T cells, natural killer cells, and γδT cells in a large patient cohort with rheumatoid arthritis: influence of age and anti-rheumatic therapy.

Objective: The aim of this cohort study was to evaluate the distribution of natural killer (NK) cells and T-cell subsets, including γδT cells, in the peripheral blood of patients with rheumatoid arthritis (RA) in a large real-life patient cohort, taking into account the patients' demographics, disease characteristics, and anti-rheumatic therapy.Method: The study recruited 508 RA patients between November 2013 and August 2015. Lymphocyte differentiation using eight-colour flow cytometry (fluorescence-activated cell sorting) of the peripheral blood was performed for all patients. Clinical data, including age, gender, disease duration, serostatus, disease activity, antibody status, immunosuppressive therapy including use of different biological disease-modifying anti-rheumatic drugs (bDMARDs) and conventional synthetic DMARDs, were retrospectively assessed using electronic patient files. Multivariate regression analysis was performed to assess the effect of these variables on T-cell, NK-cell, and γδT-cell counts.Results: The median patient age was 61.0 years and 74.1% were female. The median disease duration of RA was 12.0 years. Median Disease Activity Score based on 28-joint count was 2.8 and 56.3% were treated with bDMARDs. There were no differences in immunosuppressive therapy between different age groups. While rituximab, abatacept, and tocilizumab had no influence on lymphocyte subdifferentiation, tumour necrosis factor (TNF) inhibitors and age significantly influenced the numbers of T cells, T-helper cells, T-NK cells, NK cells, and γδT cells.Conclusion: Age and TNF-inhibition therapy influence lymphocyte subdifferentiation in patients with RA. It may be prudent to use age- and therapy-adjusted standard values for lymphocyte subsets during clinical trials and treatment of RA.

Control of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak in a day-care institution.

This article describes an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in two institutions for multi-handicapped children in Copenhagen. The aim of the study was to determine whether it was possible to eradicate MRSA in a setting with multi-handicapped children and staff where there was a high degree of physical interaction. This was a prospective interventional uncontrolled cohort study that took place from January 2003 to March 2005. All individuals in close contact with the two institutions and/or in close contact with an MRSA-colonized subject from the outbreak were included in the study: 38 children, 60 staff members and 12 close relatives of colonized subjects. Infection control measures included screening all individuals. When MRSA infection or colonization was found, an attempt was made to eradicate MRSA, staff education was undertaken and attempts were made to determine the route of transmission. Eleven individuals were found to be positive for MRSA (10.0%). All isolates were identical by pulsed-field gel electrophoresis and harboured the staphylococcal cassette chromosome mec (SCCmec) type IV. All colonized and infected individuals were associated with a single room in one of the institutions. MRSA was eradicated from all the colonized and infected subjects. This study shows that it is possible to control an MRSA outbreak in institutions for multi-handicapped children where there is a high degree of physical contact.

[Serum enzymes in grade I hypertensive patients before and following a change in nutrition in relation to polyene acid and electrolyte content]. / Serumenzyme von Hypertoniepatienten im Schweregrad I vor und nach einer veränderten Ernährung in bezug auf Polyensäuregehalt und Ionengehalt.

In patients with mild hypertension the blood pressure reduction through n-3 fatty acids can be improved by an additional increase of the potassium intake. Metabolic processes can be followed up by determinating serum enzymes. By an additional daily intake of 200 g fish for 14 days the metabolism is hardly changed. The increased activity of the transaminases after diet is probably a consequence of the increased protein intake.

[15N tracer technical studies of protein metabolism in arteriosclerosis patients]. / 15N-Tracertechnische Untersuchungen zum Eiweissstoffwechsel bei Arteriosklerosepatienten.

The incorporation of the stable isotope 15N in plasma proteins and blood cells after oral application of 3 g 15NH4Cl (95 At% 15N) per 70 kg body weight was followed up in 11 patients with ischemic heart disease or peripheral arteriosclerosis and in 7 healthy control subjects. Preliminary results indicate that the turnover of plasma protein, especially fibrin, is elevated in patients with arteriosclerosis. Investigations of platelets intimate a decreased turnover of platelet protein in patients with arteriosclerosis as compared to control subjects. Possible reasons for these alterations are discussed.

