Effects of endogenous D-alanine synthesis and autoinhibition of Bacillus anthracis germination on in vitro and in vivo infections

Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous D-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous D-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with D-alanine production, was prevented by an alanine racemase inhibitor, and required L-alanine. Interestingly, endogenous production of inhibitory levels of D-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of D-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of D-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.     

Herpes simplex virus infection causes cellular beta-amyloid accumulation and secretase upregulation

It is uncertain whether environmental factors contribute to the formation of senile plaques and neurofibrillary tangles, the abnormal features that define the Alzheimer's disease (AD) brain. We previously proposed that herpes simplex virus type 1 (HSV1) is a strong risk factor for AD when it is present in the brains of people who possess the type 4 allele of the apolipoprotein E gene (APOE-epsilon4); however a direct biochemical link between viral infection and the development of the AD pathological features has never previously been examined. Here we show that infection of cultured neuronal and glial cells with HSV1 leads to a dramatic increase in the intracellular levels of beta-amyloid (Abeta) 1-40 and 1-42, whilst levels of amyloid precursor protein (APP) in cells decrease. Similarly, Abeta1-42 deposits are present in mouse brain after HSV1 infection. In the cultured cells the mechanism involves increased Abeta production, rather than merely greater retention of cellular Abeta, as levels of beta-site APP-cleaving enzyme (BACE-1) and of nicastrin, a component of gamma-secretase, both increase in HSV1-infected cells. These novel data show that HSV1 can directly contribute to the development of senile plaques.     

Understanding respiratory syncytial virus (RSV) vaccine-enhanced disease

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and children worldwide. In addition, RSV causes serious disease in elderly and immune compromised individuals. RSV infection of children previously immunized with a formalin-inactivated (FI)-RSV vaccine is associated with enhanced disease and pulmonary eosinophilia that is believed to be due to an exaggerated memory Th2 response. As a consequence, there is currently no licensed RSV vaccine and detailed studies directed towards prevention of vaccine-associated disease are a critical first step in the development of a safe and effective vaccine. The BALB/c mouse model of RSV infection faithfully mimics the human respiratory disease. Mice previously immunized with either FI-RSV or a recombinant vaccinia virus (vv) that expresses the attachment (G) glycoprotein exhibit extensive lung inflammation and injury, pulmonary eosinophilia, and enhanced disease following challenge RSV infection. CD4 T cells secreting Th2 cytokines are necessary for this response because their depletion eliminates eosinophilia. Intriguing recent studies have demonstrated that RSV-specific CD8 T cells can inhibit Th2-mediated pulmonary eosinophilia in vvG-primed mice by as yet unknown mechanisms. Information gained from the animal models will provide important information and novel approaches for the rational design of a safe and efficacious RSV vaccine.     

Biophysical characterization of vaccinia virus thymidine kinase substrate utilization

To provide information for the development of new antiviral compounds that inhibit orthopoxviruses, further characterization of the kinetics and thermodynamics that underlie substrate utilization reactions of vaccinia virus thymidine kinase (VVTK) has been undertaken. The kinetics of 2'deoxythymidine phosphorylation by VVTK and the thermodynamics of complex formation between VVTK and the substrate 2' deoxythymidine were determined using spectroscopic and calorimetric techniques. These studies demonstrated that kinetic parameters for 2' deoxythymidine phosphorylation by VVTK were 25 microM and 0.2s(-1) for K(m) and k(cat), respectively. The enthalpy change, Delta H, for the enzyme catalyzed reaction is -18.1 kcal/mol. Thermodynamic studies for the formation of the enzyme substrate complex demonstrated a binding affinity (K(a)) of 4 x 10(4)M(-1), an enthalpy change for binding (Delta H) of -17.4 kcal/mol, and a reaction stoichiometry of two molecules of substrate binding to each enzyme tetramer. Kinetic and thermodynamic data were in agreement (K(a) approximately 1/K(m)) and showed similarities to literature values reported for herpes simplex virus thymidine kinase (HSV-TK) and human thymidine kinase 1 (hTK1) with respect to k(cat) but not with respect to K(m). The K(m) value found for VVTK in this study is nearly two orders of magnitude larger than the values reported for the hTK1 and the HSV TK enzymes.     

The Effect of Soluble Peptide Sequences on Neurite Extension on 2D Collagen Substrates and Within 3D Collagen Gels

The extension of neurites on 2D collagen-coated substrates and within 3D collagen gels is based on various chemical and mechanical environmental factors. However, extrapolating results from 2D studies to 3D environments are difficult, especially with regard to neural outgrowth. The aim of this study was to investigate the effects of inhibitory molecules on nerve growth in 3D environments as compared to 2D surfaces. E9 chick dorsal root ganglion cells were seeded within collagen gels as well as onto collagen-coated glass and were exposed, for 24 h, to one of three experimental peptide sequences; arginine-glycine-aspartic acid-threonine (RGDT), cyclo(RGD-d-Phe-Val) (cRGD), or aspartic acid-glycine-glutamic acid-alanine (DGEA). In 3D collagen gels, only the cRGD peptide sequence reduced neurite extension across a variety of gel concentrations. In contrast, on 2D surfaces, both RGD peptides reduced the number of cells expressing neurites, but cRGD still exhibited superior inhibition of neurite expression. Further evaluation of cRGD results revealed that the peptide altered neurite growth vs. stiffness to a more linear relationship that is more typical of non-adhesive environments. Overall, these results further demonstrate the importance of peptide confirmation and sequence when investigating cell behavior in 3D environments.     

