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.

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.

Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides.

We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-beta is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.

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.

Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins.

Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF) were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent homologous with the GM-CSF recently cloned from murine lung messenger RNA.

Constitutive production of macrophage colony-stimulating factor by human ovarian and breast cancer cell lines.

Many nonhematologic tumors produce growth factors that may influence cellular proliferation either by autocrine or by paracrine mechanisms. In the current study, human tumor cell lines were investigated for the constitutive production of macrophage colony-stimulating factor (M-CSF). Culture supernatants obtained from cell lines were analyzed using a radioimmunoassay and a radioreceptor assay specific for M-CSF. Among the various cell types analyzed, all the ovarian cell lines and a majority of the breast cancer cell lines secreted significant amount of an M-CSF-like factor. Treatment of mouse bone marrow cultures with culture supernatants from ovarian cancer cells stimulated the production of macrophage colonies. Analysis of total cellular RNA obtained from the ovarian cell lines by Northern blot showed multiple sizes of M-CSF transcripts with an abundance of a 4.2-kb message. The relative amount of M-CSF transcripts correlated with the level of immunoreactive material seen in the culture supernatants.

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.

The t-unique coding domain is important to the transformation maintenance function of the simian virus 40 small t antigen.

The small t antigen (t) of simian virus 40, a 174-amino-acid-containing protein, when present together with the other early viral protein, large T antigen (T), plays an important role in the maintenance of simian virus 40-induced neoplastic phenotype in certain cells. Indeed, each protein functions in a complementary manner in this process. The t coding unit is composed of two segments, a 5' region of 246 nucleotides which is identical to that of the corresponding 5' region of the T coding unit and a 3' segment of 276 nucleotides which is unique. Two mutant, t-encoding genomes, one bearing a missense and the other a nonsense mutation at the same point in the t-unique coding region were constructed in vitro and found to be defective in their ability to dissolve the actin cytoskeleton of rat fibroblasts and to complement T in the growth of mouse fibroblasts in soft agar. Therefore, the unique segment of the t gene encodes a portion of the t molecule which is essential to its transformation maintenance function.

Expression monitoring by hybridization to high-density oligonucleotide arrays.

The human genome encodes approximately 100,000 different genes, and at least partial sequence information for nearly all will be available soon. Sequence information alone, however, is insufficient for a full understanding of gene function, expression, regulation, and splice-site variation. Because cellular processes are governed by the repertoire of expressed genes, and the levels and timing of expression, it is important to have experimental tools for the direct monitoring of large numbers of mRNAs in parallel. We have developed an approach that is based on hybridization to small, high-density arrays containing tens of thousands of synthetic oligonucleotides. The arrays are designed based on sequence information alone and are synthesized in situ using a combination of photolithography and oligonucleotide chemistry. RNAs present at a frequency of 1:300,000 are unambiguously detected, and detection is quantitative over more than three orders of magnitude. This approach provides a way to use directly the growing body of sequence information for highly parallel experimental investigations. Because of the combinatorial nature of the chemistry and the ability to synthesize small arrays containing hundreds of thousands of specifically chosen oligonucleotides, the method is readily scalable to the simultaneous monitoring of tens of thousands of genes.

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.

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.

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.

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.

Solution structure and peptide binding of the SH3 domain from human Fyn.

BACKGROUND: The Src family of tyrosine kinases is involved in the propagation of intracellular signals from many transmembrane receptors. Each member of the family contains two domains that regulate interactions with other molecules, one of which is the Src homology 3 (SH3) domain. Although structures have previously been determined for SH3 domains, and ideas about peptide-binding modes have been proposed, their physiological role is still unclear. RESULTS: We have determined the solution structure of the SH3 domain from the Src family tyrosine kinase Fyn in two forms: unbound and complexed with a peptide corresponding to a putative ligand sequence from phosphatidylinositol 3' kinase. Fyn SH3 shows the typical SH3 topology of two perpendicular three-stranded beta sheets and a single turn of 3(10) helix. The interaction of SH3 with three potential ligand peptides was investigated, demonstrating that they all bind to the same site on the molecule. A previous model for ligand binding to SH3 domains predicts binding in one of two orientations (class I or II), each characterized by a consensus sequence. The ligand with the closest match to the class I consensus sequence bound with highest affinity and in the predicted orientation. CONCLUSIONS: The Fyn SH3 domain has a well-defined structure in solution. The relative binding affinities of the three ligand peptides and their orientation within the Fyn SH3 complex were consistent with recently proposed models for the binding of 'consensus' polyproline sequences. Although the affinities of consensus and non-consensus peptides are different, the degree of difference is not very large, suggesting that SH3 domains bind to polyproline peptides in a promiscuous manner.

Additive effects of steel factor and antisense TGF-beta 1 oligodeoxynucleotide on CD34+ hematopoietic progenitor cells.

