T-lymphocyte and glycemic status after vitamin D treatment in type 1 diabetes: A randomized controlled trial with sequential crossover.

BACKGROUND: Type 1 diabetes mellitus (T1D) is mediated by autoaggressive T effector cells with an underlying regulatory T-cell (Treg) defect. Vitamin D deficiency is highly prevalent in T1D, which can aggravate immune dysfunction. High-dose vitamin D treatment may enhance Tregs and improve metabolism in T1D patients. METHODS: In a randomized double-blind placebo-controlled trial with crossover design, patients received either for 3 months cholecalciferol 4000 IU/d followed by 3 months placebo or the sequential alternative. Thirty-nine T1D patients (19 women and 20 men) completed the trial. RESULTS: Primary outcome was a change of Tregs, secondary HbA1C, and insulin demand. Effects were evaluated based on intra-individual changes between treatment and placebo periods for outcome measures. Exploratory analyses included vitamin D system variant genotyping and C-peptide measurements. Median 25(OH)D3 increased to 38.8 ng/ml with males showing a significantly stronger increase (p = .003). T-lymphocyte profiles did not change significantly (p > 2); however, the intra-individual change of Tregs between males and females was different with a significantly stronger increase in men (p = .017), as well as between genotypes of the vitamin D receptor (Apa, Taq, and Bsm: genotypes aa, TT, and bb; p = .004-0.015). Insulin demands declined significantly (p = .003-.039) and HbA1C improved (p < .001). Random C-peptide levels were low but rising (median, 0.125 ng/ml; range, 0.02-0.3) in 6 patients. No toxicity was observed. CONCLUSION: A daily vitamin D dose of 4000 IU for 3 months was well tolerated and enhanced Tregs in males. Glucometabolic control improved in all. Subsequent larger trials need to address ß-cell function and genotyping for individualized vitamin D doses.

Effect of a randomised controlled vitamin D trial on insulin resistance and glucose metabolism in patients with type 2 diabetes mellitus.

The aim of our study was to investigate the influence of a 6-month vitamin D supplementation in patients with noninsulin-requiring type 2 diabetes mellitus. We included 86 patients in a placebo-controlled, randomised, double-blind study. During 6 months patients received Vigantol oil once a week corresponding to a daily dose of 1904 IU or placebo oil, followed by 6 months of follow-up. At start and at 3-month intervals 25OHD, PTH, body mass index, HbA1c, insulin, C-peptide, and homeostasis model assessment-index were measured. The primary outcome was a change in fasting blood glucose and insulin levels. After 6 months of therapy, the verum group's 25OHD had increased to a median of 35 ng/ml in comparison to the placebo group (median 20 ng/ml, p<10-6). PTH tended to decrease in the verum group (25.5 pg/ml vs. 35.0 pg/ml, p=0.08). After 6 months of therapy, 31 patients (78%) achieved a 25OHD concentration of >20 ng/ml. Their HbA1c was significantly lower at baseline (p=0.008) and after therapy (p=0.009) than in patients with 25OHD below 20 ng/ml. C-Peptide, insulin, and HOMA-index did not change significantly in the verum group but fasting insulin was positively correlated with 25OHD concentrations after 6 months of therapy in both groups. There were no significant effects of vitamin D with a daily dose of 1904 IU on metabolic parameters in type 2 diabetes. However, the correlative findings of this study suggest a link of the 25OHD status and metabolic function in type 2 diabetes. Whether vitamin D therapy with higher doses can improve glucose metabolism needs to be investigated in follow-up trials.

Efficacy and longevity of newly developed catnip oil microcapsules against stable fly oviposition and larval growth.