IgE expression pattern in lung: relation to systemic IgE and asthma phenotypes

BACKGROUND: IgE-mediated responses contribute to allergy and asthma. Little is understood regarding the relationship of tissue IgE to systemic IgE, inflammation or clinical outcomes. OBJECTIVES: To evaluate local IgE expression and cellular inflammation in the proximal and distal lung of normal subjects and subjects with asthma of varying severity and relate those tissue parameters to systemic IgE levels, atopy, lung function, and history of severe exacerbations of asthma. METHODS: Tissue from more than 90 subjects with eosinophilic (SAeo(+)) and noneosinophilic (SAeo(-)) severe asthma, mild asthma and normal subjects were immunostained for IgE, signal-amplifying isoform of IgE receptor (FcepsilonRIbeta) and markers of mast cells, eosinophils, and lymphocytes. Tissue expression of IgE, FcepsilonRIbeta, cellular inflammation, serum IgE, and atopy were compared. Regression models were used to determine the relationship of local and systemic IgE to lung function and severe exacerbations of asthma. RESULTS: Mast cell-bound IgE was present along airways but absent in lung parenchyma. Although the groups were similar in systemic/serum IgE and atopy, local/tissue IgE was highest in SAeo(+) and correlated with eosinophils and lymphocytes (r(s) = 0.52, P < .0001; and r(s) = 0.23, P = .03, respectively). Higher local IgE was associated with better lung function, but also with more severe exacerbations of asthma. CONCLUSION: Local IgE appears to be primarily a component of responses within the mucosal immune compartment and is related to cellular inflammation, lung function, and clinical outcomes in asthma. CLINICAL IMPLICATIONS: Local/airway IgE-related processes rather than systemic markers of atopy may be relevant in determining clinical outcomes in asthma.     

Kinetic Modeling of Contrast-Enhanced MRI: An Automated Technique for Assessing Inflammation in the Rheumatoid Arthritis Wrist

In recent years, development of rheumatoid arthritis (RA) drug therapy has been more directly targeted to counteract specific mechanisms of inflammation, and it is now believed that early aggressive treatment with disease modifying drugs is important to inhibit future structural joint damage. The development of these new treatments has increased the need for methodologies to assess disease activity in RA and monitor the effectiveness of drug therapy. Unlike X-ray, which shows only structural bone damage, magnetic resonance imaging (MRI) can depict soft tissue damage and synovitis, the primary pathology of RA. Recent studies have also indicated that MRI is sensitive to pathophysiologic changes that may predate radiographic erosions and may predict future joint damage. In this study, we have developed a computer automated analysis technique for MR wrist images that provides an objective measure of RA synovitis. This method applies a two-compartment pharmacokinetic model to every voxel of a dynamic contrast-enhanced MRI (DCE-MRI) dataset and outputs resulting parametric images. The aim of this technique is to not only objectively quantify the severity of rheumatoid synovitis, but to also locally determine where areas of serious disease activity are situated through kinetic modeling of blood-tissue exchange. Preliminary results show good correlation to early enhancement rate, which has previously been shown to be a useful clinical marker of RA activity. However, the use of tracer kinetic modeling methods potentially provides more specific information regarding underlying RA physiology. This approach could provide a useful new tool in RA patient management and could substantially improve RA therapeutic studies by calculating objective biomarkers of the disease state.     

Hardware assessment using the multi-module, multi-resolution system (M3R): a signal-detection study

The multi-module, multi-resolution system (M3R) is used for hardware assessment in objective, task-based signal detection studies in projection data. A phantom capable of generating multiple realizations of a random textured background is introduced. Measured backgrounds from this phantom are used along with simulated lumpy and uniform backgrounds to investigate signal-to-noise ratio as a function of exposure time. Results are shown to agree with theoretical predictions, exhibiting a power-law like dependence previously seen for studies performed either in simulation or without an imaging system, and help validate the use of simulated lumpy backgrounds in observer studies. A second study looks at signal-detection performance, measured by AUC (area under the receiver operating characteristic curve), in lumpy backgrounds for 20 M3R aperture combinations as a function of lump size and signal size. Observer performance reveals an improvement in AUC for certain ranges of signal and lump combinations through the use of multiplexed, multiple-pinhole apertures, indicating a need for task-specific aperture optimization. The channelized Hotelling observer is used with Laguerre-Gauss channels for both observer studies. Methods for selection of number of channels and channel width are discussed.