Vaccination of cattle with the N terminus of LppQ of Mycoplasma mycoides subsp. mycoides results in type III immune complex disease upon experimental infection.

Contagious bovine pleuropneumonia (CBPP) is a serious respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides. Current vaccines against CBPP induce short-lived immunity and can cause severe postvaccine reactions. Previous studies have identified the N terminus of the transmembrane lipoprotein Q (LppQ-N') of M. mycoides subsp. mycoides as the major antigen and a possible virulence factor. We therefore immunized cattle with purified recombinant LppQ-N' formulated in Freund's adjuvant and challenged them with M. mycoides subsp. mycoides. Vaccinated animals showed a strong seroconversion to LppQ, but they exhibited significantly enhanced postchallenge glomerulonephritis compared to the placebo group (P = 0.021). Glomerulonephritis was characterized by features that suggested the development of antigen-antibody immune complexes. Clinical signs and gross pathological scores did not significantly differ between vaccinated and placebo groups. These findings reveal for the first time the pathogenesis of enhanced disease as a result of antibodies against LppQ during challenge and also argue against inclusion of LppQ-N' in a future subunit vaccine for CBPP.

Treatment of patients with the hypereosinophilic syndrome with mepolizumab

BACKGROUND: The hypereosinophilic syndrome is a group of diseases characterized by persistent blood eosinophilia, defined as more than 1500 cells per microliter with end-organ involvement and no recognized secondary cause. Although most patients have a response to corticosteroids, side effects are common and can lead to considerable morbidity. METHODS: We conducted an international, randomized, double-blind, placebo-controlled trial evaluating the safety and efficacy of an anti-interleukin-5 monoclonal antibody, mepolizumab, in patients with the hypereosinophilic syndrome. Patients were negative for the FIP1L1-PDGFRA fusion gene and required prednisone monotherapy, 20 to 60 mg per day, to maintain a stable clinical status and a blood eosinophil count of less than 1000 per microliter. Patients received either intravenous mepolizumab or placebo while the prednisone dose was tapered. The primary end point was the reduction of the prednisone dose to 10 mg or less per day for 8 or more consecutive weeks. RESULTS: The primary end point was reached in 84% of patients in the mepolizumab group, as compared with 43% of patients in the placebo group (hazard ratio, 2.90; 95% confidence interval [CI], 1.59 to 5.26; P<0.001) with no increase in clinical activity of the hypereosinophilic syndrome. A blood eosinophil count of less than 600 per microliter for 8 or more consecutive weeks was achieved in 95% of patients receiving mepolizumab, as compared with 45% of patients receiving placebo (hazard ratio, 3.53; 95% CI, 1.94 to 6.45; P<0.001). Serious adverse events occurred in seven patients receiving mepolizumab (14 events, including one death; mean [+/-SD] duration of exposure, 6.7+/-1.9 months) and in five patients receiving placebo (7 events; mean duration of exposure, 4.3+/-2.6 months). CONCLUSIONS: Our study shows that treatment with mepolizumab, an agent designed to target eosinophils, can result in corticosteroid-sparing for patients negative for FIP1L1-PDGFRA who have the hypereosinophilic syndrome. (ClinicalTrials.gov number, NCT00086658 [ClinicalTrials.gov].).

A novel blood-sparing agent in cardiac surgery? First in-patient experience with the synthetic serine protease inhibitor MDCO-2010: a phase II, randomized, double-blind, placebo-controlled study in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass

