Salmonella pathogenicity island 4-mediated adhesion is coregulated with invasion genes in Salmonella enterica

Salmonella pathogenicity island 4 (SPI4) encodes a type I secretion system and the cognate substrate protein, SiiE. We have recently demonstrated that SiiE is a giant nonfimbrial adhesin involved in the adhesion of Salmonella enterica serovar Typhimurium to polarized epithelial cells. We also observed that under in vitro culture conditions, the synthesis and secretion of SiiE coincided with the activation of Salmonella invasion genes. These observations prompted us to investigate the regulation of SPI4 genes in detail. A novel approach for the generation of reporter gene fusions was employed to generate single-copy chromosomal fusions to various genes within SPI4, and the expression of these fusions was investigated. We analyzed the regulation of SPI4 genes and the roles of various regulatory systems for SPI4 expression. Our data show that the expression of SPI4 genes is coregulated with SPI1 invasion genes by the global regulator SirA. Expression of a SPI4 gene was also reduced in the absence of HilA, the central local regulator of SPI1 gene expression. Both SirA and HilA functions were required for the secretion of SiiE and the SPI4-mediated adhesion. Our data demonstrate that SPI4-mediated adhesion, as well as SPI1-mediated invasion, are tightly coregulated by the same regulatory circuits and induced under similar environmental conditions.     

Hypoxia modulates the barrier and coagulant function of cultured bovine endothelium. Increased monolayer permeability and induction of procoagulant properties.

Exposure of cultured endothelium to environments with low concentrations of oxygen, in the range of those observed in pathophysiologic hypoxemic states in vivo, compromises cellular barrier and coagulant function. An atmosphere with PO2 approximately 14 mm Hg was not lethally toxic to endothelial cultures, but cells became larger and exhibited small intercellular gaps. At low oxygen concentrations, passage of macromolecular tracers through hypoxic endothelial monolayers was accelerated in a time- and dose-dependent manner, presumably by a paracellular pathway via the gaps. Cell surface coagulant properties of the endothelium were also perturbed. At PO2 approximately 14 mm Hg thrombomodulin antigen and functional activity on the cell surface were diminished by 80-90%, and Northern blots demonstrated suppression of thrombomodulin mRNA. The decrease in thrombomodulin was twice as great compared with the general decline in total protein synthesis in hypoxia. In addition, expression of a direct Factor X activator developed under hypoxic conditions; the activator was membrane-associated and expressed on the surface of intact cultures, Ca-dependent, inhibited by HgCl2 but not PMSF, and had Km approximately 25 micrograms/ml for the substrate at pH 7.4. Synthesis of the activator was blocked by inclusion of cycloheximide, but not warfarin, in the culture medium. These results demonstrate that endothelial function is perturbed in a selective manner in the presence of low concentrations of oxygen, providing insights into mechanisms which may contribute to vascular dysfunction in hypoxemic states.     

Acute hepatitis C: high rate of both spontaneous and treatment-induced viral clearance

BACKGROUND & AIMS: Acute hepatitis C virus infection accounts for approximately 20% of cases of acute hepatitis today. The aim of this study was to define the natural course of the disease and to contribute to the development of treatment strategies for acute hepatitis C virus. METHODS: The diagnosis of acute hepatitis C virus in 60 patients was based on seroconversion to anti-hepatitis C virus antibodies or clinical and biochemical criteria and on the presence of hepatitis C virus RNA in the first serum sample. RESULTS: Fifty-one of 60 (85%) patients presented with symptomatic acute hepatitis C virus. In the natural (untreated) course of acute symptomatic hepatitis C (n = 46), spontaneous clearance was observed in 24 patients (52%), usually within 12 weeks after the onset of symptoms, whereas all asymptomatic patients (n = 9) developed chronic hepatitis C. The start of antiviral therapy (interferon-alpha with or without ribavirin) beyond 3 months after the onset of acute hepatitis induced sustained viral clearance in 80% of treated patients. CONCLUSIONS: The management of acute hepatitis C has to take into account the high rate of spontaneous viral clearance within 12 weeks after the onset of symptomatic disease. Treatment of only those patients who remain hepatitis C virus RNA positive for more than 3 months after the onset of disease led to an overall viral clearance (self-limited and treatment induced) in 91% of patients, and unnecessary treatment was avoided in those with spontaneous viral clearance. Patients with asymptomatic acute hepatitis C virus infection are unlikely to clear the infection spontaneously and should be treated as early as possible.     

