Markers of Chlamydia pneumoniae and human cytomegalovirus infection in patients with chronic peripheral vascular disease and their relation to inflammation, endothelial dysfunction and changes in lipid metabolism

Our aim was to detect markers of Chlamydia pneumoniae (CPN) and human cytomegalovirus (HCMV) infection in patients with peripheral vascular occlusive disease and to follow markers of inflammation, endothelial dysfunction and lipid metabolism alteration in patients with active infection. CPN genome was detected in 9 (47.4 %) patients by at least one PCR method. Serological markers of acute CPN infection were found in 5 (26.3 %) subjects; each of them showed also positivity in at least one of the PCR methods. HCMV DNA were detected in 2 (10.5 %) patients; HCMV-specific antibodies were detected in 14 (73.7 %) subjects, however only in IgG subclass. Subjects with HCMV PCR positivity thus showed no serological markers of active HCMV infection. Laboratory findings of acute CPN infection were associated with increased plasma levels of Lp(a), triacylglycerols, atherogenic index of plasma and E-selectin (p < 0.05). No significant differences were found in the other markers, including plasma levels of total cholesterol, ferritin, homocysteine, oxidized LDL, IL-6, IL-8, IL-18, TNF-α, soluble forms of VCAM-1 and ICAM-1, von Willebrand factor, C-reactive protein, and plasma nitrites & nitrates. Frequent presence of chlamydial DNA in atheromatous plaques from patients with peripheral vascular disease was confirmed. HCMV DNA was detected only sporadically and with positivity in anamnestic anti-HCMV antibodies (IgG) only, indicating a rare presence of latent virus rather than active replication. Patients with laboratory markers of acute CPN infection exhibited more pronounced alterations in lipid metabolism and endothelial dysfunction.     

Organic co-solvents affect activity,stability and enantioselectivity of haloalkane dehalogenases

Haloalkane dehalogenases are microbial enzymes with a wide range of biotechnological applications, including biocatalysis. The use of organic co-solvents to solubilize their hydrophobic substrates is often necessary. In order to choose the most compatible co-solvent, the effects of 14 co-solvents on activity, stability and enantioselectivity of three model enzymes, DbjA, DhaA, and LinB, were evaluated. All co-solvents caused at high concentration loss of activity and conformational changes. The highest inactivation was induced by tetrahydrofuran, while more hydrophilic co-solvents, such as ethylene glycol and dimethyl sulfoxide, were better tolerated. The effects of co-solvents at low concentration were different for each enzyme-solvent pair. An increase in DbjA activity was induced by the majority of organic co-solvents tested, while activities of DhaA and LinB decreased at comparable concentrations of the same co-solvent. Moreover, a high increase of DbjA enantioselectivity was observed. Ethylene glycol and 1,4-dioxane were shown to have the most positive impact on the enantioselectivity. The favorable influence of these co-solvents on both activity and enantioselectivity makes DbjA suitable for biocatalytic applications. This study represents the first investigation of the effects of organic co-solvents on the biocatalytic performance of haloalkane dehalogenases and will pave the way for their broader use in industrial processes.     

Comparison of four routinely used vitamin D automated immunoassays

BackgroundTo compare four automated immunoassays for the measurement of 25(OH)-vitamin D (25-OHD) and to assess the impact on the results obtained from a healthy population.MethodsWe analysed 100 serum samples on Unicel DxI 800 (Beckman Coulter), Architect i1000 (Abbott), Cobas e411 (Roche) and Liaison XL (DiaSorin). Passing-Bablok regression and Bland-Altman plots were used for method comparison. In order to categorise the obtained values, results were categorised into the following groups: 0-25 nmol/L, 25-50 nmol/L, 50-75 nmol/L and above 75 nmol/L and compared. The percentage of samples below 75 nmol/L, and below 50 nmol/L was then calculated for every method.ResultsAccording to paired comparisons, each method differs from others (p<0.0001) except Cobas vs Architect, which do not show a statistically significant difference (p=0.39). The strongest correlation was found between Liaison and Architect (ρ=0.94, p<0.0001). The percentage of samples below the recommended value of 75 nmol/L were: 70% (Architect), 92% (Liaison), 71% (Cobas) and 89% (Unicel). The percentage of samples below the value of 50 nmol/L were: 17% (Architect), 55% (Liaison), 28% (Cobas) and 47% (Unicel).ConclusionsThe observed differences stem from the use of different analytical systems for 25-OHD concentration analysis and can result in different outcomes. The recommended values should be established for each assay in accordance with the data provided by the manufacturer or in the laboratory, in accordance with proper standardisation.     

HLA-DR7-Specific monoclonal antibodies and a chimpanzee anti-DR7 serum detect different epitopes on the same molecule

We describe here two monoclonal antibodies with HLA-DR7 serologic specificity. The antibodies, SFR16-DR7M, a cytotoxic rat IgM antibody of high affinity, and SFR16-DR7G, a noncytotoxic antibody of the rat IgG 2a class, react with only DR7-positive cells in radioimmunoassay. The cytotoxic activity of SFR16-DR7M correlates completely with the presence of the DR7 specificity, and segregates with the DR7-bearing haplotype in a family. SFR16-DR7M precipitates a class II molecule with the electrophoretic characteristics of DR molecules from LG-10, an HLA-DR7 homozygous cell line. SFR16-DR7G completely inhibits the cytotoxicity of SFR16-DR7M, but only partially inhibits the cytotoxicity of a chimpanzee antiserum with DR7 specificity, Gay/Swei. In binding-inhibition studies, binding of SFR16-DR7M to LG-10 cells is only partially inhibited by the chimpanzee antiserum and vice versa. Both SFR16-DR7M and Gay/Swei reciprocally deplete the same class II molecules from a ³⁵S-methionine-labeled detergent-solubilized membrane preparation of the LG-10 cell line. The chimpanzee serum Gay contains antibodies reactive with epitopes on separated DR7 beta chains, while both SFR16-DR7M and SFR16-DR7G bind only to DR7 alpha-beta complexes. These data suggest that at least two allogeneic epitopes exist which result in the same serologic specificity, and that these epitopes differ in their requirement for alpha-beta complex formation.