Purification and characterization of a saliva-interacting cell-wall protein from Streptococcus mutans serotype f by using monoclonal-antibody immunoaffinity chromatography.

A rat monoclonal antibody, LO SM2, of the immunoglobulin M class, specific for a saliva receptor (SR) from Streptococcus mutans serotype f, was able to precipitate the SR from crude cell-wall-associated antigens (WEA) of this bacteria in presence of a detergent mixture. We have then used the technique of monoclonal-antibody immunoaffinity chromatography to purify the S. mutans SR. Pure SR was obtained from a crude WEA fraction with a single chromatographic step. The active SR could be eluted from the column in a highly purified form with 0.2 M-glycine/HC1, pH 2.8. The final yield was about 32% in terms of binding activity. Characterization of the SR by crossed immunoelectrophoresis, sodium dodecyl sulphate- or 4-30%-native-gradient-polyacrylamide-gel electrophoresis showed that the receptor is a single polypeptide chain of Mr approx. 74000. Native or denaturated forms of the SR adsorbed on to a solid support, such as nitrocellulose, are recognized by monoclonal antibody LO SM2, and both forms are still able to bind the ligand, saliva.     

Human IgG and Streptococcus mutans SR protein contain cross-reactive epitopes

Antigen B, a glycoprotein present on the cell surface of "mutans streptococci," mediates bacterial adherence to teeth surfaces and has been implicated in cross-reactivity with human heart components. Elevated levels of anti-IgG antibodies were generally found in sera of rabbits immunized with protein SR, a B-related protein from Streptococcus mutans serogroup f, or recombinant protein SR (rSR). These anti-IgG antibodies could be involved in the previously mentioned heart cross-reactivities. Results from immunoblots and ELISA analyses demonstrate that these anti-IgG antibodies recognize common epitopes on SR, rSR, and human IgG2 and IgG4 probably located on the Fab region. Furthermore, control experiments with biotinylated human IgG show that the cross-reactions between IgG and SR were not mediated by an FcR mechanism. Direct competition between rSR and human IgG in binding to anti-IgG or anti-SR antibodies confirm that S. mutans SR protein possesses Ag mimicry with human IgG. Our studies provide some evidence that S. mutans SR protein and human IgG H chains share autoimmune epitopes which could play a role in the induction of anti-IgG antibodies and therefore could explain the enhancement of anti-IgG antibody levels observed in rabbits immunized with either S. mutans whole cells or purified B-related Ag.     

Pheochromocytoma mimicking an acute myocardial infarction

We report a 42-year-old female who presented with retrosternal pain, dyspnoea and nausea. Electrocardiography suggested a recent anterior myocardial infarction. However, emergency coronary angiography showed normal blood flow through all the coronary arteries. Paroxysmal hypertension raised the suspicion of a pheochromocytoma. Indeed, abdominal ultrasonography and computed tomography revealed a mass in the left adrenal gland. Elevated levels of plasma and urine catecholamines supported the diagnosis of pheochromocytoma. Left adrenalectomy was performed without complications and pathological examination revealed a 5.5 cm pheochromocytoma. After surgery, all antihypertensive medication was discontinued and the blood pressure returned to normal within several days. Currently, the patient is asymptomatic, has normal catecholamine levels and the electrocardiographic signs of ischaemia have resolved entirely. This case illustrates that a rare clinical entity such as pheochromocytoma should be considered in the differential diagnosis of acute coronary syndrome. (Neth Heart J 2007; 15:248-51.)     

Treatment with apolipoprotein A-1 mimetic peptide reduces lupus-like manifestations in a murine lupus model of accelerated atherosclerosis

Introduction

The purpose of this study was to evaluate the effects of L-4F, an apolipoprotein A-1 mimetic peptide, alone or with pravastatin, in apoE^-/-Fas^-/-C57BL/6 mice that spontaneously develop immunoglobulin G (IgG) autoantibodies, glomerulonephritis, osteopenia, and atherosclerotic lesions on a normal chow diet.

Methods

Female mice, starting at eight to nine weeks of age, were treated for 27 weeks with 1) pravastatin, 2) L-4F, 3) L-4F plus pravastatin, or 4) vehicle control, followed by disease phenotype assessment.

