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Biomedical subjects

T Brennan

Publications and source records attributed to T Brennan.

At least 19 recordsLinked to original sources

Delay in seeking emergency care.

OBJECTIVE: To determine whether patient clinical and socioeconomic characteristics predict patient delay in coming to the emergency department (ED). METHODS: Adult ED patients at five urban teaching hospitals were surveyed regarding self-reported delay in coming to the ED. Delay was measured by self-perception as well as by the number of days ill and unable to work. Patient socioeconomic and clinical characteristics were obtained by survey questionnaire and chart review. Cross-sectional analysis within a prospective study of 4,094 consecutive patients was performed using a subset of 1,920 patients (84% eligible rate) to whom questionnaires were administered. RESULTS: Overall, 32% of the patients completing the survey reported delay in seeking ED care. Of these patients reporting delay, 71% thought their problem would go away or was not serious. Patients who were older, had higher acuity, or were frequent ED users reported less delay in coming to the ED, while patients without a regular physician or who were African American reported more delay. Perception of increased number of days ill prior to visiting the ED was reported by frequent ED users and those with worse baseline physical function, while patients who had higher acuity reported fewer days ill prior to coming to the ED. CONCLUSIONS: A patient's decision to delay coming to the ED often reflects a belief that his or her illness is either self-limited or not serious. The decision to delay correlates with patient characteristics and access to a regular physician. The correlates of delay in seeking ED care may depend on the delay measure used. Better understanding of patients at risk for delaying care may influence interventions to reduce delay.

Adult↗

West Nile virus serosurvey and assessment of personal prevention efforts in an area with intense epizootic activity: Connecticut, 2000.

West Nile virus (WNV) can cause large outbreaks of febrile illness and severe neurologic disease. This study estimates the seroprevalence of WNV infection and assesses risk perception and practices regarding potential exposures to mosquitoes of persons in an area with intense epizootics in 1999 and 2000. A serosurvey of persons aged > or = 12 years was conducted in southwestern Connecticut during October 10-15, 2000, using household-based stratified cluster sampling. Participants completed a questionnaire regarding concern for and personal measures taken with respect to WNV and provided a blood sample for WNV testing. Seven hundred thirty persons from 645 households participated. No person tested positive for WNV (95% CI: 0-0.5%). Overall, 44% of persons used mosquito repellent, 56% practiced > or = two personal precautions to avoid mosquitoes, and 61% of households did > or = two mosquito-source reduction activities. In multivariate analyses, using mosquito repellent was associated with age < 50 years, using English as the primary language in the home, being worried about WNV, being a little worried about pesticides, and finding mosquitoes frequently in the home (P<0.05). Females (OR = 2.0; CI = 1.2-2.9) and persons very worried about WNV (OR = 3.8; CI = 2.2-6.5) were more likely to practice > or = two personal precautions. Taking > or = two mosquito source reductions was associated with persons with English as the primary language (OR = 2.0; CI = 1.1-3.5) and finding a dead bird on the property (OR = 1.8; CI = 1.1-2.8). An intense epizootic can occur in an area without having a high risk for infection to humans. A better understanding of why certain people do not take personal protective measures, especially among those aged > or = 50 years and those whose primary language is not English, might be needed if educational campaigns are to prevent future WNV outbreaks.

Adolescent↗

Utilization of new technologies in drug trials and discovery.

It has become widely accepted that individual genetic variation is a prime determinant in both disease susceptibility and toxic response to therapeutic agents and xenobiotics. Emerging genetic sequence data and phenotype association studies are expected to enable disease risk prediction and guide subsequent therapeutic approaches in individual cases. However, making a good match between an individual genetic profile, disease risk prediction, and appropriate therapeutic intervention will require genotyping many polymorphic sites in large numbers of genes or single nucleotide polymorphism sites throughout the genome. Additionally, each polymorphism will have to be associated with a phenotype. Presumably, a composite phenotype may be predicted by integrating anticipated contributions from each polymorphism contributing to the complex genotype. Methods for executing such large-scale genotyping studies are rapidly evolving and becoming available. DNA microarray technology applied in hybridization-based genotyping assays is particularly well suited to respond to the accelerating pace of polymorphism discovery and the associated demand for highly parallel genotyping capability.