BACKGROUND Antifibrinolytics have been used for 2 decades to reduce bleeding in cardiac surgery. MDCO-2010 is a novel, synthetic, serine protease inhibitor. We describe the first experience with this drug in patients. METHODS In this phase II, double-blind, placebo-controlled study, 32 patients undergoing isolated primary coronary artery bypass grafting with cardiopulmonary bypass were randomly assigned to 1 of 5 increasing dosage groups of MDCO-2010. The primary aim was to evaluate pharmacokinetics (PK) with assessment of plasmatic concentrations of the drug, short-term safety, and tolerance of MDCO-2010. Secondary end points were influence on coagulation, chest tube drainage, and transfusion requirements. RESULTS PK analysis showed linear dosage-proportional correlation between MDCO-2010 infusion rate and PK parameters. Blood loss was significantly reduced in the 3 highest dosage groups compared with control (P = 0.002, 0.004 and 0.011, respectively). The incidence of allogeneic blood product transfusions was lower with MDCO-2010 4/24 (17%) vs 4/8 (50%) in the control group. MDCO-2010 exhibited dosage-dependent antifibrinolytic effects through suppression of D-dimer generation and inhibition of tissue plasminogen activator-induced lysis in ROTEM analysis as well as anticoagulant effects demonstrated by prolongation of activated clotting time and activated partial thromboplastin time. No systematic differences in markers of end organ function were observed among treatment groups. Three patients in the MDCO-2010 groups experienced serious adverse events. One patient experienced intraoperative thrombosis of venous grafts considered possibly related to the study drug. No reexploration for mediastinal bleeding was required, and there were no deaths. CONCLUSIONS This first-in-patient study demonstrated dosage-proportional PK for MDCO-2010 and reduction of chest tube drainage and transfusions in patients undergoing primary coronary artery bypass grafting. Antifibrinolytic and anticoagulant effects were demonstrated using various markers of coagulation. MDCO-2010 was well tolerated and showed an acceptable initial safety profile. Larger multi-institutional studies are warranted to further investigate the safety and efficacy of this compound.

Association of oliguria with the development of acute kidney injury in the critically ill

Urine output (UO) criterion may increase the sensitivity of the definition of acute kidney injury (AKI). We determined whether the empirically derived definition for oliguria(<0.5 ml/kg/h) is independently associated with adverse outcome. Data analysis included hourly recorded UO from the prospective, multicenter FINNAKI study conducted in 16 Finnish intensive care units. Confounder-adjusted association of oliguria of different severity and duration primarily with the development of AKI defined by creatinine criterion (Cr-AKI) or renal replacement therapy(RRT) was assessed. Secondarily, we determined the association of oliguria with 90-day mortality. Of the 1966 patients analyzed for the development of AKI, 454 (23.1%) reached this endpoint. Within this AKI cohort, 312 (68.7%)developed Cr-AKI, 21 (4.6%) commenced RRT without Cr-AKI, and 121 (26.7%) commenced RRT with Cr-AKI. Episodes of severe oliguria (<0.1 ml/kg/h) for more than 3 h were independently associated with the development of Cr-AKI or RRT. The shortest periods of consecutive oliguria independently associated with an increased risk for 90-day mortality were 6–12 h of oliguria from 0.3 to <0.5 ml/kg/h, over 6 h of oliguria from 0.1 to <0.3 ml/kg/h, and severe oliguria lasting over 3 h.Thus, our findings underlie the importance of hourly UO measurements.

Vaccination with the recombinant chimeric antigen recNcMIC3-1-R induces a non-protective Th2-type immune response in the pregnant mouse model for N. caninum infection

The major route of transmission of Neospora caninum in cattle is transplacentally from an infected cow to its progeny. Therefore, a vaccine should be able to prevent both the horizontal transmission from contaminated food or water and the vertical transmission. We have previously shown that a chimeric vaccine composed of predicted immunogenic epitopes of NcMIC3, NcMIC1 and NcROP2 (recNcMIC3-1-R) significantly reduced the cerebral infection in BALB/c mice. In this study, mice were first vaccinated, then mated and pregnant mice were challenged with 2×10(6)N. caninum tachyzoites at day 7-9 of pregnancy. Partial protection was only observed in the mice vaccinated with a tachyzoite crude protein extract but no protection against vertical transmission or cerebral infection in the dams was observed in the group vaccinated with recNcMIC3-1-R. Serological and cytokine analysis showed an overall lower cytokine level in sera associated with a dominant IL-4 expression and high IgG1 titers. Thus, the Th2-type immune response observed in the pregnant mice was not protective against experimental neosporosis, in contrary to the mixed Th1-/Th2-type immune response observed in the non-pregnant mouse model. These results demonstrate that the immunomodulation that occurs during pregnancy was not favorable for the protection against N. caninum infection conferred by vaccination with recNcMIC3-1-R.

