Human phagocytic cell response to histamine derived from potential probiotic strains of Lactobacillus reuteri

Histamine derived from lactobacilli isolates is considered to be a potential immunomodulator able to interact with the host immune system. We tested the effect of pure histamine (0.413?mM) together with the effect of cell-culture supernatants (CCS) containing different concentration of histamine produced by two of Lactobacillus reuteri isolates on the activities of antioxidant enzyme, as well as on the phagocytic activity of human leucocytes (HL). Phagocytic activity represents the non-specific immune response of HL homogenate, in vitro. Analysed histamine-producers were represented by a goatling isolate named L. reuteri KO5 and a lamb isolate named L. reuteri E and histamine production was determined using HPLC method connected with UV detection. Concretely, the samples contained the mixture of isolated HL and the addition of lactobacilli CCS at three different final concentrations of histamine ～ 0.1, 1.8 and 5.4?mM. It was found that pure histamine (0.413?mM) did not significantly influence the oxidant-antioxidant balance in HL demonstrated by unchanged degree of HL lipid peroxidation. However, at the same time, the final activity of catalase and superoxide dismutase were significantly changed (p?≤?0.001).Moreover, the phagocytic index (p?≤?0.01), lysozyme (p?≤?0.05) and peroxidase activity (p?≤?0.001), and production of IL-1β significantly decreased. CCS containing different concentration of produced histamine were also able to modulate the host non-specific immune response together with the enzymatic activity of SOD and catalase too. However, our findings indicated that the impact of lactobacilli histamine is strictly strain-dependent and concentration dependent. Moreover, it seems that histamine is not the only one lactobacilli metabolite, which may play an important role in overall immunomodulatory and antioxidant potential of tested lactobacilli.     

Rationale, design, and methods for the risk evaluation and communication health outcomes and utilization trial (REACH OUT)

OBJECTIVE: To test the primary study hypothesis that a physician-delivered coronary heart disease risk evaluation and communication program can lower patients' predicted 10-year risk of myocardial infarction or death due to coronary heart disease by 10% within 6 months compared to usual care. DESIGN: Prospective, parallel group, open-label, controlled, cluster-randomized multinational trial; the study site is the unit of randomization. SETTING: Patients were recruited from 106 general practices located in nine European countries. PATIENTS: Men and women aged 45 to 64 (N=1500) with a documented history of hypertension (treated or untreated), systolic blood pressure > or =140 mmHg (or > or =130 mmHg in the presence of renal or kidney disease), no history of cardiovascular disease, and a predicted 10-year risk of myocardial infarction or death due to coronary heart disease > or =10%. INTERVENTION: Sites were randomized to deliver a physician-directed coronary heart disease risk communication and education program or usual care. The intervention program included informing patients of their 10-year risk of myocardial infarction or death due to coronary heart disease, educating patients about modifiable risk factors and their control, and three follow-up phone calls by a physician or study nurse. MAIN OUTCOME MEASURE: Predicted 10-year risk of myocardial infarction or death due to coronary heart disease at 6 months. CONCLUSIONS: REACH OUT will evaluate a novel, patient-focused, physician-implemented application of coronary heart disease risk equations. Results of the study will be of practical relevance to physicians, health care organizations, and those who issue clinical guidelines for the reduction of cardiovascular risk.     

Increased plasma levels of NT-proANP and NT-proBNP as markers of cardiac dysfunction in septic patients

The family of natriuretic peptides comprises several structurally related 22-53-amino acid peptides, such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which are vasoactive peptides with vasodilator and diuretic properties and play an important role in cardiovascular homeostasis. The salutary cardiovascular effects of natriuretic peptides suggest that ANP and BNP may have a pathophysiological significance in the cardiac dysfunction of septic patients. We determined plasma levels of the stable N-terminal prohormone forms of ANP (NT-proANP) and BNP (NT-proBNP) as well as troponin I (TNI) as a marker of myocardial cell injury by ELISA methods in 19 septic patients and 19 healthy controls at day one of severe sepsis. Left ventricular ejection fraction (LVEF) was determined on day 1 of severe sepsis by echocardiography. Significantly higher concentrations of NT-proANP were measured in non-survivors (mean = 13415 pmol/l +/- SEM = 4295) and survivors (mean = 7386 pmol/l +/- SEM = 1807) as compared to controls (mean = 1404 pmol/l +/- SEM = 181; p<0.001). Levels of NT-proBNP were also significantly higher in non-survivors (mean = 3439 pmol/l +/- SEM = 1246; p<0.05) and survivors (mean = 1009 pmol/l +/- SEM = 263; p<0.001) as compared to controls (mean = 200 pmol/l +/- SEM = 24) and correlated well with an increase in TNI-levels (r = 0.71; p<0.001). NT-proANP and NT-proBNP may serve as useful laboratory markers to indicate myocardial dysfunction and may help to differentiate between survivors and non-survivors of severe sepsis.     