The stable fly, Stomoxys calcitrans (Diptera: Muscidae), is one of the most important pests of cattle and costs U.S. cattle producers billions of dollars in losses annually. In this study, the efficacy of catnip oil encapsulated in gelatin in oviposition deterrence and larval growth inhibition in stable flies was examined under laboratory conditions. More than 98% inhibition of stable fly larval growth and female oviposition was observed in larval and oviposition media treated with encapsulated catnip oil (0.5 g). Further, dose-response tests showed that as little as 0.1 g of encapsulated catnip oil provided > 85% oviposition deterrence. The release of nepetalactones from the capsules was more rapid when the capsules were placed on a moist substrate rather than a dry substrate. Encapsulated catnip oil also exhibited antibacterial activity, supporting the hypothesis that its inhibition of larval growth may be based on its killing of the bacteria on which larvae feed. The use of encapsulated catnip oil can provide an alternative control strategy for stable fly management.

Response of radiation-induced fibrosarcoma-1 in mice to cyclophosphamide monitored by in vivo 31P nuclear magnetic resonance spectroscopy.

In vivo 31P nuclear magnetic resonance spectroscopy has been used to examine the RIF-1 fibrosarcoma in mice during untreated growth and following chemotherapy with cyclophosphamide. Levels of inorganic phosphate increase relative to phosphocreatine or nucleoside triphosphates during early untreated growth. After the tumor reaches a volume of approximately 1 g, no further decrease in energy level is observed. Following treatment with cyclophosphamide, tumor phosphorus metabolite ratios and pH are significantly altered, compared to untreated age-matched controls. During the growth delay period following chemotherapy there is a significant reduction in the ratio of inorganic phosphate to other phosphate metabolites, compared to age-matched controls. In addition, a more alkaline pH is observed in the tumors of treated animals. When the growth delay period ends, nuclear magnetic resonance spectra return to pretreatment patterns. The magnitude of the differences in 31P nuclear magnetic resonance spectral parameters between treated animals and untreated controls is dose dependent. However, doses of cyclophosphamide above 200 mg/kg do not result in earlier spectroscopic alterations, nor in larger effects by Day 3 after treatment, even though clonogenic cell killing and growth delay are greater at these higher doses.

In vivo 31P nuclear magnetic resonance spectroscopy of subcutaneous 9L gliosarcoma: effects of tumor growth and treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea on tumor bioenergetics and histology.

In vivo 31P nuclear magnetic resonance spectroscopy was used to examine the bioenergetics of the rat 9L gliosarcoma during untreated growth and in response to chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea. Tumor growth was associated with a decline in the phosphocreatine and nucleoside triphosphate resonances, consistent with an increase in tumor hypoxia during untreated growth. Following chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (10 mg/kg), tumor levels of phosphocreatine and nucleoside triphosphate rebounded while the level of inorganic phosphate in the tumor declined. Histological comparison of treated and untreated tumor sections 4 days posttreatment showed that the treated tumor had a lower proportion of necrotic cells, a higher proportion of viable cells, and a 5-fold higher level of interstitial space than the control tumor.

31P-nuclear magnetic resonance studies of the effect of recombinant human interleukin 1 alpha on the bioenergetics of RIF-1 tumors.

The effect of a single injection of human recombinant interleukin 1 alpha (IL-1 alpha) on s.c. RIF-1 tumors in mice was studied by in vivo 31P nuclear magnetic resonance spectroscopy. Spectra were obtained before and up to 24 h after IL-1 alpha. At 2, 4, 6, and 8 h after IL-1 alpha injection, RIF-1 tumors exhibited a reduction in bioenergetic status compared to untreated controls. The Pi to beta-nucleoside triphosphate and the phosphomonoester to beta-nucleoside triphosphate ratios increased, while the phosphocreatine to Pi and phosphodiester to phosphomonoester ratios decreased. Tumor blood flow, estimated by 86RbCl uptake, decreased within 30 min after IL-1 alpha treatment. Minimum perfusion was detected at 4 h, with recovery between 6 and 12 h after IL-1 alpha treatment. Histological sections of the RIF-1 tumors revealed intravascular congestion by 2 h, extravascular hemorrhage by 4 h, and necrosis by 12 h after treatment with IL-1 alpha. The time course of bioenergetic changes in RIF-1 tumors determined by 31P-NMR spectroscopy was found to parallel the reduction and subsequent recovery of tumor blood flow.

