Application of mycobacterial proteomics to vaccine design: improved protection by Mycobacterium bovis BCG prime-Rv3407 DNA boost vaccination against tuberculosis

Information from comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guerin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six M. tuberculosis DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol M. tuberculosis infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost "on top" of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.     

Sequence of Echinochloa hoja blanca tenuivirus RNA-5

The sequence is presented of RNA-5 of Echinochloa hoja blanca tenuivirus, a second tenuivirus associated with rice cultivation in Latin America (after rice hoja blanca virus). The RNA is 1334 nucleotides long and contains in the complementary sense RNA a single long open reading frame. The deduced amino acid sequence of this open reading frame shows that it encodes a highly basic and hydrophilic 44 kD protein (pc5) with about 50% similarity to the pc5 protein of maize stripe virus (MStV). This and other features of the RNA are discussed.The GenBank accession number of the sequence reported in this paper is L47430.     

Efficacy of Amodiaquine in uncomplicated falciparum malaria in Nigeria in an area with high-level resistance to Chloroquine and Sulphadoxine/Pyrimethamine

Falciparum Malaria is hyperendemic in southern Nigeria and chloroquine resistance is an increasing problem. Therefore, the parasitological and haematological response to treatment with amodiaquine was studied in children under 5 years during a 14-day follow-up. Of 105 children who accomplished the study (out of 114 who were enrolled), 95.3% were parasite-negative on thick blood film on day 7, which decreased to 89.5% on day 14. The haemoglobin levels increased on average by 1.3% on day 14 (±1.9) and more pronounced in children with anaemia < 10 g/dl on enrolment. The number of patients with adverse events (mainly pruritus and nausea) was few. This study shows that amodiaquine is effective, safe and affordable in an area with high resistance to chloroquine.     

Genomic gain of PIK3CA and increased expression of p110alpha are associated with progression of dysplasia into invasive squamous cell carcinoma

The regulation of apoptosis in atherosclerosis is not completely defined. The aim of this study was to determine the expression of Bcl-2, Bcl-x, Bax, and Bak in relation to apoptosis in advanced atherosclerotic lesions. In atherectomy (15), endarterectomy (10), and control non-atherosclerotic segments of renal (2) and of coronary and carotid (5) arteries, the extent of apoptosis was determined using TdT dUTP nick end labelling (TUNEL) and nuclear morphology (karyorrhexis/pyknosis) and expression of apoptosis regulators by immunohistochemistry and western blot analysis on paraffin-embedded material. In all specimens, the atherosclerotic involvement was advanced: grade V (n=18) and grade VI (n=7). The apoptotic index was high (mean 30%) in advanced lesions compared with controls (<2%) and smooth muscle cells (SMCs) were the predominant cell type undergoing apoptosis. In all TUNEL-positive apoptotic cells, Bax and Bak were present, while Bcl-x was absent. Bcl-2 was absent in a majority of these cells, but occasional TUNEL-positive cells expressed Bcl-2. In non-apoptotic cells, Bcl-x was present and western blot detected only the long isoform, Bcl-xL, from the plaques. In conclusion, increased Bax and Bak coupled with lack/paucity of Bcl-2 and Bcl-xL are associated with SMC apoptosis in advanced lesions. Bcl-xL in non-apoptotic cells appears to contribute to prolonged cell survival.     

Moraxella catarrhalis--infected alveolar epithelium induced monocyte recruitment and oxidative burst

The recruitment of monocytes appears to be a crucial factor for inflammatory lung disease. Alveolar epithelial cells contribute to monocyte influx into the lung, but their impact on monocyte inflammatory capacity is not entirely clear. We thus analyzed the modulation of monocyte oxidative burst by A549 and isolated human alveolar epithelial cells. Epithelial infection with Moraxella catarrhalis induced monocyte adhesion, transepithelial migration, and superoxide generation, whereas stimulation with lipopolysaccharide, tumor necrosis factor-alpha, interleukin-1beta, or interferon-gamma induced adhesion or transmigration, but failed to initiate monocyte burst. The effect of microbial challenge was mimicked by phorbol myristate acetate and inhibited by the protein kinase C inhibitor bisindoylmaleimide. Furthermore, evidence for a role of platelet-activating factor-signaling in monocytes is presented. Monocyte burst was neither induced by supernatant nor affected by fixation of A549 cells, excluding the contribution of epithelium-derived soluble factors but emphasizing the mandatory role of intercellular contact. The employment of blocking antibodies, however, denied a role for the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, or CD11b/CD18 and CD49d/CD29. In essence, infection of alveolar epithelial cells with M. catarrhalis might amplify the inflammatory capacity of invading monocytes eliciting their superoxide production. The epithelial response to this microbial challenge thus clearly differed from that to proinflammatory cytokines.     