Left ventricular shape index assessed by gated stress myocardial perfusion SPECT: initial description of a new variable.

BACKGROUND: Ventricular remodeling is predictive of congestive heart failure (CHF). We aimed to automatically quantify a new myocardial shape variable on gated myocardial perfusion single photon emission computed tomography (SPECT) (MPS) and to evaluate the association of this new SPECT parameter with the risk of hospitalization for CHF. METHODS AND RESULTS: A computer algorithm was used to measure the 3-dimensional (3D) left ventricular (LV) shape index (LVSI), derived as the ratio of maximum 3D short- and long-axis LV dimensions, for end systole and end diastole. LVSI normal limits were obtained from stress technetium 99m sestamibi MPS images of 186 patients (60% of whom were men) (control subjects) with a low likelihood of CAD (< 5%). These limits were tested in a consecutive series of 93 inpatients (85% of whom were men) having MPS less than 1 week after hospitalization, of whom 25 were hospitalized for CHF exacerbation. Variables associated with CHF hospitalization were tested by receiver operating characteristic curve and multivariate logistic regression analyses. LVSI repeatability was assessed in 52 patients with ischemic cardiomyopathy who had sequential stress MPS within 60 days after the initial MPS without clinical events in the interval between MPS studies. Control subjects had lower end-systolic and end-diastolic LVSIs compared with patients with CHF and those without CHF (P < .001). Receiver operating characteristic curve areas for the prediction of hospitalization as a result of CHF were similar for LV ejection fraction and end-systolic LVSI. End-systolic and end-diastolic LVSIs were independent predictors of CHF hospitalization by multivariate analysis; however, end-systolic LVSI had the greatest added value among all tested variables. Repeatability was excellent for both end-systolic LVSI (R2 = 0.85, P < .0001) and end-diastolic LVSI (R2 = 0.82, P < .001). CONCLUSION: LVSI is a promising new 3D variable derived automatically from gated MPS providing highly repeatable ventricular shape assessment. Preliminary findings suggest that LVSI might have clinical implications in patients with CHF.     

Comprehensive Evaluation of the Effects of Enalapril on Matrix Metalloproteinases Levels in Hypertension

Purpose

Angiotensin-converting enzyme inhibitors (ACEi) may downregulate matrix metalloproteinases (MMPs). We examined whether enalapril affects MMP-2, MMP-8, and MMP-9 levels and activity, and their endogenous inhibitors (tissue inhibitors of MMPs, TIMP-1 and TIMP-2) levels in hypertensive patients. Moreover, we assessed the effects of enalaprilat on MMP-9 and TIMP-1 secretion by human endothelial cells (HUVECs).

Methods

Thirty-eight hypertensive patients received enalapril for 8 weeks and were compared with thirty-eight normotensive controls. Blood samples were collected at baseline and after treatment. Plasma ACE activity was determined by a fluorimetric assay. Plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 were measured by ELISA and gelatin zymography. A fluorogenic peptide cleavage assay was used to measure MMP activity. HUVECs cells were stimulated by phorbol-12-myristate-13-acetate (PMA) and the effects of enalaprilat (10^?10 to 10^?6?M) on MMP-9 and TIMP-1 levels were determined.

Results

Enalapril decreased blood pressure and ACE activity in hypertensive patients (P?<?0.05), but had no effects on plasma MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 levels, or MMP activity. Enalaprilat had no effects on PMA-induced increases in MMP-9 and TIMP-1 secretion by HUVECs or on MMP activity.

Conclusions

We show consistent evidence, both in vivo and in vitro, that enalapril does not affect MMPs and TIMPs levels in hypertensive patients.