Diana R. Cundell

Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcβ1‐3Gal, GalNAcβ1‐4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H2O2 and CO2. This mutant produced decreased concentrations of H2O2 and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down‐regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo

PAF Receptor Anchors Streptococcus Pneumoniae to Activated Human Endothelial Cells

Streptococcus pneumoniae can produce asymptomatic colonization or aggressive sepsis. We sought to differentiate the molecular mechanisms of these disparate courses. Cytokine or thrombin activation of human vascular endothelial cells and type II pneumocytes enhanced pneumococcal adherence relative to resting cells. Adherence and subsequent invasion was dramatically reduced by PAF receptor antagonists. Cells transfected with the PAF receptor gained the ability to support pneumococcal adherence. PAF or PAF receptor antagonists inhibited attachment and invasion. Adherence involved phosphorylcholine on the pneumococcal teichoic acid. Virulent pneumococci target the PAF receptor on activated human cells, a necessary step to facilitate subsequent invasion.

The effect of aerial parts of Echinacea on the circulating white cell levels and selected immune functions of the aging male Sprague–Dawley rat

Echinacea is one of the most widely used over-the-counter herbal preparations that purport to improve immune system function, especially when taken as a short course of therapy (6-8 weeks). Since many purchasers are older individuals, a double-blind, placebo-controlled study was performed to investigate whether Echinacea could affect total and differential white cell counts, phagocytic activity and interleukin (IL-2) levels in 12-month-old, healthy, male Sprague-Dawley rats when administered over an 8-week period. Echinacea (50 mg/kg of aerial parts) mixed with peanut butter or peanut butter alone was fed to 16 rats, which were receiving regular food and water ad libitum. Cell counts and immune functions were determined on rat tail vein blood on a weekly basis. Echinacea significantly increased circulating total white cell counts during the first 2 weeks of administration, and IL-2 levels during the final 5 weeks of the study period (p<0.05). Differential counts were altered during the entire 8-week study, with mononuclear cells significantly increased to the detriment of granulocytes (p<0.05). No such changes were observed in animals given peanut butter alone. No difference was observed in phagocytic function between animals given Echinacea or peanut butter alone. These studies suggest that aerial components of Echinacea affect both mononuclear cell levels and circulating IL-2 levels in older animals.

Generation of an intelligent medical system, using a real database, to diagnose bacterial infection in hospitalized patients.

The initial diagnosis of bacterial infections in the absence of laboratory microbiological data requires physicians to use clinical algorithms based on symptoms, patient history and infection site. Optimization of such algorithms would be achieved by including as many variables associated with bacterial infection as possible. Demographic data are easily available and frequently used to sub-group human populations. A prospective investigation was, therefore, undertaken to examine the influence of demographic variables on bacterial infection rates, using data obtained from 173 patients presenting to Albert Einstein Medical Center. Data was randomly selected from 149 of these patients and used to generate fuzzy rules to model an intelligent medical system. To test the accuracy of this system at determining bacterial infection, based solely on demographic data, the program was given the remaining 24 patients information. All 18 patients with either streptococcal, staphylococcal or Escherichia coli infections were correctly diagnosed. Non-E.coli GNR were misdiagnosed as E. coli infections in two patients resulting in an overall prediction rate for the 24 patients of 91.66%. This study suggests that the direct correlation of demographic variables with a predisposition to bacterial infection allow the design of an intelligent medical system, which shows great future potential as a diagnostic tool for all physicians.

Development of Anti-Adhesion Carbohydrate Drugs for Clinical Use

Specific adherence of many human pathogens is mediated by microbial adhesin proteins that recognize sugar chains attached to host cell surface glycoconjugates. More than twenty years ago a strategy was proposed for preventing or interrupting the progression of bacterial and viral infection by using soluble monosaccharides or oligosaccharides as competitive inhibitors of microbial adherence. Since then, investigations from many laboratories into the detailed structures of both microbial adhesin molecules and the carbohydrate epitopes they recognize on mammalian cells have at least partially elucidated the molecular basis for carbohydrate mediated adherence for dozens of microbial species. As new enzyme-based technologies for manufacturing oligosaccharides have emerged during the past few years, commercial scale production of oligosaccharides has become economically feasible. Proceeding from this scientific and technological platform, we are developing a class of drugs known as carbohydrate adhesion ligand homologs - inhibitors of microbial adherence comprised of free oligosaccharides chemically identical to sugar chain adhesin ligands carried on surface glycoconjugates of host mucosal cells.

Using fuzzy sets to analyze putative correlates between age, blood type, gender and/or race with bacterial infection

Previous studies have suggested that the demographic variables of age and blood type may serve as risk factors for infection by specific bacterial species. Since both demographic variables and bacterial species are defined using generally accepted parameters, they constitute highly suitable variables for the generation of a fuzzy logic program. A prospective study was therefore undertaken to examine the influence of age, blood type, gender and race on bacterial infection rates using a real database generated from 187 bacteremic patients admitted to Albert Einstein Medical Center. A fuzzy logic program was created using 155 randomly selected patients data with four input (demographic variables) and four output classes (infections with staphylococci, streptococci, Escherichia coli or non-E. coli gram negative rods (non-E.coli GNR)). To see whether bacterial infection could be predicted based on demographic data alone, the program was tested using the remaining 32 patients data. The program was able to correctly determine the bacterial output group of 27 of 32 randomly selected patients, giving an overall correlation of 84.38%. This study suggests that the direct correlation of demographic variables with a predisposition to bacterial infection allow the design of an intelligent medical system, which shows great future potential as a powerful diagnostic tool for all physicians.

An investigation of differential skin colonization of neonates by staphylococci, using an artificial neural network-based system

An intelligent system to examine the influence of the demographic variables of gender, blood type, and race on the distribution of skin staphylococci bacteria on neonates was designed. The system was based on a supervised neural network. Our study suggests that demographic variables are major factors influencing staphylococcal distribution.

Using Intelligent Systems in Predictions of the Bacterial Causative Agent of an Infection

In this study, we designed a fuzzy logic system to examine the influence of the demographic variables of age, blood type, gender and race on bacterial infection rates using a medical database assembled over 17 months from patients presenting to Albert Einstein Medical Center. The intelligent system was created using 155 patients, randomly selected from the database, and consisted of four input categories of demographic variables and four output categories of bacterial infection (streptococci, staphylococci, Escherichia coli and non-E. coli gram negative rods). The remaining 32 patients were used to assess the programs ability to correctly determine bacterial infection when provided only with demographic data. Our intelligent system correctly assigned the bacterial output group in 27 of these 32 patients, giving an overall correlation of 84.4%. These studies suggest that demographic variables are major factors influencing bacterial infection. Such a system may, therefore, hold promise as a diagnostic tool.