Disposition and Elimination of Meropenem in Cerebrospinal Fluid of Hydrocephalic Patients with External Ventriculostomy

The broad antibacterial spectrum and the low incidence of seizures in meropenem-treated patients qualifies meropenem for therapy of bacterial meningitis. The present study evaluates concentrations in ventricular cerebrospinal fluid (CSF) in the absence of pronounced meningeal inflammation. Patients with occlusive hydrocephalus caused by cerebrovascular diseases, who had undergone external ventriculostomy (n = 10, age range 48 to 75 years), received 2 g of meropenem intravenously over 30 min. Serum and CSF were drawn repeatedly and analyzed by liquid chromatography-mass spectroscopy. Pharmacokinetics were determined by noncompartmental analysis. Maximum concentrations in serum were 84.7 ± 23.7 μg/ml. A CSF maximum (C_maxCSF) of 0.63 ± 0.50 μg/ml (mean ± standard deviation) was observed 4.1 ± 2.6 h after the end of the infusion. C_maxCSF and the area under the curve for CSF (AUC_CSF) depended on the AUC for serum (AUC_S), the CSF-to-serum albumin ratio, and the CSF leukocyte count. Elimination from CSF was considerably slower than from serum (half-life at β phase [t_1/2β] of 7.36 ± 2.89 h in CSF versus t_1/2β of 1.69 ± 0.60 h in serum). The AUC_CSF/AUC_S ratio for meropenem, as a measure of overall CSF penetration, was 0.047 ± 0.022. The AUC_CSF/AUC_S ratio for meropenem was similar to that for other β-lactam antibiotics with a low binding to serum proteins. The concentration maxima of meropenem in ventricular CSF observed in this study are high enough to kill fully susceptible pathogens. They may not be sufficient to kill bacteria with a reduced sensitivity to carbapenems, although clinical success has been reported for patients with meningitis caused by penicillin-resistant pneumococci and Pseudomonas aeruginosa.     

Analysis of intracellular storage and regulated secretion of 3 von Willebrand disease-causing variants of von Willebrand factor

The rapid exocytosis of von Willebrand factor (VWF) in response to vascular injury can be attributed to the fact that VWF is stored in the Weibel-Palade bodies (WPBs) of endothelial cells. We describe a system for examining the ability of VWF to drive both the formation of a storage compartment and the function of that compartment with respect to regulated secretion. Transient transfection of HEK293 cells with wild-type human VWF cDNA leads to the formation of numerous elongated organelles that resemble WPBs. These "pseudo-WPBs" exhibit the internal structure, as well as the ability to recruit membrane proteins including P-selectin, of bona fide WPBs. Finally, VWF was efficiently secreted upon stimulation by phorbol ester. We used this system to examine 3 VWF mutations leading to von Willebrand disease that affect VWF multimerization and constitutive secretion. Surprisingly we find that all 3 mutants can, to some extent, make pseudo-WPBs that recruit appropriate membrane proteins and that are responsive to secretagogues. The most striking defects are a delay in formation and a reduction in the length and number of pseudo-WPBs in proportion to the clinical severity of the mutation. Studies of pseudo-WPB formation in this system thus yield insights into the structure-function relationships underpinning the ability of VWF to form functional WPBs.     

Fatty acids differentially influence phosphatidylinositol 3-kinase signal transduction in endothelial cells: impact on adhesion and apoptosis

In contrast to n-6 fatty acids like arachidonic acid (AA), the anti-inflammatory potential of n-3 fatty acids such as docosahexaenoic acid (DHA) has been demonstrated. We examined the phosphatidylinositol (PI)3-kinase dependent effects of AA versus DHA on monocyte rolling, adhesion and transmigration through inflammatory activated human umbilical venous endothelial cells (HUVEC) as well as on apoptosis, to investigate the impact on vascular inflammation. HUVEC were pre-incubated with AA, DHA or sham, and stimulated with VEGF, TNF-alpha or staurosporine. Rolling and adhesion were investigated by means of a parallel flow chamber; transmigration was performed in a static assay. Activation of PI3-kinase was measured as phosphorylation of protein kinase B (Akt). Apoptosis was determined by caspase-3 activity and annexin-V analysis. Pre-incubation of HUVEC with DHA markedly decreased TNF-alpha-induced monocyte rolling, adhesion, and transmigration, although expression of endothelial adhesion molecules was unchanged. In contrast, AA increased TNF-alpha-induced rolling. Both fatty acids did not alter TNF-alpha-mediated upregulation of the adhesion molecules ICAM-1, VCAM-1, and E-selectin. The divergent effects of AA and DHA were abrogated with PI3-kinase inhibitors. After pre-incubation with DHA, VEGF-, TNF-alpha- and staurosporine-induced phosphorylation of Akt was decreased when compared to AA. DHA pre-incubation significantly increased staurosporine-induced apoptosis. In addition, DHA in comparison to AA augmented staurosporine-mediated increase in caspase-3 activity. In conclusion, DHA-induced a reduction in rolling, adhesion and transmigration of monocytes through inflammatory activated HUVEC that is in part PI3-kinase dependent. PI3-kinase driven phosphorylation of Akt and apoptosis of HUVEC as contribution to the resolution of inflammation is differentially modulated by DHA versus AA.