MicroRNAs 143 and 150 in whole blood enable detection of T-cell immunoparalysis in sepsis.

BACKGROUND: Currently, no suitable clinical marker for detection of septic immunosuppression is available. We aimed at identifying microRNAs that could serve as biomarkers of T-cell mediated immunoparalysis in sepsis. METHODS: RNA was isolated from purified T-cells or from whole blood cells obtained from septic patients and healthy volunteers. Differentially regulated miRNAs were identified by miRNA Microarray (n = 7). Validation was performed via qPCR (n = 31). RESULTS: T-cells of septic patients revealed characteristics of immunosuppression: Pro-inflammatory miR-150 and miR-342 were downregulated, whereas anti-inflammatory miR-15a, miR-16, miR-93, miR-143, miR-223 and miR-424 were upregulated. Assessment of T-cell effector status showed significantly reduced mRNA-levels of IL2, IL7R and ICOS, and increased levels of IL4, IL10 and TGF-ß. The individual extent of immunosuppression differed markedly. MicroRNA-143, - 150 and - 223 independently indicated T-cell immunoparalysis and significantly correlated with patient's IL7R-/ICOS-expression and SOFA-scores. In whole blood, composed of innate and adaptive immune cells, both traits of immunosuppression and hyperinflammation were detected. Importantly, miR-143 and miR-150 - both predominantly expressed in T-cells - retained strong power of discrimination also in whole blood samples. CONCLUSIONS: These findings suggest miR-143 and miR-150 as promising markers for detection of T-cell immunosuppression in whole blood and may help to develop new approaches for miRNA-based diagnostic in sepsis.

Influence of continuous combined estradiol-norethisterone acetate preparations on insulin sensitivity in postmenopausal nondiabetic women.

OBJECTIVE: Estrogen-progestogen replacement therapy (HRT) may be associated with deterioration of insulin sensitivity in comparison to estrogens alone, which tend to improve insulin sensitivity in postmenopausal women. Insulin sensitivity with the use of continuous combined 17-beta estradiol/norethisterone acetate (E2/NETA) preparations has not been examined before in postmenopausal women. DESIGN: In a double-blind randomized parallel study, we evaluated the effect of 2 mg E2/1 mg NETA (high dose E2/NETA), 1 mg E2/0.5 mg NETA (low dose E2/NETA), or placebo (P) on the insulin sensitivity index (SI) in three groups (18 women/group) of postmenopausal nondiabetic women (follicle stimulating hormone [FSH] > 40 mIU/mL, mean +/- SD) aged 56 +/- 3 years, BMI 25 +/- 4 kg/m2, cholesterol 233 +/- 42 mg/dL, and triglycerides 87 +/- 36 mg/dL. Insulin sensitivity was measured by means of a two-step hyperinsulinemic euglycemic glucose clamp (insulin infusion rate, 0.25 and 1.0 mU/kg/min for 120 min each) at baseline and after 3 months of daily administration of high dose E2/NETA, low dose E2/NETA, or P. Analysis was performed assuming equivalence of start-end changes of insulin sensitivity among treatment groups (Anderson-Hauck test). RESULTS: SI was 7.7 +/- 2.9, 7.5 +/- 3.4, 6.8 +/- 2.2 at baseline and 6.3 +/- 3.0, 7.9 +/- 2.5, 7.1 +/- 3.1 mL/min/m2 per mu U/mL 3 months after the administration of high dose E2/NETA, low dose E2/NETA, and P, respectively. The low dose E2/NETA group had start-to-end changes of SI which were equivalent to the P group (0.4 [95% confidence interval [CI] -0.8; 1.7] vs. 0.4 [-0.3; 1.0]) (p = 0.02). For the high dose E2/NETA group, equivalence could not be shown with either the P (p = 0.89) or with the low dose E2/NETA group (p = 0.90). SI within the high dose E2/NETA group decreased by -1.5 (95% CI -2.7; -0.2) mL/min/m2 per mu U/mL. HbAlc decreased from 5.3 +/- 0.3 to 5.1 +/- 0.3% within the high dose E2/NETA group (p < 0.03) and remained unchanged within the low dose E2/NETA and P group. Fasting plasma glucose, fasting serum insulin, and C-peptide, as well as triglycerides and BMI were comparable among the groups at baseline and after 3 months. Total cholesterol decreased by 12% and 8% in women treated with high dose and low dose E2/NETA (p < 0.02), respectively, and remained unchanged within the P group. CONCLUSIONS: These results indicate that 3 months use of a low dose continuous E2/NETA preparation does not change insulin sensitivity in postmenopausal women. At high dose of E2/NETA, a modest decrease seems possible. The effects of E2/NETA on other parameters of carbohydrate and lipid metabolism are neutral or favorable.