First foot prints of chemistry on the shore of the Island of Superheavy Elements

Chemistry has arrived on the shore of the Island of Stability with the first chemical investigation of the superheavy elements Cn, 113, and 114. The results of three experimental series leading to first measured thermodynamic data and qualitatively evaluated chemical properties for these elements are described. An interesting volatile compound class has been observed in the on-line experiments for the elements Bi and Po. Hence, an exciting chemical study of their heavier transactinide homologues, elements 115 and 116 is suggested.

Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

The transcriptional regulator megakaryoblastic leukemia-1 mediates serum response factor-independent activation of tenascin-C transcription by mechanical stress

The extracellular matrix protein tenascin-C (TNC) is up-regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain-induced TNC expression depends on RhoA-actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia-1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c-fos was abolished. Cyclic strain failed to induce TNC in MKL1-deficient but not in SRF-deficient fibroblasts, and strain-induced TNC expression strongly depended on the SAP domain of MKL1. Promoter-reporter and chromatin immunoprecipitation experiments unraveled a SAP-dependent, SRF-independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.

Treatment of NASH with ursodeoxycholic acid: cons

Non-alcoholic steatohepatitis (NASH) has a prevalence of 1% in Western countries. Its causes as well as its medical treatment are, to date, still debated. Recently, studies of agents suggested to have antiapoptotic, insulin-sensitizing or anti-inflammatory effects in patients with NASH have been conducted, one of which is ursodeoxycholic acid (UDCA), a tertiary bile acid. Between 1994 and 2008, four prospective randomized, double-blind, placebo-controlled studies of the treatment of NASH with UDCA were conducted. The first study, by Lindor et al., compared the impact of 13-15 mg/kg/day of UDCA to a placebo. The second study by Dufour et al. had an additional third arm that administered combination therapy with UDCA and vitamin E. The third and fourth studies by Leuschner et al. and by Ratziu et al. evaluated high doses of UDCA at 25-35 mg/kg/day, and used liver biopsies and serum liver enzyme levels to evaluate the impact of UDCA. With the exception of Ratziu et al.'s study, which was lacking a second liver biopsy, none of these studies showed any significant differences in the treatment of NASH with UDCA compared with a placebo. However, Dufour et al. did observe a significant improvement of NASH with the combination (UDCA/VitE) vs placebo therapy, whereas UDCA monotherapy was not effective in the treatment of NASH. Nevertheless, the effects of other bile acids and combination therapies need to be explored.

Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations

We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution to investigate interaction and binding of α-tochoperol transfer protein (α-TTP) to phosphatidylinositol phosphate lipids (PIPs). Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP-PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein/ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED) phenotypes. Specifically, R221 is main residue responsible for the stabilization of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP.

Vaccination of mice with chitosan nanogel-associated recombinant NcPDI against challenge infection with Neospora caninum tachyzoites

The effects of nanogel encapsulation of recombinant NcPDI (recNcPDI) following vaccination of mice by intranasal or intraperitoneal routes and challenge infection with Neospora caninum tachyzoites were investigated. Nanogels were chitosan based, with an alginate or alginate-mannose surface. None of the mice receiving recNcPDI intraperitoneal (i.p.) (without nanogels) survived, whereas intranasal (i.n.) application protected 9 of 10 mice from disease. Association of recNcPDI with nanogels improved survival of i.p. vaccinated mice, but nanogels without recNcPDI gave similar protection levels. When nanogels were inoculated via the i.n. route, 80% of the mice were protected. Association of recNcPDI with the alginate-coated nanogels protected all mice against disease. Quantification of the cerebral parasite burden showed a significant reduction of parasite numbers in most experimental groups vaccinated i.n., except those vaccinated with alginate-mannose nanogels with or without recNcPDI. For i.p. vaccinated groups, no significant differences in cerebral infection densities were measured, but there was a reduction in the groups vaccinated with recNcPDI associated with both types of nanogels. Analysis of the immune responses of infected mice indicated that association of recNcPDI with nanogels altered the patterns of cytokine mRNA expression profiles, but had no major impact on the antibody subtype responses. Nevertheless, this did not necessarily relate to the protection.