Potentiation of interleukin 1 alpha mediated antitumor effects by ketoconazole.

In the present studies, the regulatory role of adrenal hormones on the antitumor activity of recombinant human interleukin 1 alpha (IL-1 alpha) was investigated. Ketoconazole, a potent but transient inhibitor of adrenal steroid hormone biosynthesis, inhibited IL-1 alpha induced increases in plasma corticosterone. In s.c. RIF-1 tumors (C3H/HeJ mice) ketoconazole potentiated IL-1 alpha induced hemorrhagic necrosis (59Fe labeled RBC uptake) and prolonged intervals of low tumor perfusion (86Rb+ uptake) and attendant depletion of tumor high energy phosphate reserves as determined by in vivo 31P nuclear magnetic resonance spectroscopy. In normal muscle and skin the ketoconazole-IL-1 alpha combination had no effect on RBC content and little or no effect on tissue perfusion. Ketoconazole potentiation of IL-1 alpha induced tumor pathophysiologies was accompanied by time and ketoconazole dose dependent potentiation of RIF-1 tumor clonogenic cell killing. Although ketoconazole at 40 mg/kg and IL-1 alpha at 25 micrograms/kg alone each produced approximately 50% clonogenic cell kill, a combined treatment (IL-1 alpha 1 h after ketoconazole) resulted in surviving fractions of approximately 1.5%. In vitro, ketoconazole and IL-1 alpha induced only additive clonogenic cell kill in primary RIF-1 explant cultures. The effect of elevated plasma corticosterone levels, induced by ketamine-acepromazine anesthesia, on IL-1 alpha responsiveness was also studied in the RIF-1 tumor model. In C3H/HeJ mice, anesthesia increased plasma corticosterone levels within 30 min, abrogated the IL-1 alpha effect on tumor perfusion, and prevented depletion of tumor high energy phosphate metabolite reserves. Our results are consistent with the hypothesis that IL-1 alpha mediated adrenal hormone responses exert a profound negative feedback on IL-1 alpha antitumor activities. Our data also indicate that adrenal steroid hormone biosynthetic pathways could provide a focus for modulation strategies to increase the efficacy of cytokine based therapeutic interventions.

Proton nuclear magnetic resonance methyl and methylene linewidths from plasma decrease during postprandial lipemia.

Narrow proton nuclear magnetic resonance (1H-NMR) linewidths from plasma have been associated with the presence of malignancy (Fossel et al., New Engl. J. Med. (1986) 315, 1369-1376). In that study, subjects and controls were not fasted. In the present study, 1H-NMR methyl and methylene linewidths were measured in plasma from normolipemic individuals without cancer both during fasting and every 90 min after eating a fat meal. Plasma lipoprotein levels were measured in order to relate results to postprandial lipemia. Methyl, methylene, and average 1H-NMR linewidths were strongly positively correlated with high-density lipoprotein levels and inversely correlated with triacylglycerol-rich lipoprotein levels in both the fasting and postprandial states. Linewidths decreased postprandially, reaching a nadir at the peak of plasma triacylglycerol levels. This study demonstrated that postprandial lipemia can lead to narrowing of plasma methyl and methylene resonances comparable to that reported for subjects with cancer.

Energy metabolism, pH changes, and lactate production in RIF-1 tumors following intratumoral injection of glucose.