Base Sequence↗

Two-dimensional parallel array technology as a new approach to automated combinatorial solid-phase organic synthesis

An automated, 96-well parallel array synthesizer for solid-phase organic synthesis has been designed and constructed. The instrument employs a unique reagent array delivery format, in which each reagent utilized has a dedicated plumbing system. An inert atmosphere is maintained during all phases of a synthesis, and temperature can be controlled via a thermal transfer plate which holds the injection molded reaction block. The reaction plate assembly slides in the X-axis direction, while eight nozzle blocks holding the reagent lines slide in the Y-axis direction, allowing for the extremely rapid delivery of any of 64 reagents to 96 wells. In addition, there are six banks of fixed nozzle blocks, which deliver the same reagent or solvent to eight wells at once, for a total of 72 possible reagents. The instrument is controlled by software which allows the straightforward programming of the synthesis of a larger number of compounds. This is accomplished by supplying a general synthetic procedure in the form of a command file, which calls upon certain reagents to be added to specific wells via lookup in a sequence file. The bottle position, flow rate, and concentration of each reagent is stored in a separate reagent table file. To demonstrate the utility of the parallel array synthesizer, a small combinatorial library of hydroxamic acids was prepared in high throughput mode for biological screening. Approximately 1300 compounds were prepared on a 10 μmole scale (3-5 mg) in a few weeks. The resulting crude compounds were generally >80% pure, and were utilized directly for high throughput screening in antibacterial assays. Several active wells were found, and the activity was verified by solution-phase synthesis of analytically pure material, indicating that the system described herein is an efficient means for the parallel synthesis of compounds for lead discovery. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗

Membrane compartmentation is required for efficient T cell activation.

The plasma membrane of mammalian cells contains detergent-resistant membrane rafts enriched in glycosphingolipids and cholesterol. Although several important signaling molecules have been found in such rafts, evidence documenting a functional role for their localization has been scarce. Using a fractionation scheme that preserves tyrosine phosphorylation, we show that T cell activation leads to a striking compartmentation in the rafts of activated T cell receptor and associated signal-transducing molecules. Conditions that reversibly disrupt raft structure either by dispersing their contents or by forcing their internalization reversibly disrupt the earliest steps of T cell activation. Thus, raft integrity is a prerequisite for efficient T cell receptor signal transduction.

Animals↗

The nucleotide sequence of Saccharomyces cerevisiae chromosome V.

Here we report the sequence of 569,202 base pairs of Saccharomyces cerevisiae chromosome V. Analysis of the sequence revealed a centromere, two telomeres and 271 open reading frames (ORFs) plus 13 tRNAs and four small nuclear RNAs. There are two Tyl transposable elements, each of which contains an ORF (included in the count of 271). Of the ORFs, 78 (29%) are new, 81 (30%) have potential homologues in the public databases, and 112 (41%) are previously characterized yeast genes.

Base Sequence↗

Virus-induced cytokines regulate circulating lymphocyte levels during primary SIV infections.

Decline in blood CD4+ lymphocytes during primary symptomatic infections with HIV is usually attributed to viral killing, and has not been considered in terms of altered lymphocyte migration and sequestration. We therefore sought to examine whether CD4+ cell loss from blood of macaques undergoing an acute primary SIV infection might be due to increased synthesis of cytokines, known to profoundly affect lymphocyte trafficking, rather than to direct lymphocyte destruction by virus. The findings indicate that rapid lymphocyte depletion following acute infection is not selective for CD4+ cells, correlates precisely with increased plasma IFN-gamma and tumor necrosis factor-alpha levels, and is reversible. CD4/CD8 ratios in lymph nodes with high viral burdens remain relatively unchanged despite lymphocyte loss from blood. Levels of cytokine mRNA measured in lymphoid organs reflect neither cytokine plasma levels nor their potential to induce sequestration. These results support a model of cytokine-induced lymphocyte extravasation to account for the acute HIV/SIV-induced CD4+ cell lymphopenia and raise questions regarding the extent to which altered lymphocyte migration plays a role in the gradual CD4+ cell depletion throughout infection.

