Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.

Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.

Determination of the molecular subtypes of diffuse large B-cell lymphomas using immunohistochemistry: a case series from the Inselspital, Bern, and a critical appraisal of this determination in Switzerland

The two major subtypes of diffuse large B-cell lymphoma (DLBCL) (germinal centre B-cell - like (GCB-DLBCL) and activated B-cell - like (ABC-DLBCL)) are defined by means of gene expression profiling (GEP). Patients with GCB-DLBCL survive longer with the current standard regimen R-CHOP than patients with ABC-DLBCL. As GEP is not part of the current routine diagnostic work-up, efforts have been made to find a substitute than involves immunohistochemistry (IHC). Various algorithms achieved this with 80-90% accuracy. However, conflicting results on the appropriateness of IHC have been reported. Because it is likely that the molecular subtypes will play a role in future clinical practice, we assessed the determination of the molecular DLBCL subtypes by means of IHC at our University Hospital, and some aspects of this determination elsewhere in Switzerland. The most frequently used Hans algorithm includes three antibodies (against CD10, bcl-6 and MUM1). From records of the routine diagnostic work-up, we identified 51 of 172 (29.7%) newly diagnosed and treated DLBCL cases from 2005 until 2010 with an assigned DLBCL subtype. DLBCL subtype information was expanded by means of tissue microarray analysis. The outcome for patients with the GCB subtype was significantly better compared with those with the non-GC subtype, independent of the age-adjusted International Prognostic Index. We found a lack of standardisation in the subtype determination by means of IHC in Switzerland and significant problems of reproducibility. We conclude that the Hans algorithm performs well in our hands and that awareness of this important matter is increasing. However, outside clinical trials, vigorous efforts to standardise IHC determination are needed as DLBCL subtype-specific therapies emerge.

Holocene flood frequency across the Central Alps – solar forcing and evidence for variations in North Atlantic atmospheric circulation

The frequency of large-scale heavy precipitation events in the European Alps is expected to undergo substantial changes with current climate change. Hence, knowledge about the past natural variability of floods caused by heavy precipitation constitutes important input for climate projections. We present a comprehensive Holocene (10,000 years) reconstruction of the flood frequency in the Central European Alps combining 15 lacustrine sediment records. These records provide an extensive catalog of flood deposits, which were generated by flood-induced underflows delivering terrestrial material to the lake floors. The multi-archive approach allows suppressing local weather patterns, such as thunderstorms, from the obtained climate signal. We reconstructed mainly late spring to fall events since ice cover and precipitation in form of snow in winter at high-altitude study sites do inhibit the generation of flood layers. We found that flood frequency was higher during cool periods, coinciding with lows in solar activity. In addition, flood occurrence shows periodicities that are also observed in reconstructions of solar activity from C-14 and Be-10 records (2500-3000, 900-1200, as well as of about 710, 500, 350, 208 (Suess cycle), 150, 104 and 87 (Gleissberg cycle) years). As atmospheric mechanism, we propose an expansion/shrinking of the Hadley cell with increasing/decreasing air temperature, causing dry/wet conditions in Central Europe during phases of high/low solar activity. Furthermore, differences between the flood patterns from the Northern Alps and the Southern Alps indicate changes in North Atlantic circulation. Enhanced flood occurrence in the South compared to the North suggests a pronounced southward position of the Westerlies and/or blocking over the northern North Atlantic, hence resembling a negative NAO state (most distinct from 4.2 to 2.4 kyr BP and during the Little Ice Age). South-Alpine flood activity therefore provides a qualitative record of variations in a paleo-NAO pattern during the Holocene. Additionally, increased South Alpine flood activity contrasts to low precipitation in tropical Central America (Cariaco Basin) on the Holocene and centennial time scale. This observation is consistent with a Holocene southward migration of the Atlantic circulation system, and hence of the ITCZ, driven by decreasing summer insolation in the Northern hemisphere, as well as with shorter-term fluctuations probably driven by solar activity. (C) 2013 Elsevier Ltd. All rights reserved.

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^