Genetic variants in the NOD2/CARD15 gene are associated with early mortality in sepsis patients

Objective: Genetic variants in the NOD2/CARD15 gene resulting in adiminished capacity to activate NF-κB in response to bacterial cell wall products have been associated with Crohn's disease (CD). Recently, we found an association between the variant Leu1007fsinsC of the NOD2/CARD15 gene (SNP13) and asignificantly increased rate of transplant related mortality (TRM) due to intestinal and pulmonary complications in stem cell transplantation (SCT). To assess apossible contribution of variants in the NOD2/CARD15 gene to sepsis related mortality (SRM) we investigated 132 prospectively characterised, consecutive patients with sepsis. Design and patients: The three most common NOD2/CARD15 variants (Arg702Trp, Gly908Arg, and Leu1007fsinsC) were determined in 132 prospectively characterised patients with sepsis attended to three intensive care units at the University of Regensburg by Taqman PCR. NOD2/CARD15 genotype and major patients' characteristics were correlated with SRM. Results: Patient groups with and without NOD2/CARD15 variants did not differ in their clinical characteristics such as median age, gender, reason for admission or APACHE score; however, SRM (day30) was increased in patients with NOD2/CARD15 coding variants (42 vs. 31%) and was highest (57%) in 8 patients carrying the Leu1007fsinsC variant (p < 0.05). Multivariate analysis demonstrated the Leu1007fsinsC genetic variant as an independent risk factor for SRM. Conclusion: Our findings indicate amajor role of NOD2/CARD15 coding variants for SRM. This may be indicative for arole of impaired barrier function and bacterial translocation in the pathophysiology of early sepsis related deat

Flow Cytometric Immunophenotyping of Mature Lymphatic Neoplasias Using Knowledge Guided Cluster Analysis

Flow cytometry is widely used for the immunological characterization of hematopoietic malignancies. Discrimination of normal and malignant cellular immunophenotypes is the most critical step in data analysis, especially if multi‐color analysis is performed on highly heterogenous cell suspensions. We therefore investigated, whether adaptive, simultaneous multiparameter gating allowed automated, operator independent analysis of data obtained from the immunophenotyping of blood or bone marrow samples with regard to the presence of non‐Hodgkin lymphoma cells. The identification of physiological and malignant cells was achieved by predefining population boundaries, based on the expectations of the population’s location in two‐dimensional dot plots. The prospective application of these predefined region boundaries in 52 blood and bone marrow samples enabled identification of lymphoma cells with regard to their presence and immunophenotype, based on the correlation of markers as defined in multiple tubes. Our data confirm that highly standardized data analysis methods can reduce the variability of analysis and support the expert in establishing a rapid classification of the sample

Flow cytometric immunophenotyping of mature lymphatic neoplasias using knowledge guided cluster analysis. Anal Cell Pathol

Flow cytometry is widely used for the immunological characterization of hematopoietic malignancies. Discrimination of normal and malignant cellular immunophenotypes is the most critical step in data analysis, especially if multi-color analysis is performed on highly heterogenous cell suspensions. We therefore investigated, whether adaptive, simultaneous multiparameter gating allowed automated, operator independent analysis of data obtained from the immunophenotyping of blood or bone marrow samples with regard to the presence of non-Hodgkin lymphoma cells. The identification of physiological and malignant cells was achieved by predefining population boundaries, based on the expectations of the population&apos;s location in two-dimensional dot plots. The prospective application of these predefined region boundaries in 52 blood and bone marrow samples enabled identification of lymphoma cells with regard to their presence and immunophenotype, based on the correlation of markers as defined in multiple tubes. Our data confirm that highly standardized data analysis methods can reduce the variability of analysis and support the expert in establishing a rapid classification of the sample

E-LDL and Ox-LDL differentially regulate ceramide and cholesterol raft microdomains in human Macrophages

Atherosclerosis is characterized by the generation of lipid-loaded macrophage-derived foam cells. To study the effect of different types of atherogenic lipoproteins, human macrophages were loaded with enzymatically degraded low density lipoprotein (E-LDL) or oxidized LDL (Ox-LDL). Cellular cholesterol content was increased by E-LDL, whereas Ox-LDL increased the ceramide content. Cell surface expression analysis by flow cytometry and confocal microscopy revealed that Ox-LDL increased ceramide and lactosylceramide expression compared to E-LDL loading and induced ceramide rafts, whereas loading with E-LDL induced cholesterol-rich microdomains. Formation of different rafts may have consequences for raft associated signaling in cholesterol homeostasis and apoptosis in human macrophages

Genetic variants in the NOD2/CARD15 gene are associated with early mortality in sepsis patients

Objective: Genetic variants in the NOD2/CARD15 gene resulting in adiminished capacity to activate NF-κB in response to bacterial cell wall products have been associated with Crohn's disease (CD). Recently, we found an association between the variant Leu1007fsinsC of the NOD2/CARD15 gene (SNP13) and asignificantly increased rate of transplant related mortality (TRM) due to intestinal and pulmonary complications in stem cell transplantation (SCT). To assess apossible contribution of variants in the NOD2/CARD15 gene to sepsis related mortality (SRM) we investigated 132 prospectively characterised, consecutive patients with sepsis. Design and patients: The three most common NOD2/CARD15 variants (Arg702Trp, Gly908Arg, and Leu1007fsinsC) were determined in 132 prospectively characterised patients with sepsis attended to three intensive care units at the University of Regensburg by Taqman PCR. NOD2/CARD15 genotype and major patients' characteristics were correlated with SRM. Results: Patient groups with and without NOD2/CARD15 variants did not differ in their clinical characteristics such as median age, gender, reason for admission or APACHE score; however, SRM (day30) was increased in patients with NOD2/CARD15 coding variants (42 vs. 31%) and was highest (57%) in 8 patients carrying the Leu1007fsinsC variant (p < 0.05). Multivariate analysis demonstrated the Leu1007fsinsC genetic variant as an independent risk factor for SRM. Conclusion: Our findings indicate amajor role of NOD2/CARD15 coding variants for SRM. This may be indicative for arole of impaired barrier function and bacterial translocation in the pathophysiology of early sepsis related deat

Alteration of the pulmonary surfactant system in full-term infants with hereditary ABCA3 deficiency

RATIONALE: ABCA3 mutations are known to cause fatal surfactant deficiency. OBJECTIVE: We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis. METHODS: Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting. RESULTS: ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively. CONCLUSION: Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis