Computer Algorithms To Detect Bloodstream Infections

Automated bloodstream infection surveillance using electronic data is an accurate alternative to surveillance using manually collected data

Phosphorylation Site Mutations Affect Herpes Simplex Virus Type 1 ICP0 Function

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol. 76:1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos 1 (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function

Development of a Clinical Data Warehouse for Hospital Infection Control

Existing data stored in a hospital's transactional servers have enormous potential to improve performance measurement and health care quality. Accessing, organizing, and using these data to support research and quality improvement projects are evolving challenges for hospital systems. The authors report development of a clinical data warehouse that they created by importing data from the information systems of three affiliated public hospitals. They describe their methodology; difficulties encountered; responses from administrators, computer specialists, and clinicians; and the steps taken to capture and store patient-level data. The authors provide examples of their use of the clinical data warehouse to monitor antimicrobial resistance, to measure antimicrobial use, to detect hospital-acquired bloodstream infections, to measure the cost of infections, and to detect antimicrobial prescribing errors. In addition, they estimate the amount of time and money saved and the increased precision achieved through the practical application of the data warehouse

US28 Is a Potent Activator of Phospholipase C during HCMV Infection of Clinically Relevant Target Cells

<div><p>Members of the cytomegalovirus family each encode two or more genes with significant homology to G-protein coupled receptors (GPCRs). In rodent models of pathogenesis, these viral encoded GPCRs play functionally significant roles, as their deletion results in crippled viruses that cannot traffic properly and/or replicate in virally important target cells. Of the four HCMV encoded GPCRs, US28 has garnered the most attention due to the fact that it exhibits both agonist-independent and agonist-dependent signaling activity and has been demonstrated to promote cellular migration and proliferation. Thus, it appears that the CMV GPCRs play important roles in viral replication <em>in vivo</em> as well as promote the development of virus-associated pathology. In the current study we have utilized a series of HCMV/US28 recombinants to investigate the expression profile and signaling activities of US28 in a number of cell types relevant to HCMV infection including smooth muscle cells, endothelial cells and cells derived from glioblastoma multiforme (GBM) tumors. The results indicate that US28 is expressed and exhibits constitutive agonist-independent signaling activity through PLC-β in all cell types tested. Moreover, while CCL5/RANTES and CX3CL1/Fractalkine both promote US28-dependent Ca⁺⁺ release in smooth muscle cells, this agonist-dependent effect appears to be cell-specific as we fail to detect US28 driven Ca⁺⁺ release in the GBM cells. We have also investigated the effects of US28 on signaling via endogenous GPCRs including those in the LPA receptor family. Our data indicate that US28 can enhance signaling via endogenous LPA receptors. Taken together, our results indicate that US28 induces a variety of signaling events in all cell types tested suggesting that US28 signaling likely plays a significant role during HCMV infection and dissemination <em>in vivo</em>.</p> </div

Lack of genetic influence on the innate inflammatory response to Helicobacter infection of the gastric mucosa

Helicobacter pylori (H. pylori) is a bacterial pathogen that resides at the gastric mucosa and has a world-wide prevalence of over 50%. Infection usually lasts for the life of the host, and although all infected individuals will develop histologic gastritis only a subset will develop symptomatic gastritis, peptic ulcer disease, gastric MALT lymphoma, or gastric adenocarcinoma. The bacterial and host factors that determine clinical outcome and influence the development of widely varying diseases have not been elucidated. We compared disease in Helicobacter-infected SCID mice on different genetic backgrounds with their corresponding immunocompetent partners to determine if the genetics of the host significantly impacts the innate inflammatory outcome, independent of variations in bacterial virulence factors. BALB/c SCID and C57BL/6 SCID mice developed equivalent histologic gastritis by eight weeks of infection. Immunocompetent BALB/c mice and C57BL/6 mice developed significantly lower or higher degrees of inflammation respectively. Innate inflammation in immunodeficient mice on the C57BL/6 background remained low even in the absence of the regulatory cytokine IL-10. These results demonstrate that adaptive immunity is not required for the generation of low level inflammation in response to Helicobacter infection and that the degree of inflammation is consistent among different genetic backgrounds. Additionally, this inflammation is limited even in the absence of regulatory T cells

US28 potentiates signaling via endogenous Gαi coupled LPA receptors in HCMV infected arterial smooth muscle cells.