Acute Disease↗

Characteristics of the CD8+ lymphocytosis during primary simian immunodeficiency virus infections.

OBJECTIVE: To investigate the source of the expanded blood CD8+ subsets during an acute primary simian immunodeficiency virus (SIV) infection of macaques and the potential role of these cells in disease progression. DESIGN AND METHODS: The primary CD8+ lymphocytosis, which occurs at 1-2 weeks following infection with SIVsmm/PBj-14, was examined in rhesus and cynomolgus macaques. Extensive subset analysis of the expanded blood CD8+ cell pool in a rhesus macaque was compared phenotypically with those in thymus, lymph nodes, spleen, ileum and lung washouts obtained at necropsy during blood lymphocytosis. The influence of the primary CD8+ cells expansion on disease progression was assessed at days 175-679 post-infection in long-term PBj-14 survivors staged according to immunological, virological and histopathological changes in their lymphoid organs. RESULT: The very rapid and transient blood lymphocytosis following infection consisted of two distinct CD45RA(low), CD8+ and CD28-, lymphocyte function-associated antigen (LFA)-1(high), CD45RA(high), CD8+ populations. These populations were present in low levels in thymus, lymph and spleen but were highly represented in mucosal tissues, such as long washout, in which CD28- LFA-1(high) CD45RA(high) CD8+ cells comprised 86% of CD8+ cells, and gut, which was predominantly CD45RA(low) CD28- CD8+ cells. A comparison of progressor and non-progressor PBj-14-infected rhesus and cynomolgus macaques also indicated that the existence or magnitude of a blood CD8+ lymphocytosis during the acute phase of infection did not by itself appear to influence or be predictive of disease progression. CONCLUSION: The marked blood CD8+ lymphocytosis observed during acute SIV infection did not result from expansion of virus-specific precursors in peripheral lymph node and did not appear to influence the rate of disease progression. The findings provide a novel explanation for the primary CD8+ cell lymphocytosis and invoke a mechanism whereby virus-induced cytokine/chemokine production in mucosal sites initiate the transient migration of a pre-existing CD8+ population into the blood from compartments such as lung and gut. Such results suggest that the magnitude of lymphocytosis may depend on the level of viral replication in mucosal tissues and the presence of other infections, for example, cytomegalovirus.

Animals↗

Deconvolution of combinatorial libraries for drug discovery: experimental comparison of pooling strategies.

An experimental evaluation of several different pooling strategies for combinatorial libraries was conducted using a library of 810 compounds and an enzyme inhibition assay (phospholipase A2). The library contained compounds with varying degrees of activity as well as inactive compounds. The compounds were synthesized in groups of three and pooled together in various formats to realize different pooling strategies. With one exception, all iterative deconvolution strategies and position scanning resulted in identification of the same compound. The results are in good agreement with the predicted outcome from theoretical and computational methods. These data support the tenet that active compounds for pharmaceutically relevant targets can be successfully identified from combinatorial libraries organized in mixtures.

Drug Evaluation, Preclinical↗

Deletion of the nef gene abrogates the ability of SIV smmPBj to induce acutely lethal disease in pigtail macaques.

Simian immunodeficiency virus (SIV) infection in macaque species is typically associated with the development of a progressive immunodeficiency disease, similar to human AIDS, resulting in death of animals in months to years after infection. In contrast, a variant virus, termed SIVsmmPBj, induces an acute disease in macaques, resulting in death in 5 to 14 days after infection. Previously, we have shown that several viral determinants contribute to the pathogenesis of this disease. The present study was undertaken to evaluate the role of Nef in the pathogenesis of SIVsmmPBj-induced acute disease. A molecular clone of SIVsmmPBj was generated that contains a deletion in the nef coding region (PBj6.6 delta nef). Virus derived from this molecular clone was tested with the parental virus, PBj6.6, in replication studies in pigtail macaque and rhesus macaque peripheral blood mononuclear cells (PBMCs). In general, PBj6.6 delta nef displayed markedly reduced replication abilities when compared with PBj6.6; the only exception being in stimulated pigtail macaque PBMCs, where replication kinetics were nearly identical. In addition, PBj6.6 delta nef was unable to induce the proliferation of peripheral blood mononuclear cells (PBMCs) in vitro, a unique characteristic of acutely pathogenic SIVsmmPBj. Inoculation of this virus into pigtail macaques resulted in infection, but did not result in any detectable acute disease. These studies suggest that Nef is an important viral determinant in the pathogenesis of SIVsmmPBj-induced disease, and further suggest that Nef plays a significant role in viral replication in vivo.

