Meta-Analysis of Linkage Studies for Complex Diseases: An Overview of Methods and a Simulation Study

Linkage genome scans for complex diseases have low power with the usual sample sizes, and hence meta‐analysis of several scans for the same disease might be a promising approach. Appropriate data are now becoming accessible. Here we give an overview of the available statistical methods and current applications. In a simulation study, we compare the power of different methods to combine multipoint linkage scores, namely Fisher's p‐value combination, the truncated product method, the Genome Search Meta‐Analysis (GSMA) method and our weighting methods. In particular, we investigate the effects of heterogeneity introduced by different genetic marker sets and sample sizes between genome scans. The weighting methods explicitly take those differences into account and have more power in the simulated scenarios than the other methods.     

Inflammatory regulation of extracellular matrix remodeling in calcific aortic valve stenosis.

BACKGROUND: Calcific aortic stenosis (AS), the most frequent heart valve disorder in developed countries, leads to the calcification and fibrous thickening of the valve. While several studies have addressed the process of valvular calcification, the molecular pathomechanisms of the extensive matrix remodeling remain unclear. Because inflammation is present in stenotic valves, we hypothesized that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) might influence cell proliferation and regulate the expression and activation of matrix metalloproteinases (MMPs)--enzymes that are thought to be involved in calcific AS. METHODS: Immunohistochemistry for leukocytes, TNFalpha, MMP-1, and the endogenous MMP inhibitor tissue inhibitor of metalloproteinase (TIMP)-1 was performed on human stenotic (n = 19) and control (n = 8) valves. Primary cultures of human aortic valve myofibroblasts were incubated with and without TNFalpha, and cell proliferation was assessed. The expression and activation of MMP-1 were detected by Western blotting and a specific MMP-1 activity assay. RESULTS: Control valves showed scattered macrophages and low expression of TNFalpha, MMP-1, and TIMP-1. In stenotic valves, leukocyte infiltration and a strong, colocalized expression of TNFalpha and MMP-1 were present, while TIMP-1 remained unchanged. Double-label immunofluorescence localized TNFalpha mainly to macrophages. In cultured human aortic valve myofibroblasts, TNFalpha stimulated proliferation and induced a time-dependent increase in MMP-1 expression and activation, while TIMP-1 remained unchanged. CONCLUSION: The results indicate that matrix remodeling in calcific AS involves the expression and activation of MMPs. Activated leukocytes, by the secretion of TNFalpha, may stimulate valvular myofibroblasts to proliferate and express MMPs, thus regulating actively the matrix remodeling in calcific AS.     

Susceptibility to beta-lactam antibiotics and production of beta-lactamase inBacteroides fragilis

Using the agar dilution technique, 231 strains of Bacteroides fragilis, isolated during a 2-year period from human infections, were identified at subspecies level and were tested for susceptibility to 13 beta-lactam antibiotics. The penicillins were benzylpenicillin, ampicillin, carbenicillin, cloxacillin, and the recently described penicillin derivatives cyclacillin, ticarcillin, and PC-904. The following cephalosporin derivatives were tested: cephaloridine, cephalothin, cephalexin, cefamandole and cefuroxime. The cephamycin C derivative cefoxitin was also included in the study. Cefoxitin was the most effective drug tested since more than 80% of the strains were inhibited by 8 microgram/ml or less, and no strain had a minimal inhibitory concentration (MIC) of more than 64 microgram/ml. There was no marked difference in sensitivity among the subspecies with exception of subspecies vulgatus, which was slightly more sensitive to all antibiotics tested. The size of the inoculum was an important factor for obtaining reproducible results in the sensitivity tests. Increased inocula resulted in markedly higher MICs for cephaloridine and cefuroxime. Production of beta-lactamase was performed on all isolates by a chromogenic cephalosporin substrate and about 90% of the strains were found to be beta-lactamase producers.     

Coagulopathy after snake bite byBothrops neuwiedi: Case report and results of in vitro experiments

Summary Coagulation studies were performed in a patient who had been bitten by a snake of the speciesBothrops neuwiedi. The patient presented with hemorrhagic necrosis at the envenomization site and considerable bleeding from venous puncture sites. He developed a severe defibrination syndrome with a clottable fibrinogen level of approximately 0.1 g/l. Fibrinogen was not measurable by clotting time assay. Fibrin degradation products were greatly elevated. Treatment with antivenom caused an anaphylactic reaction within ten minutes and serum sickness after three days. In vitro experiments revealed thatB. neuwiedi venom directly activates Factors II and X, but does not activate Factor XIII. In vivo consumption of Factor XIII afterB. neuwiedi envenomization is ascribed to the action of Factor IIa. At low venom concentrations clotting is initiated by activation of prothrombin by the venom either directly or via Factor X activation. Treatment with heparin might be beneficial in coagulopathy secondary to snake bite by reducing circulating active thrombin. The venom contains thrombinlike proteases which cause slow clotting of fibrinogen, and plasmin-like components causing further proteolysis of fibrinogen and fibrin. Antivenom has no effect on the proteolytic action of the snake venom. The in vivo effects of antivenom are presumably caused by acceleration of the elimination of venom components from the circulation. Intravenous administration of antivenom caused normalization of blood coagulation parameters within 48 h.     

Expression of bone sialoprotein and bone morphogenetic protein-2 in calcific aortic stenosis

BACKGROUND AND AIM OF THE STUDY: Calcific aortic stenosis, the major heart valve disease encountered in the elderly, leads to massive calcium deposition in the valve leaflets that morphologically resembles bone formation. Recent studies have demonstrated the expression of various bone-associated proteins in stenotic valves, suggesting that valvular calcification may be an actively regulated process. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, and bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor cytokine superfamily, are known to participate in the regulation of bone development and maturation. Their pathogenetic role in calcific aortic stenosis is unknown. METHODS: Using an immunoperoxidase technique and antibodies against BSP and BMP-2, the expression of BSP and BMP-2 was examined in 16 human aortic valves with calcific aortic stenosis obtained at valve replacement, and in seven normal autopsy controls without signs of aortic stenosis. RESULTS: By semiquantitative scoring, stenotic valves showed a significantly increased staining of BSP in cells and extracellular matrix as compared to control valves (2.7 +/- 0.1 versus 0.6 +/- 0.2 score units, p <0.001). Marked BMP-2 expression was detected in stenotic valves, mostly in cell-rich areas associated with focal calcium deposits, but no specific staining for BMP-2 was detected in control valves (1.5 +/- 0.2 versus 0.0 +/- 0.0 score units, p <0.001). CONCLUSION: These results demonstrate for the first time that BSP and BMP-2 are differentially expressed in normal aortic valves and in aortic stenosis, thereby supporting the concept that valvular calcification might be based on an actively regulated process involving BSP and BMP-2.