Results

In preliminary studies, dysfunctional, proinflammatory high-density lipoproteins (piHDL) were decreased six hours after a single L-4F, but not scrambled L-4F, injection in eight- to nine-week old mice. After 35 weeks, L-4F-treated mice, in the absence/presence of pravastatin, had significantly smaller lymph nodes and glomerular tufts (P_{L, LP} < 0.05), lower serum levels of IgG antibodies to double stranded DNA (dsDNA) (P_L < 0.05) and oxidized phospholipids (oxPLs) (P_{L, LP} < 0.005), and elevated total and vertebral bone mineral density (P_{L, LP} < 0.01) compared to vehicle controls. Although all treatment groups presented larger aortic root lesions compared to vehicle controls, enlarged atheromas in combination treatment mice had significantly less infiltrated CD68⁺ macrophages (P_LP < 0.01), significantly increased mean α-actin stained area (P_LP < 0.05), and significantly lower levels of circulating markers for atherosclerosis progression, CCL19 (P_{L, LP} < 0.0005) and VCAM-1 (P_L < 0.0002).

Conclusions

L-4F treatment, alone or with pravastatin, significantly reduced IgG anti-dsDNA and IgG anti-oxPLs, proteinuria, glomerulonephritis, and osteopenia in a murine lupus model of accelerated atherosclerosis. Despite enlarged aortic lesions, increased smooth muscle content, decreased macrophage infiltration, and decreased pro-atherogenic chemokines in L-4F plus pravastatin treated mice suggest protective mechanisms not only on lupus-like disease, but also on potential plaque remodeling in a murine model of systemic lupus erythematosus (SLE) and accelerated atherosclerosis.     

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation.     

Interferon-gamma increases monocyte HLA-DR expression without effects on glucose and fat metabolism in postoperative patients.

Tissue injury is associated with decreased cellular immunity and enhanced metabolism. Immunodepression is thought to be counteracted by interferon (IFN)-gamma, which increases human leukocyte antigen (HLA)-DR expression. Hypermetabolism could be enhanced by IFN-gamma because cytokines induce a hypermetabolic response to stress. In healthy humans, IFN-gamma enhanced HLA-DR expression without effects on glucose and fat metabolism. In the present study, we evaluated whether IFN-gamma lacks potential harmful side effects on metabolic and endocrine pathways while maintaining its beneficial effects on the immune system under conditions in which the inflammatory response system is activated. In 13 patients scheduled for major surgery, we studied HLA-DR expression on peripheral blood monocytes before surgery and postoperatively randomized the patients into an intervention and a placebo group. Subsequently, we evaluated the effects of a single dose of IFN-gamma vs. saline on short-term monocyte activation, glucose and lipid metabolism, and glucose and lipid regulatory hormones. HLA-DR expression on monocytes was restored from postoperative levels of 54% (42-60%; median and interquartiles) to 92% (91-96%) 24 h after IFN-gamma administration but stayed low in the placebo-treated patients. IFN-gamma did not affect glucose metabolism (plasma glucose, rate of appearance and disappearance of glucose) and lipid metabolism (plasma glycerol, plasma free fatty acids, and rates of appearance and disappearance of glycerol). IFN-gamma had no effect on plasma cortisol, adrenocorticotropic hormone, growth hormone, insulin, C-peptide, glucagon, epinephrine, and norepinephrine concentrations. We conclude that IFN-gamma exerts a favorable effect on cell-mediated immunity in patients after major surgery without effects on glucose and lipid metabolism.     

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background  

Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.     

Endogenous cholesterol synthesis is associated with VLDL-2 apoB-100 production in healthy humans

Subjects with high plasma cholesterol levels exhibit a high production of VLDL apolipoprotein B-100 (apoB-100), suggesting that cholesterol is a mediator for VLDL production. The objective of the study was to examine whether endogenous cholesterol synthesis, reflected by the lathosterol-cholesterol ratio (L-C ratio), affects the secretory rates of different VLDL subfractions. Ten healthy subjects were studied after overnight fasting. During a 10 h primed, constant infusion of 13C-valine (15 micromol/kg/h), enrichment was determined in apoB-100 from ultracentrifugally isolated VLDL-1 and VLDL-2 by gas chromatography mass spectrometry. The synthesis rates of VLDL-1 apoB-100 and VLDL-2 apoB-100, catabolism, and transfer were estimated by compartmental analysis. Mean VLDL-1 apoB-100 pool size was 90 +/- 15 mg, and mean VLDL-2 apoB-100 pool size was 111 +/- 14 mg. Absolute synthesis rate of VLDL-1 apoB-100 was 649 +/- 127 mg/day and 353 +/- 59 mg/day for VLDL-2 apoB-100. There was a strong association between the absolute synthesis rate of VLDL-2 apoB-100 and L-C ratio (r 2 = 0.61, P < 0.01). In contrast, no correlation was observed between L-C ratio and absolute synthesis rate of VLDL-1 apoB-100 (r 2 = 0.302, P = 0.09). In conclusion, these data provide additional support for an independent regulation of VLDL-1 apoB-100 and VLDL-2 apoB-100 production.Endogenous cholesterol synthesis is correlated only with the VLDL-2 apoB-100 production.