Serum levels of endothelial injury markers creatine kinase-BB and soluble thrombomodulin during human liver transplantation.

Endothelial damage within the sinusoids of the liver probably plays a key role in primary liver dysfunction following transplantation. The aim of this work was to study the serum levels of two potential markers of endothelial damage, creatine kinase-BB and soluble thrombomodulin, during human graft revascularization. Thirteen human liver grafts were preserved in UW solution (mean time: 13.8 h). Creatine kinase-BB and transaminase activities and soluble thrombomodulin levels were measured: 1) in effluent and 2) in serum samples sequentially collected before revascularization, then during the first 120 min of revascularization and first post-operative week. No correlation was observed between serum values (peak) and effluent values. In serum, pre-operative creatine kinase-BB activities were correlated with soluble thrombomodulin levels (p = 0.01). Both increased significantly during the first minutes of the revascularization, then decreased markedly. In contrast, AST activity was maximal at day 1. This detectable and early release of creatine kinase-BB and soluble thrombomodulin in blood is in keeping with the early occurence of endothelial damage. Together with previous data, these findings suggest that serum determination of these two markers may be a useful tool in the assessment of endothelial injury in liver transplantation.

Influence of age, sex and ABO blood group on activated partial thromboplastin time.

To evaluate the influence of age, sex and ABO blood group on the activated partial thromboplastin time (APTT), we performed this test in 642 preoperative ambulant adult subjects. There was a significant negative correlation (R = 0.31, p < 0.001) between age and APTT. Sex and ABO blood group had a significant influence on APTT, with lower mean in females (30.9 s) and non-O subjects (30.7 s) than in males (31.6 s) and O subjects (32.0 s) (p = 0.015 and < 0.001, respectively). The largest difference between the upper cut-off values of APTT determined in patient subgroups defined on age, sex and ABO blood group is observed for non-O females over 40 years (36.9 s) and O males under 40 years (41.1 s). These results emphasize the laboratory's difficulties to define valid APTT normal range and thus to detect true mild coagulation disorders in preoperative asymptomatic patients.

Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture.

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was conducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhesion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27 kDa proteins. Indeed, adhesion to Caco-2 cell monolayers of C. difficile strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficile may utilize blood components as adhesins to adhere to human intestinal cultured cells.

[Characterization of anti-mitochondrial antibodies type 2 (anti-M2): comparison of two techniques, western blotting and indirect immunofluorescence]. / Caractérisation des anticorps antimitochondries de type 2 (anti-M2): comparaison de deux techniques, immunotransfert et immunofluorescence indirecte.

About 94% of patients with typical features of primary biliary cirrhosis (PBC) have been shown to be anti-M2 positive. Today the relevance of anti-M2 antibodies as a diagnostic marker of PBC is well established. The usual method of detection is by indirect immunofluorescence with cryostat sections of rat organs. In our laboratory we have developed a second identification technique for these antibodies: Western-blotting. To compare immunofluorescence and immunoblotting results, we selected sera from 252 patients: 142 sera from patients with documented PBC, 50 from patients with another hepatic disease, 10 from patients with haematological lupus and 50 from healthy blood donors. We characterized antimitochondrial antibody M2 by the presence of one or more of five antigenic determinants: 65-70 kDa (a), 52-54 kDa (b), 44 kDa (c), 23-26 kDa (d) and 16-20 kDa (e). This technique is especially useful as a backup method intention for identifying a very slight or atypical fluorescence pattern.

Use of purified F1845 fimbrial adhesin to study localization and expression of receptors for diffusely adhering Escherichia coli during enterocytic differentiation of human colon carcinoma cell lines HT-29 and Caco-2 in culture.

Whole diffusely adhering Escherichia coli (DAEC) C1845 cells bearing the F1845 adhesive factor bind diffusely to differentiated human colon carcinoma cell lines HT-29 and Caco-2. By using antibodies directed against the purified fimbrial adhesin F1845 factor, the expression of the DAEC F1845-specific brush border receptors in the polarized human intestinal HT-29 and Caco-2 epithelial cells was studied by indirect immunofluorescence. A low level of DAEC F1845 receptors in undifferentiated intestinal cells was detected; they were localized in a cluster of cells. DAEC F1845 receptors were expressed at a high level in differentiated HT-29 and Caco-2 cells. DAEC F1845 receptors were expressed at a strikingly high level in the apical domains of the cells and developed during enterocytic differentiation in culture, in parallel with the apical expression of the intestinal brush border hydrolase, sucrase-isomaltase.