Amino Acid Sequence↗

An automated multiplex oligonucleotide synthesizer: development of high-throughput, low-cost DNA synthesis.

An automated oligonucleotide synthesizer has been developed that can simultaneously and rapidly synthesize up to 96 different oligonucleotides in a 96-well microtiter format using phosphoramidite synthesis chemistry. A modified 96-well plate is positioned under reagent valve banks, and appropriate reagents are delivered into individual wells containing the growing oligonucleotide chain, which is bound to a solid support. Each well has a filter bottom that enables the removal of spent reagents while retaining the solid support matrix. A seal design is employed to control synthesis environment and the entire instrument is automated via computer control. Synthesis cycle times for 96 couplings are < 11 min, allowing a plate of 96 20-mers to be synthesized in < 5 hr. Oligonucleotide synthesis quality is comparable to commercial machines, with average coupling efficiencies routinely > 98% across the entire 96-well plate. No significant well-to-well variations in synthesis quality have been observed in > 6000 oligonucleotides synthesized to date. The reduced reagent usage and increased capacity allow the overall synthesis cost to drop by at least a factor of 10. With the development of this instrument, it is now practical and cost-effective to synthesize thousands to tens of thousands of oligonucleotides.

Amides↗

Synthesis, antiviral activity and enzymatic phosphorylation of 9-phosphonopentenyl derivatives of guanine.

(E)-9-(5-Phosphonopent-4-enyl)guanine and (E)-9-[3-(hydroxymethyl)-5- phosphonopent-4-enyl]guanine which bear a vinyl phosphonate moiety as a mimic of the phosphate group were synthesized. Their activities against human immunodeficiency virus type-1 (HIV-1), herpes simplex virus type-1 (HSV-1) and human cytomegalovirus (HCMV) were evaluated in vitro in parallel with those of 9-(5-phosphonopentyl)guanine and 9-(5,5-difluoro-5- phosphonopentyl)guanine. Both vinyl phosphonates exhibited anti-HIV-1 and anti-HCMV activities, whereas the methyl- and difluoromethyl phosphonate analogues were inactive. The selectivity index, calculated as the ratio of the toxicity for the host cells (50% reduction in cell viability or in [methyl-3H]thymidine incorporation) to the 50% inhibitory concentration for HIV-1 replication, was the highest for (E)-9-[3-(hydroxymethyl)-5-phosphonopent-4-enyl]guanine. The acyclonucleotide analogues were also studied as substrates of guanylate kinase, an enzyme believed to play a critical role in the conversion of acyclic phosphate and phosphonate derivatives of guanine to their antivirally active diphosphate derivatives. (E)-9-(5-Phosphonopent-4- enyl)guanine and (E)-9-[3-(hydroxymethyl)-5-phosphonopent-4-enyl]guanine were good substrates of guanylate kinase, being phosphorylated with efficiencies of 14 and 36% of that determined for GMP, respectively. These results contrast with the poor efficiency found for 9-(5-phosphonopentyl)guanine (0.3%) and the lack of phosphorylation of 9-(5,5-difluoro-5-phosphonopentyl)guanine by guanylate kinase (Navé et al. (1992) Arch. Biochem. Biophys. 295, 253-257). The role of the vinyl phosphonate group in the expression of the anti-HIV-1 activity of the phosphonopentenyl derivatives of guanine is discussed.

Animals↗