Platelet fibrinogen: subcellular localization by means of immunofluorescent studies in normals and in congenital afibrinogenemia.

We have studied the site of fibrinogen localization in normal platelets and in the platelets of a patient with congenital afibrinogenemia (CA). The methods employed were: direct immunofluorescence technique (DIT) and indirect immunofluorescence technique (IIT). By means of the DIT normal platelets were shown to have a clear peripheral staining. Such staining disappeared after treatment with proteolytic enzymes and after specific blocking experiments. Such peripheral staining of platelets was absent in congenital afibrinogenemia even after fibrinogen infusion. By means of the IIT platelets were shown to have a considerable amount of fibrinogen. Such protein was demonstrated to represent an important part of platelet surface, since intact platelets were able to absorb completely a specific antifibrinogen antiserum.     

Silybin combined with phosphatidylcholine and vitamin E in patients with nonalcoholic fatty liver disease: a randomized controlled trial

The only currently recommended treatment for nonalcoholic fatty liver disease (NAFLD) is lifestyle modification. Preliminary studies of silybin showed beneficial effects on liver function. Realsil (RA) comprises the silybin phytosome complex (silybin plus phosphatidylcholine) coformulated with vitamin E. We report on a multicenter, phase III, double-blind clinical trial to assess RA in patients with histologically documented NAFLD. Patients were randomized 1:1 to RA or placebo (P) orally twice daily for 12 months. Prespecified primary outcomes were improvement over time in clinical condition, normalization of liver enzyme plasma levels, and improvement of ultrasonographic liver steatosis, homeostatic model assessment (HOMA), and quality of life. Secondary outcomes were improvement in liver histologic score and/or decrease in NAFLD score without worsening of fibrosis and plasma changes in cytokines, ferritin, and liver fibrosis markers. We treated 179 patients with NAFLD; 36 were also HCV positive. Forty-one patients were prematurely withdrawn and 138 patients analyzed per protocol (69 per group). Baseline patient characteristics were generally well balanced between groups, except for steatosis, portal infiltration, and fibrosis. Adverse events (AEs) were generally transient and included diarrhea, dysgeusia, and pruritus; no serious AEs were recorded. Patients receiving RA but not P showed significant improvements in liver enzyme plasma levels, HOMA, and liver histology. Body mass index normalized in 15% of RA patients (2.1% with P). HCV-positive patients in the RA but not the P group showed improvements in fibrogenesis markers. This is the first study to systematically assess silybin in NAFLD patients. Treatment with RA but not P for 12 months was associated with improvement in liver enzymes, insulin resistance, and liver histology, without increases in body weight. These findings warrant further investigation.     

Remarkably fast and selective aromatization of Hantzsch esters with MoOCl4 and MoCl5: a chemical model for possible biologically relevant properties of molybdenum-containing enzymes

Mo(VI) and Mo(V) salts both react selectively with Hantzsch esters to produce substitute pyridines in good-to-excellent yield (75-99%). The remarkable reactivity and selectivity of MoOCl(4) under reflux of acetonitrile and MoCl(5) in dichloromethane at room temperature encouraged us to propose that molybdenum-containing enzymes (such as xanthine or aldehyde oxidase) also participate to some degree in the metabolism of 1,4-dihydropyridine drugs in the liver analogous to NADH in the respiratory chain.     

Regulation of biomechanical signals by NF-kappaB transcription factors in chondrocytes

Physical therapies and exercise are beneficial not only for physiological recovery in inflamed or injured joints, but also for promoting a homeostatic equilibrium in healthy joints. Human joints provide the pivot points and physiological hinges essential for ambulation and movement to the body, and it is this mobility that in return promotes the health of the joints. But how mobilization regulates the joint microenvironment at the molecular level has remained enigmatic for many years. Recent advances in joint biomechanics and molecular approaches have facilitated an enriched understanding of how joints operate. Consequently, the mechanisms active during joint inflammation that lead to arthritic conditions, both in vivo in animal models, and in vitro at cell and tissue levels, have become increasingly detailed and defined. These efforts have produced mounting evidences supporting the premise that biomechanical signals play a fundamental role in both the etiopathogenesis of arthritic diseases and in the physiological restoration of joints. This report aims to summarize current peer-reviewed literature and available experimental data to explain how the signals generated by mechanical forces/joint mobilization generate beneficial effects on inflamed articular cartilage, and to propose the basis for using appropriate physical therapies for the optimal benefit to the patient suffering from joint associated injuries.     

A highly efficient biomimetic aromatization of Hantzsch-1,4-dihydropyridines with t-butylhydroperoxide, catalysed by iron(III) phthalocyanine chloride

Rapid aromatization of Hantzsch-1,4-DHPs with t-butylhydroperoxide catalysed by iron(III) phthalocyanine chloride is described. The reaction proceeds smoothly at room temperature within 1-35 min and the products of high purity were isolated in excellent yields. To explain the reactivity of this catalytical system plausible mechanism have been proposed to involve formation of high-valent oxoferryl species as in cytochrome P450 itself.     