Control of focal adhesion dynamics by material surface characteristics

The mechanisms of cell adhesion to the extracellular matrix (ECM) which are of fundamental importance for function, survival, and growth of cells involve the formation of focal adhesions to facilitate integrin signaling. Recently, it became evident that focal adhesions are not stable but move to enable cell migration and ECM formation. We examined the number, size, and dynamic behavior of focal adhesions in living MG-63 osteoblastic cells, which were cultured on titanium surfaces with different roughnesses and on stainless steel (SS). As a marker for focal adhesions we used GFP-tagged vinculin, a cytoskeletal protein. Focal adhesions were smaller on titanium and on SS than on collagen-coated glass coverslips. The corundum-blasted rough surface of titanium induced the smallest adhesions. On all the surfaces that we have tested, we observed a mobility of focal adhesions. On collagen-coated coverslips focal adhesions moved with a speed of 60 nm/min. The speed was reduced on titanium and still more restricted on SS. The topography did not affect the mobility of focal adhesions. We conclude that on the material surfaces that we have studied a reduced mobility of focal adhesions may strengthen the linkages between cell and ECM but impair the ability to dynamically organize and remodel the ECM. The results may have a great impact in the functional evaluation of tailored biomaterial surfaces for the application in tissue engineering.     

Analysis of gene expression patterns in small amounts of human ventricular myocardium by a multiplex RNase protection assay

 End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology, clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the β₁-adrenoceptor, the stimulatory G protein α-subunit (G_sα), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), β-myosin heavy chain (β-MHC), and the atrial natriuretic peptide (ANP). We extracted 10.7±2.1 μg total RNA from three myocardial biopsies taken in vitro. All of the six genes were measurable in duplicate in a total of 7 μg RNA. mRNAs of β₁-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively, whereas β-MHC and G_sα mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and G_sα levels obtained by Northern blot analysis with 7.5 μg total RNA showed a good correlation between the two methods. The RNase protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies. Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure. Received: 12 May 1997 / Accepted: 8 September 1997     

The effect of tourniquet release timing on perioperative blood loss in simultaneous bilateral cemented total knee arthroplasty: a prospective randomized study.

Today the use of pneumatic tourniquet is commonly accepted in total knee arthroplasty (TKA) to reduce perioperative blood loss. There are a few prospective randomised and nonrandomised studies that compare the effect of tourniquet release timing in cementless or cemented unilateral TKA. However, many of these studies show an inadequate reporting and methodology. This randomized prospective study was designed to investigate the efficiency of tourniquet release timing in preventing perioperative blood loss in a simultaneous bilateral TKA study design. To our knowledge, this is the first study of its kind, in which the effect of tourniquet release timing on perioperative blood loss was investigated in simultaneous bilateral cemented TKA to compare both techniques intraindividually. In 20 patients (40 knees) one knee was operated with tourniquet release and hemostasis before wound closure, and the other knee with tourniquet release after wound closure and pressure dressing. We found no significant difference in total blood loss between both techniques (p=0.930), but a significant difference in operating time (p=0.035). There were no postoperative complications at a follow-up of 6 month. Other studies report an increase the blood loss in early tourniquet release and an increase the risk of early postoperative complications in deflation of tourniquet after wound closure. In this study we found no significant difference in perioperative blood loss and no increase of postoperative complications. Therefore, we recommend a tourniquet release after wound closure to reduce the duration of TKA procedure and to avoid possible risks of extended anaesthesia.     

Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts.To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17±4 g hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8±2.3 to 45.1±3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7±89.8 to 2,181±135.4 mmHg/s in the contraction rate, and 713±60.2 to 1,364±137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.