Substrate stimulation of 7 alpha-hydroxylase, an enzyme located in the cholesterol-poor endoplasmic reticulum.

We examined the role of cholesterol in altering the activity of the microsomal cytochrome P-450 enzyme, cholesterol-NADPH:oxygen oxidoreductase (cholesterol 7 alpha-hydroxylase). Liposomes were used to deliver cholesterol to hepatic microsomes. Formation of 7 alpha-hydroxycholesterol was quantitated by isotope dilution/gas chromatography-mass spectrometry. As the liposomal cholesterol/phospholipid molar ratio increased, 7 alpha-hydroxylase activity increased, whereas the activity of another microsomal cytochrome P-450 enzyme, ethylmorphine N-demethylase, decreased. To determine if the degree of stimulation was affected by the endogenous activity (without liposomes), microsomes, from rats fed chow alone or chow containing cholestyramine, taurocholate, or cholesterol were challenged with cholesterol-enriched liposomes. The degree of stimulation was dependent upon the endogenous activity: cholestyramine-fed much greater than cholesterol = chow control greater than taurocholate-fed. To determine if cholesterol stimulates 7 alpha-hydroxylase by increasing membrane viscosity, microsomes were incubated with liposomes having the same cholesterol/phospholipid molar ratio as microsomes, but different viscosities. Dipalmitoylphosphatidylcholine (high viscosity) liposomes increased microsomal viscosity and decreased 7 alpha-hydroxylase activity. In contrast, dioleoylphosphatidylcholine (low viscosity) liposomes decreased microsomal viscosity and increased enzyme activity. Since greater viscosity inhibits 7 alpha-hydroxylase, cholesterol cannot stimulate the enzyme by increasing membrane viscosity. The data suggest that cholesterol stimulates production of 7 alpha-hydroxycholesterol by providing substrate.

[Assessment of prognosis in acute myocardial infarct after reaching the maximum enzyme activity]. / Prognoseeinschätzung des akuten Myokardinfarktes nach dem Erreichen der maximalen Enzymaktivität.

A new method is described for estimation of the prognosis of acute myocardial infarction progress due to temporary decrease in the isoenzymes, i.e. malic dehydrogenase (MDH), aspartate aminotransferase (ASAT), lactic dehydrogenase (LDH), and the decrease in the creatine kinase (CK). It is shown that the time of enzymatic activity is an essential factor. If 120 hours of decrease in enzymatic activity of MDH, LDH, and CK are exceeded, the prognosis of the myocardial infarction deteriorates dramatically.

[Enzyme liberation after electrical His bundle ablation]. / Enzymfreisetzung nach Anwendung der elektrischen His-Bündel-Ablation.

The release of isoenzymes, i.e. aspartate aminotransferase (ASAT), lactic dehydrogenase (LDH), malic dehydrogenase (MDH), and of creatine kinase (CK) after electric His bundle ablation is presented. The enzyme release is increasing with the amount of energy applied during the intervention. Mitochondrial MDH and ASAT were found in six of the seven patients. Therefore, the size of myocardial necrosis by ablation can be estimated by the sequence of the release of the enzymes, and the isoenzymes, respectively.

[Changes in protein metabolism in arteriosclerosis patients]. / Veränderter Eiweissstoffwechsel bei Arteriosklerosepatienten.

The sequence of the incorporation of the stable isotope 15N in plasma protein and fibrinogen has been followed by an 15N labelling test in 11 patients with arteriosclerosis and a control group of 7 persons. The investigation showed a higher turnover of plasma protein and especially of fibrinogen in the patients with arteriosclerosis.

[Recording protein metabolism and cell proliferation with the stable isotope 15N]. / Erfassung des Eiweissstoffwechsels und der Zellproliferation mit dem stablien Isotop 15N.