We previously demonstrated that TGF-beta 1 antisense oligodeoxynucleotides can release early CD34+ bone marrow progenitors from quiescence, and increase the numbers of mixed and large erythroid colonies. As Steel Factor (SF) has a similar effect on colony formation by CD34+ cells, we tested whether this factor acts by blocking the inhibitory effects of TGF-beta. That this is not generally the case was demonstrated by the finding that the combination of TGF-beta 1 antisense with SF in cultures of CD34+ bone marrow cells yielded enhanced colony formation that was more than additive when compared to cultures containing the single agents. This combination also yielded enhanced colony formation by CD34+ umbilical cord blood cells, but in this case, the effect was slightly less than additive. Thus in cord blood, some, but not all, of the progenitors that are maintained in quiescence by TGF-beta can be triggered into cycle by SF. However, the absolute number of CFU-GEMMs found in the antisense TGF-beta plus SF cultures of cord blood was 4-fold higher than that found in comparable bone marrow cultures. These data correlate well with our previous observations that umbilical cord blood contains 4-fold more CD34+ CD38- cells, the population found to respond to TGF-beta 1 antisense oligodeoxynucleotides.

Specific Th1 cell lines that confer protective immunity against experimental Borrelia burgdorferi infection in mice.

Although humoral responses to Borrelia burgdorferi (Bb) have been shown to be protective in some animal models of Lyme disease, the role of T cells in this disease is less well understood. This work describes three Bb-specific T cell lines that prevent disease progression in syngeneic mice. The T cell lines were generated in C3H mice immunized with Bb in complete Freund's adjuvant. All lines were Bb-specific, CD4+, TCRalphabeta+, and they proliferated and produced interferon-gamma and interleukin-2 on stimulation with Bb. Injection of the cell lines into naive C3H recipients significantly reduced the number of organisms recoverable from the blood and tissues of infected mice and protected them from developing Bb-induced periarthritis. These studies demonstrated that Th1 cells can confer resistance to Bb infection in susceptible mice and suggested that the timing of this T cell response may be critical for determining disease outcome.

Effect of local ultraviolet irradiation on infections of mice with Candida albicans, Mycobacterium bovis BCG, and Schistosoma mansoni.

In this study, we investigated whether mice given ultraviolet (UV)-B (280-320 nm) radiation in doses sufficient to alter cutaneous immune cells and impair the induction of contact hypersensitivity would also have impaired resistance to infectious agents administered at the site of UV irradiation. C3H mice were exposed to 400 J/m2 UVR from FS40 sunlamps on four consecutive days. Immediately after the last UV treatment, groups of mice were injected subcutaneously with Candida albicans, injected intradermally (ID) with Mycobacterium bovis bacillus Calmette-Guerin (BCG), or infected percutaneously with Schistosoma mansoni in UV-irradiated skin. The induction of the delayed hypersensitivity response to C. albicans and BCG, as assessed by footpad swelling, was unaffected by UV irradiation. However, the number of viable mycobacteria recovered from the lymphoid organs of BCG-infected mice was increased significantly in the UV-irradiated animals for a period of more than 2 months. Low-dose UV irradiation of the skin at the site of infection did not influence the number of S. mansoni parasites recoverable from the internal organs of mice that had been infected with cercariae percutaneously 6 weeks earlier. We conclude that the ability of UV radiation to impair the development of cell-mediated immunity to antigens introduced in a UV-irradiated site is not universal and depends on the particular antigen administered. We hypothesize that the involvement of epidermal Langerhans cells as the primary antigen-presenting cells in the induction of cell-mediated immunity may be the critical factor in determining whether a particular immune response will be affected by local UV irradiation.

Design, synthesis, and evaluation of nonpeptidic inhibitors of human rhinovirus 3C protease.

The design, synthesis, and biological evaluation of reversible, nonpeptidic inhibitors of human rhinovirus (HRV) 3C protease (3CP) are reported. A novel series of 2,3-dioxindoles (isatins) were designed that utilized a combination of protein structure-based drug design, molecular modeling, and structure-activity relationship (SAR). The C-2 carbonyl of isatin was envisioned to react in the active site of HRV 3CP with the cysteine responsible for catalytic proteolysis, thus forming a stabilized transition state mimic. Molecular-modeling experiments using the apo crystal structure of human rhinovirus-serotype 14 (HRV-14) 3CP and a peptide substrate model allowed us to design recognition features into the P1 and P2 subsites, respectively, from the 5- and 1-positions of isatin. Attempts to optimize recognition properties in the P1 subsite using SAR at the 5-position were performed. In addition, a series of ab initio calculations were carried out on several 5-substituted isatins to investigate the stability of sulfide adducts at C-3. The inhibitors were prepared by general synthetic methods, starting with commercially available 5-substituted isatins in nearly every case. All compounds were tested for inhibition of purified HRV-14 3CP. Compounds 8, 14, and 19 were found to have excellent selectivity for HRV-14 3CP compared to other proteolytic enzymes, including chymotrypsin and cathepsin B. Selected compounds were assayed for antiviral activity against HRV-14-infected HI-HeLa cells. A 2.8 A cocrystal structure of derivative 19 covalently bound to human rhinovirus-serotype 2 (HRV-2) 3CP was solved and revealed that the isatin was situated in essentially the same conformation as modeled.

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, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements.

The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC90 approximately 0.10 microM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.