Lack of effects of the beta3-adrenoreceptor agonist UL-TG 307 on insulin sensitivity and insulin secretion in Type 2 diabetic patients.

The effects of a novel beta3-adrenoreceptor agonist, UL-TG 307, on insulin sensitivity and insulin secretion, lipid metabolism, and body weight were investigated. Thirteen diet treated male Type 2 diabetic patients participated in a randomized, double-blind, placebo controlled cross-over trial with two 14 day administration periods with placebo and UL-TG 307 (24 mg daily). After each administration period insulin secretion was assessed by means of an OGTT and insulin sensitivity was measured by an hyperinsulinaemic euglycaemic glucose clamp. Lipid metabolism was evaluated by measuring non-esterified fatty acid, glycerol, and triglyceride serum concentrations at the end of each administration period. Treatment with UL-TG 307 did not improve insulin sensitivity (insulin sensitivity index (S(I)): UL-TG 307 2.5 -/+ 0.6 (mean +/- SD) vs. placebo 2.2 +/- 0.8 ml/min*m2 per microU/ml) nor increased insulin secretion (area under the serum insulin profile AUC0-240/plasma glucose AUC0-240: UL-TG 307 8.8 +/- 7.4 vs. placebo 8.3 +/- 6.4 microU/ ml/mmol/l). No differences in lipid metabolism, metabolic control, and body weight were observed. We conclude that two weeks' administration of the beta3-adrenoreceptor agonist UL-TG 307 in a daily dose of 24 mg did not lead to any significant effect in diet treated type 2 diabetic patients.

Pharmacokinetic and pharmacodynamic properties of long-acting insulin analogue NN304 in comparison to NPH insulin in humans.

NN304 is a long-acting insulin analogue that is acylated with a 14-C-fatty acid chain. Protraction of action of this novel insulin analogue is due not to slow absorption after subcutaneous administration but to reversible binding to albumin. We investigated the pharmacokinetic and pharmacodynamic properties of insulin analogue NN304 (0.3 and 0.6 U/kg) in comparison to NPH insulin (0.3 and 0.6 IU/kg) in 10 healthy volunteers performing a randomised, double-blind, cross-over, placebo-controlled glucose clamp study. During the observation period of 24 hours the areas under the insulin curve for NPH[0.3 IU/kg] vs. NPH[0.6 IU/kg] were 60 vs. 102 nmol min l(-1) (p<0.01) and for insulin analogue NN304[0.3 U/kg] vs. NN304[0.6 U/kg] 490 vs. 932 nmol min l(-1) (p <0.001), suggesting a clear dose-response relationship for both NPH insulin and NN304. The amount of disposed glucose (area under the curve of glucose infusion) differed with statistical significance between the five treatments and was highest with NPH[0.6 IU/kg] (2671 mg/kg) and lowest with placebo (265 mg/kg). However, area under the curve of glucose infusion after treatment with NN304 was only 36% (dose of 0.3 U/kg) and 24% (dose of 0.6 U/kg) of that observed with corresponding doses of NPH insulin. Moreover, increasing dosages of NN304 failed to demonstrate a significant dose-response with regard to the area under the curve of glucose infusion. This study demonstrates that the principle of protracted insulin action of NN304 by reversible binding to albumin is effective in humans albeit at a much lower rate of glucose utilisation when compared to NPH insulin. Thus, in contrast to animal studies NN304 and NPH insulin can not be considered equipotent in humans.

Effect of the rapid-acting insulin analogue insulin aspart on quality of life and treatment satisfaction in patients with Type 1 diabetes.

AIMS: To compare quality of life (QoL) and treatment satisfaction in patients with Type 1 diabetes receiving the rapid-acting insulin analogue, insulin aspart (IAsp), with that in patients receiving soluble human insulin (HI). METHODS: In this 6-month, multinational, randomized, open-label trial, 424 patients from German-speaking countries were subjected to psychometric assessment before and after randomization (ratio 2 : 1) to basal-bolus treatment with either IAsp (n = 283) or HI (n = 141). Patients on HI were advised to keep an injection-meal interval of 30 min, whereas patients on IAsp were advised to inject immediately before meals. Treatment satisfaction and diabetes-related QoL were assessed using validated instruments to measure the domains of patients' individual treatment goals, physical complaints, worries about the future, social relations, leisure time flexibility, daily hassles, diet restrictions, burdens and fear of hypoglycaemia, blood glucose fluctuations, self-efficacy, and fear of insulin analogues. RESULTS: After 6 months, IAsp was associated with significantly greater improvement in treatment satisfaction than HI in two different scales (P < 0.01), and in QoL with respect to diet restrictions (P < 0.01). Improved satisfaction was mainly due to increased dietary and leisure time flexibility (P < 0.0001). Twenty-three percent of the IAsp group vs. 14% of the HI group achieved small but important improvements of total QoL (between-group difference, P < 0.06). The number needed to treat (NNT) with IAsp for an important increase in QoL was calculated to be 10. Regression analyses of potential predictors of improvement in QoL highlighted patients intensely striving for physical strength (P < 0.01; NNT = 7) and patients feeling less protected against hypoglycaemia (P < 0.005; NNT = 8) as being the most likely to benefit from IAsp. CONCLUSIONS: Under these study conditions, IAsp improved treatment satisfaction and quality of life regarding diet restrictions when compared with human insulin. The 'numbers needed to treat' for important quality of life benefits indicate that the effect of IAsp in this regard is not trivial.