The metabolic consequences of increased glucose availability were examined in subcutaneous RIF-1 tumors in vivo, using 13C and 31P NMR spectroscopy. Significant increases in the levels of nucleotide triphosphates and phosphocreatine relative to low energy phosphates and in tumor pH were observed within 30 min following injection of 1 g/kg of glucose directly into the tumor. These changes did not occur following an equivalent intratumoral dose of the non-metabolizable sugar alcohol, mannitol. When [1-13C]-glucose was administered, [3-13C]-lactate and [3-13C]-alanine were the only labeled metabolites detected in the in vivo 13C NMR spectra during the period of bioenergetic improvement. Biochemical analysis revealed a substantial increase in tumor and plasma glucose concentration, but no increase in either tumor or plasma lactate, consistent with the absence of acidosis. Evaluation of the distribution of glucose in the tumor by quantitative autoradiography of [1-14C]-2-deoxyglucose administered with the glucose indicated that, on average, 7 mM of the added glucose distributed over the entire tumor within 10 min. The significant improvement in overall metabolic status of the tumors following glucose administration is attributed to the existence of substrate limited regions within the tumor.

Influence of monovalent cations on the binding of a charged and an uncharged ('carbo'-)muscarinic antagonist to muscarinic receptors.

1. The effect of the buffer concentration on binding of [3H]-N-methylscopolamine to muscarinic receptors M2 was tested in rat heart. Tracer binding was of low affinity in a 20 mM imidazole buffer (pKD 8.3), inhibited by an increase from 10 to 100 mM of the sodium phosphate buffer concentration (pKD 9.92 to 9.22), slightly inhibited by an increase of the Tris/HC1 buffer concentration from 20 to 100 mM (pKD 9.70 to 9.47) and unaffected by an increase of the histidine/HC1 buffer concentration from 20 to 100 mM (pKD 9.90 to 9.82). We chose the last buffer to analyse the effect of ions on antagonists binding to cardiac M2 receptors and to transiently expressed wild-type and (Y533-->F) mutant m3 muscarinic receptors in COS-7 cells. 2. Equilibrium [3H]-N-methylscopolamine binding to cardiac M2 receptors was inhibited, apparently competitively, by monovalent salts (LiCl > or = NaCl > or = KCl). In contrast, binding of the uncharged 3,3-dimethylbutan-1-ol ester of diphenylglycolic acid (BS-6181) was facilitated by addition of monovalent salts (LiCl > or = NaCl > or = KCl) to the binding buffer. This cation binding pattern is consistent with interaction with a large, negative field strength binding site, such as, for instance, a carboxylic acid. 3. In the presence of 100 mM NaCl, [3H]-N-methylscopolamine had a similar affinity for the wild-type m3 receptor (pKD 9.85) and for a (Y533-->F) mutant m3 receptor (pKD 9.68). However, in the absence of added salts, the tracer had a significantly lower affinity for the mutated (pKD 10.19) as compared to the wild-type (pKD 10.70) m3 receptor. BS-6181 had a significantly lower affinity for the (Y533-->F) mutant m3 muscarinic receptor, as compared to the wild-type m3 receptor, both in the absence (pKD 6.19-6.72) in the presence (pKD 6.48-7.40) of 100 mM NaCl. The effects of NaCl on binding of the uncharged ester and of [3H]-N-methylscopolamine to the m3 receptor were decreased by the mutation. 4. Taken together, these results support the hypothesis that monovalent cations from the buffer may interact with the cation binding site of the receptors (an aspartate residue in the third transmembrane helix of muscarinic receptors). Buffer cations may inhibit competitively the binding of (charged) muscarinic ligands having a tertiary amine or ammonium group, while facilitating the receptor recognition by uncharged, isosteric 'carbo-analogues'. Mutation of the (Y533-->F) of the m3 receptor decreased the affinity of the receptor for positive charges, including the sodium ion.

Stereoselective interaction of uncharged esters at four muscarinic receptor subtypes.