Investigations of nitrogen and protein metabolism with the stable isotope 15N were carried out on 11 patients with atherosclerosis and 7 healthy control subjects. After oral application of 3 g 15NH4Cl (95 At% 15N) per 70 kg body weight the incorporation of the isotope 15N in plasma proteins and blood cells and the 15N elimination in urine were followed up. Retardations of 15N elimination, an accelerated incorporation of 15N in fibrin and a retarded 15N incorporation in platelet protein were observed in patients with atherosclerosis. The described method enables complex assertions about protein metabolism of the whole body and so represents a possibility to evaluate objectively the influence of an intervention on metabolism.

[Prognosis assessment of acute myocardial infarction on the basis of temporal changes of isoenzyme activities]. / Prognoseeinschätzung des Akuten Myokardinfarktes aufgrund zeitlicher Veränderungen von Isoenzymaktivitäten.

A new method is described for determining the prognosis of acute myocardial infarction (AMI) progress due to temporal increase in the isoenzyme, i.e. malic dehydrogenase (MDH), aspartate aminotransferase (ASAT), lactic dehydrogenase (LDH), and the increase in the creatine kinase (CK). It is shown that the time of enzymatic activity is an essential factor. If 25 hours of increase in enzymatic activity of MDH and CK are exceeded, AMI deteriorates dramatically.

[Effect of the amount of polyunsaturated acids fed to swine on the enzymes, thrombocytes and erythrocyte parameters]. / Einfluss der Gabe von PC-Säuren an Schweinen auf Enzyme und Thrombozyten und Erythrozytenparameter.

We examined in pigs after PC feeding the activities of the succinate dehydrogenase and the isocitrate dehydrogenase in the mitochondria of the liver, the fatty acid pattern of the liver, and the cell metabolism measured on the basis of selected parameters of thrombocytes and erythrocytes. A high PC proportion in the feed caused changes of the investigated parameters, in particular changes in the protein metabolism and the lipid pattern.

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes.

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.

[Malate dehydrogenase isoenzymes in myocardial infarction]. / Izofermenty malatdegidrogenazy pri infarkte miokarda.

Plasma CPK activity and the activity of isoenzymes MDH, AST and LDH were assessed in 60 patients with myocardial infarction of different severity, with reference to the time since the onset of the attack. The peaks of CPK and MDH-C activity were reached sooner than those of LDH-M and AST-C, while the CPK and MDH-C curves were similar. The severity of the disease showed correlation to later onset of enzyme peaks and markedly delayed decrease in the respective values. Increased activity of mitochondrial isoenzymes and blood LDH-M provided additional information on the severity of the disease. Delayed normalization of these activities was associated with a poor diagnosis.

An improved assay for cholesterol 7 alpha-hydroxylase activity using phospholipid liposome solubilized substrate.

A persistent problem in measurement of cholesterol 7 alpha-hydroxylase (7 alpha-OHase) activity by isotope incorporation has been solubilization of cholesterol substrate. Solubilization with Tween 20, for example, resulted in a 75% reduction in 7 alpha-OHase activity after a 60 min incubation of substrate with microsomes. Incorporation of cholesterol substrate into small, unilamellar phospholipid vesicles (liposomes) prevented this effect, resulting in a 50% increase in activity over the same 60 min incubation at optimal concentrations. Using cholesterol in liposomes as substrate, standard assay conditions were determined to be: preparation of liposomes with 180 microM cholesterol substrate and 0.5 mg phospholipid/assay; incubation of these liposomes with 0.5 mg microsomal protein at 37 C for 60 min; addition of a NADPH generating system to start the reaction, and incubation at 37 C for 30 min before stopping the reaction and determining the amount of 7 alpha-hydroxycholesterol formed. In addition to preventing the detergent-related inhibition of the enzyme, liposome-solubilized substrate also reduced the variation among replicates from a coefficient of 45% with Tween 20 to 4.2% with phospholipid. This method provides a sensitive and reliable alternative to methods which require more sophisticated equipment and allows total control of substrate concentration in a form readily accessible to the enzyme.