Time-action profile of the soluble, fatty acid acylated, long-acting insulin analogue NN304.

AIMS: To compare the pharmacokinetic and pharmacodynamic properties of subcutaneously injected NN304, a novel long-acting insulin analogue, to NPH-insulin during euglycaemic glucose clamps in 11 healthy volunteers. METHODS: On three study days NN304 was injected in three different doses (0.15, 0.3, 0.6 U/kg body weight), while NPH-insulin (0.3 U/kg) was injected in identical dose on two other days. RESULTS: Injection of NN304 resulted in a linear and proportional increase in total NN304 concentrations (AUC0-1440 min: 0.15 U/kg: 344+/-43, 0.3 U/kg: 666+/-82, 0.6 U/kg: 1295+/-210 nmol/l; P<0.001). Maximal concentrations (609+/-140, 1046+/-283, 2033+/-460 pmol/l; P<0.001) were reached after 4-6 h. The metabolic response (expressed as maximal glucose infusion rates (GIR)) induced by subcutaneous injection of NN304 did not show the pronounced peak seen with NPH-insulin in an identical dose: GIRmax 3.2+/-1.1 vs. 4.4+/-1.8 mg/kg/min (P<0.05 for 0.3 U/kg NN304 vs. NPH-insulin; mean of both study days with NPH-insulin, all others not significant). NN304 also showed a slower onset of action, as indicated by a significantly higher tmax (446+/-162 vs. 359+/-175 min) and lower AUC0-240min (0.5+/-0.3 vs. 0.8+/-0.4 g/kg/240min; P<0.05, respectively). The three different doses of NN304 induced a significantly different glucose consumption in the first 720 min after injection (AUC0-720 min 1.1+/-0.6, 1.9+/-0.8, 1.7+/-0.8 g/kg; P<0.05 for 0.15 U/kg), but not over the whole study period (AUC0-1440 min 1.8+/-1.1, 3.1+/-1.3, 2.8+/-1.4 g/kg). CONCLUSIONS: Injection of NN304 at different doses resulted in an increase in total NN304 concentration in a linear dose-response effect and a more even metabolic effect than NPH-insulin. However, we found no clear dose-response in its metabolic effect.

Intramuscular versus subcutaneous injection of soluble and lispro insulin: comparison of metabolic effects in healthy subjects.

The aim of this study was to compare the glucodynamic effects of soluble insulin and the rapid acting insulin analogue insulin lispro after subcutaneous (s.c.) and intramuscular (i.m.) injection. Twelve healthy male volunteers (age 26.8 +/- 1.7 years, BMI 23.2 +/- 2.3 kg m(-2); mean +/- SD) participated in this single-centre, open-labelled, euglycaemic glucose clamp study on four different days. Soluble insulin or insulin lispro (0.2 U kg(-1)) were injected s.c. or i.m. into the thigh by syringe. The glucodynamic effects were assessed by registering the glucose infusion rates necessary to maintain blood glucose at 5.0 mmol l(-1) for the subsequent 420 min. Intramuscular injection of soluble insulin led to an earlier peak of metabolic action when compared to s.c. administered soluble insulin (tmax 138 +/- 29 vs 179 +/- 34 min; p < 0.05). The maximal metabolic effect and metabolic activity during the first 2 h after i.m. and s.c. injection of soluble insulin were comparable (GIRmax 9.7 +/- 2.3 vs 7.8 +/- 2.3 mg kg(-1) min(-1); n.s., AUC0-120min 0.60 +/- 0.18 vs 0.50 +/- 0.15 g kg(-1) 120 min; n.s.). Subcutaneous administration of insulin lispro led to a metabolic effect resembling that induced by i.m. application of soluble insulin (tmax 116 +/- 26 vs 138 +/- 29 min; n.s., GIRmax 11.1 +/- 2.3 mg vs 9.7 +/- mg kg(-1) min(-1); n.s.). However, the overall metabolic response during the first 2 h after injection was higher with s.c. insulin lispro (AUC0-120min 0.81 +/- 0.26 vs 0.60 +/- 0.18 g kg(-1) 120 min; p < 0.05). The glucodynamic activity of i.m. applied insulin lispro was comparable to that of lispro s.c.. Following i.m. injection of soluble insulin, the metabolic activity peaked more rapidly than with s.c. administration. In contrast, the metabolic effect of insulin lispro was similar with either route. The time-action profile of i.m. injected soluble insulin lies between that of s.c. applied soluble insulin and insulin lispro.