Src Kinases Are Required for a Balanced Production of IL-12/IL-23 in Human Dendritic Cells Activated by Toll-Like Receptor Agonists

Background

Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC.

Principal Findings

In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation.

Conclusions

We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases.     

Chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis

Psychological stress affects different physiological processes including haematopoiesis. However, erythropoietic effects of chronic psychological stress remain largely unknown. The adult spleen contains a distinct microenvironment favourable for rapid expansion of erythroid progenitors in response to stressful stimuli, and emerging evidence suggests that inappropriate activation of stress erythropoiesis may predispose to leukaemic transformation. We used a mouse model to study the influence of chronic psychological stress on erythropoiesis in the spleen and to investigate potential mediators of observed effects. Adult mice were subjected to 2 hrs daily restraint stress for 7 or 14 consecutive days. Our results showed that chronic exposure to restraint stress decreased the concentration of haemoglobin in the blood, elevated circulating levels of erythropoietin and corticosterone, and resulted in markedly increased number of erythroid progenitors and precursors in the spleen. Western blot analysis revealed significantly decreased expression of both erythropoietin receptor and glucocorticoid receptor in the spleen of restrained mice. Furthermore, chronic stress enhanced the expression of stem cell factor receptor in the red pulp. Moreover, chronically stressed animals exhibited significantly increased expression of bone morphogenetic protein 4 (BMP4) in the red pulp as well as substantially enhanced mRNA expression levels of its receptors in the spleen. These findings demonstrate for the first time that chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis and leads to the prolonged activation of stress erythropoiesis pathways. Prolonged activation of these pathways along with an excessive production of immature erythroid cells may predispose chronically stressed subjects to a higher risk of leukaemic transformation.     

Biochemical markers of endothelial activation in primary hyperparathyroidism.

Patients with primary hyperparathyroidism (PHPT) have impaired vasodilation both dependent and independent of endothelium. The aims of our study were to measure three different biochemical markers of endothelial activation, i. e., plasma thrombomodulin, soluble(s) E-selectin, and von Willebrand factor, in PHPT patients before and one year after successful parathyroidectomy, and to distinguish the potential effect of hypercalcemia and/or high parathyroid hormone from that of major cardiovascular risk factors (diabetes mellitus, hyperlipidemia, hypertension, obesity, smoking habit) on endothelial function. Twenty consecutive patients with PHPT subdivided into two groups according to the absence (n = 8) or presence (n = 12) of one or more risk factors, and fifteen healthy normocalcemic subjects were studied. Baseline thrombomodulin levels were similar in the groups with and without risk factors, and in controls. In contrast, sE-selectin and von Willebrand factor were higher in PHPT patients with risk factors than in those without risk factors (p < 0.05 and p < 0.01, respectively) and controls (p < 0.01). Neither thrombomodulin nor sE-selectin changed after parathyroidectomy in either PHPT group. Plasma von Willebrand factor decreased (p < 0.01) in patients without risk factors, while persisting at high levels in patients with risk factors. In conclusion, in spite of a limitation due to the small number of patients, our study suggests that classic cardiovascular risk factors seem to be the main determinants for the high plasma levels of sE-selectin and vWF in PHPT. Together with unaltered thrombomodulin and sE-selectin levels, a plasma vWF decrease after parathyroidectomy might reflect a specific mechanism of its endothelial calcium- and/or PTH-stimulated secretion in some PHPT patients without risk factors. Whether a vWF reduction after parathyroidectomy may be used as a biochemical index for improved endothelial function in PHPT patients without risk factors has yet to be demonstrated in larger studies.     

Prevalence and level of Listeria monocytogenes and other Listeria sp. in ready-to-eat minimally processed and refrigerated vegetables

Minimally processed and refrigerated vegetables can be contaminated with Listeria species bacteria including Listeria monocytogenes due to extensive handling during processing or by cross contamination from the processing environment. The objective of this study was to examine the microbiological quality of ready-to-eat minimally processed and refrigerated vegetables from supermarkets in Osijek, Croatia. 100 samples of ready-to-eat vegetables collected from different supermarkets in Osijek, Croatia, were analyzed for presence of Listeria species and Listeria monocytogenes. The collected samples were cut iceberg lettuces (24 samples), other leafy vegetables (11 samples), delicatessen salads (23 samples), cabbage salads (19 samples), salads from mixed (17 samples) and root vegetables (6 samples). Listeria species was found in 20 samples (20 %) and Listeria monocytogenes was detected in only 1 sample (1 %) of cut red cabbage (less than 100 CFU/g). According to Croatian and EU microbiological criteria these results are satisfactory. However, the presence of Listeria species and Listeria monocytogenes indicates poor hygiene quality. The study showed that these products are often improperly labeled, since 24 % of analyzed samples lacked information about shelf life, and 60 % of samples lacked information about storage conditions. With regard to these facts, cold chain abruption with extended use after expiration date is a probable scenario. Therefore, the microbiological risk for consumers of ready-to-eat minimally processed and refrigerated vegetables is not completely eliminated.     

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.