal relations between gene function and gene expression that can guide analysis of uncharacterized worm genes, and demonstrate a shift in expression from evolutionarily conserved genes to worm-specific genes over the course of development.

Substituted benzamide inhibitors of human rhinovirus 3C protease: structure-based design, synthesis, and biological evaluation.

A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease was identified using structure-based design. The design, synthesis, and biological evaluation of these inhibitors are reported. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3CP substrate. alpha,beta-Unsaturated cinnamate esters irreversibly inhibited the 3CP and displayed antiviral activity (EC(50) 0.60 microM, HRV-16 infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 A cocrystal structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.

Structure-based design of ketone-containing, tripeptidyl human rhinovirus 3C protease inhibitors.

Tripeptide-derived molecules incorporating C-terminal ketone electrophiles were evaluated as reversible inhibitors of the cysteine-containing human rhinovirus 3C protease (3CP). An optimized example of such compounds displayed potent 3CP inhibition activity (K = 0.0045 microM) and in vitro antiviral properties (EC50=0.34 microM) when tested against HRV serotype-14.

Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes.

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.