[Analysis of the effect of selection in a therapy study]. / Analyse der Selektionseffekte einer Therapiestudie.

282 patients with SCLC were treated for the first time at the Chest-Hospital Heidelberg-Rohrbach from July 1981 until November 1983. Only 85 were integrated into a German randomized multicenter study of combined chemo- and radiotherapy, the other 197 were treated with individual therapeutic regimens. Generally, randomized and non-randomized patients were similar with respect to the prognostic factors and survival, but the group of excluded patients was more heterogeneous. Further analysis revealed prognostic relevance to surgical treatment, emergency treatment, various contra-indications and a poor general condition, but patients older than 70 years should not be generally excluded. General implications for clinical trials are that the clinical and methodological relevance of each criterion of exclusion should be critically examined and the baseline data on all excluded patients collected prospectively.

Expression of phase-specific haemolymph polypeptides in a laboratory strain and field catches of Schistocerca gregaria.

Uvarov's theory of locust phase polymorphism implies a differential phase-specific gene expression. It was the goal of the present investigation to provide data on the molecular level for this concept. We used the technique of 2D gel electrophoresis to generate haemolymph polypeptide maps from mature locust males. Under the given conditions we identified 238 polypeptide spots by their molecular weight and isoelectrical point. Isolated and crowded males from the Mainz strain, all originating from crowded ancesters, differed in 20 spots: three were solitary-specific and 17 crowded-specific. Field catches of solitary and gregarious S. gregaria in Mauritania revealed that those males showed the phase-specific expression of the same 20 polypeptide spots as the Mainz strain. (It should be mentioned that the field catches and the Mainz animals differed in nine spots, but none of them was phase-specific.) As a crosscheck we reared originally solitary field catches for two generations under crowded conditions. This resulted in the repression of one solitary-specific spot and the expression of 14 gregarious/crowded-specific spots. Since JH conceivably plays a decisive role in the manifestation of the solitary phase we applied the JH analogue fenoxycarb to crowded Mainz males. 15 days after the treatment nine of the 17 gregarious-specific polypeptide spots were repressed. The fact that experimental manipulations-limited by time-cannot alter the expression of all phase-specific polypeptides may reflect the observation that, in nature, some phase characteristics can change within hours whereas others need several generations. The results provide evidence that a number of haemolymph polypeptides are expressed and repressed, respectively, in relation to phase.

Post-prandial administration of the insulin analogue insulin aspart in patients with Type 1 diabetes mellitus.

AIMS: In intensified insulin therapy, the recent development of short-acting insulin analogues with a very rapid onset of action forces a new discussion in terms of the optimal injection-meal interval. This study evaluated prandial glycaemia in patients with Type 1 diabetes following the subcutaneous injection of soluble human insulin (HI) and the insulin analogue insulin aspart (IAsp) at different injection-meal intervals and investigated whether administration of IAsp after the meal might provide satisfactory metabolic control. METHODS: In a randomized, double-blind, double-dummy, four-period crossover study, 20 Type 1 diabetic patients were investigated. Prandial insulin was administered 15 min before the start of the meal (HI(-15min)), immediately before the meal (HI(0min); IAsp(0min)) and 15 min after the start of the meal (IAsp(+15min)). RESULTS: Plasma glucose excursions from baseline levels during the 4 h (PGexc) were highest with HI(0min) (17.9 mmol.l(-1).h; P < 0.05 vs. other treatments) and were not statistically different for HI(-15min), IAsp(0min) and IAsp(15min) (13.6, 11.9 and 14.2 mmol.l(-1).h, respectively). Maximum concentration of plasma glucose (PGmax) was lowest with IAsp(0min) (11.2 mmol/l; P < 0.05 vs. other treatments). PGmax was comparable with HI(-15min), HI(0min) and IAsp(+15min) (13.3, 14.1 and 13.2 mmol/l, respectively). CONCLUSIONS: With regard to prandial glycaemia IAsp(+15min) is as effective as HI(-5min) and superior to HI(0min). Thus, post-prandial dosing of the insulin analogue IAsp offers an attractive and feasible therapeutic option for well-controlled patients with Type 1 diabetes mellitus.