<p>A) HASMCs were infected with the indicated viruses at a MOI of 3. At 48 hpi, cells were labeled with Fluo-4 AM, stimulated with 1uM lysophosphatidic acid (LPA) and calcium flux was measured using a FlexStation II fluorometer. The results are also presented graphically (B) The results are presented graphically and represent the mean+/−S.E. of three to seven independent experiments performed in duplicate. C) To determine if the US28 effect was due to LPA signaling through endogenous Gαi proteins, we pretreated cells with the Gαi inhibitor PTx prior to stimulation with LPA. PTx completely abrogated the effects of US28 on LPA signaling indicating the involvement of Gαi in this process. The calcium traces represent change in relative fluorescence units (ΔRFU) for each condition tested and are representative of three to seven independent experiments performed in duplicate. **p<0.05..</p

US28 is a potent activator of signaling even at very low expression levels.

<p>A) Development of a HEK-based Tet-regulatable US28 expression system and analysis of DOX regulated changes in US28 protein levels. US28 expression was titrated using DOX concentrations ranging from 1 pg/ml to 1000 pg/ml. pUS28 expression was analyzed by western blot and the results indicate that 100 pg/ml DOX blocks US28 expression >90%, while 1000 pg/ml DOX blocks US28 expression >99%. Cellular GAPDH expression in whole cell lysates is shown for control purposes. B) US28 stimulated PLC-β activity was then analyzed over the range of pUS28 expression levels. Interestingly, while 100 pg/ml DOX blocks pUS28 expression >90%, the corresponding drop in US28 stimulated PLC-β was ∼45%. Similarly, while 1000 pg/ml blocks pUS28 expression >99%, the corresponding drop in US28 stimulated PLC-β was ∼75%. CCL5/RANTES was used at a concentration of 10 nM.</p

US28 expression and PLC-β signaling in fibroblasts and endothelial cells infected with TB40/E recombinant viruses.

<p>A) HS68 HFFs infected at MOIs of 0.2, 1, and 5 (left panel) or HUVECs infected at MOIs of 0.6, 3, and 15 (right panel) were analyzed by western blot for IE1/2 or β-actin expression at 48 hpi. B) Extracts prepared from HFFs infected at a MOI of 5 (left panel) or HUVECs infected at a MOI of 15 (right panel) were analyzed by immunoprecipitation/western blot for pUS28 expression at 48 hpi. C) HS68 HFFs (left panel) or HUVECs (right panel) infected with increasing MOIs as described in panel A were analyzed for PLC-β activity at 48 hpi. In each cell type, PLC-β activation was measured by labeling cells 24 hpi with 1 µCi/ml 3H-myoinositol followed by isolation of total inositol phosphates at 48 hpi. PLC-β activity is represented as the percent conversion of input myoinositol into inositol phosphates.</p

pUS28 is expressed in smooth muscle and glioblastoma cells.

<p>A) HASMCs and U373MG were infected with FIX-US28Flag at a MOI of 3 and pUS28 expression was analyzed by immunoprecipitation/western blot at the indicated times post-infection (upper panels). IE1/IE2 expression was analyzed by western blot and is shown for reference (lower panels). B) HASMCs and U373MG were infected with the indicated viruses at a MOI of 3 and pUS28 expression was analyzed by immunoprecipitation/western blot (upper panels) and IE1/IE2 expression was analyzed by western blot (lower panels). In each case, pUS28 expression was detected by IP for the Flag epitope, followed by immunoblot with a Flag antibody.</p