We investigated the binding and pharmacological properties of the esters of 3,3-dimethylbutan-1-ol (the carbon analogue of choline) with either diphenylglycolic acid, (R)-phenylcyclohexylglycolic acid, or (S)-phenylcyclohexylglycolic acid [BS-6181, (R)-BS-7826 and (S)-BS-7826, respectively] at muscarinic M1, M2, M3 (Hm3) and M4 receptors. The three uncharged compounds were muscarinic receptor antagonists, with pA2 or pKi values between 7.9 and 5.6. The achiral ester BS-6181 displayed highest affinity for M1, M3 (Hm3) and M4 receptors (pA2 or pKi = 7.2-7.6) and lower affinity for M2 receptors (pA2 or pKi = 6.7 and 6.8). The four muscarinic receptor subtypes were able to distinguish between the two enantiomers of the cyclohexyl derivative of BS-6181 [(R)- and (S)-BS-7826], with a preference for the (R)-isomer (up to 79-fold). Interestingly, the (S)-enantiomer of BS-7826, being the distomer, was found to be M4 selective (pKi/M4 = 6.9; pA2 or pKi/M1-M3 (Hm3) = 5.6-6.2). These results indicate that uncharged compounds may (stereo)selectively bind to muscarinic receptors via hydrophobic interactions. Thus, an ionic bond between muscarinic ligands and an anionic site of the receptor is not absolutely necessary for recognition of muscarinic receptors.

Effect of membrane lipid environment on the activity of bovine adrenal 3-oxo-delta 5-steroid isomerase.

The 3-oxo-delta 5-steroid isomerase (EC 5.3.3.1) activity from bovine adrenal cortex microsomes can be extracted in soluble form by the use of appropriate detergents, although recovery of enzyme activity is low (ca. 2%). Activity is restored upon removal of detergent and reconstitution of the enzyme into phospholipid vesicles. Both Km and Vmax of 3-oxo-delta 5-steroid isomerase of intact microsomes increase as the pH is raised from 7.5 to 9.5, with a particularly sharp increase (6- to 8-fold) above pH 8.5. The kinetic parameters of a detergent-solubilized isomerase preparation show little increase from pH 7.5 to 9.0, but isomerase reconstituted into artificial phospholipid vesicles demonstrates a 6- to 10-fold increase in both Km and Vmax over this pH range. Addition of Ca++ (1 mM) enhances the pH dependence of both Km and Vmax of the membrane-bound isomerase, causing a slight rise in Vmax/Km.

The value of heparin concentration monitoring in off-pump coronary bypass surgery.

BACKGROUND/OBJECTIVE: In recent years, a remarcable increase in off-pump coronary bypass surgery (OPCAB) was observed. The identical anticoagulation treatment in cardiac surgery with cardiopulmonary bypass (CPB) and OPCAB makes the hemodynamic management in OPCAB procedures essential, since a perioperative compensation of blood loss is difficult and the hemodynamic stability has to be maintained by infusions. The aim of this study was to evaluate the circulating heparin concentration measurement in OPCAB patients by using the heparin/protamine titration method with Hepcon/HMSplus (HMS). PATIENTS AND METHOD: In 8 patients (3F/5M) undergoing elective OPCAB, the calculated heparin dose was administered and heparin concentration was registered together with activated clotting time (ACT) 5 min after administration. Measurements were carried out in 45 min periods and additional heparin was administered if the measured heparin concentration was lower than the calculated. The protamin dose was also automatically calculated by HMS. RESULTS: The mean operation time was 155 +/- 36 min (80-210) with 2.3 bypass grafts per patient. There was a significant difference between the demand for heparin as determined by HMS and the conventionally calculated value (p<0.05). Similarly, the calculated and measured concentration of heparin diverged significantly from each other (p<0.05), independent of ACT. In spite of an initially higher administration of heparin as calculated by HMS, the concentration of circulating heparin was below the estimated value in 7 patients which made a further administration of heparin necessary. The 24 h postoperative blood loss was 550 +/- 176 mL (300-850). No rethoracotomy for bleeding was necessary. CONCLUSIONS: HMS could be a useful method for appropriate anticoagulative treatment in OPCAB procedures, if there is a sufficient hemodynamic management with restrictive administration of infusions guaranted during surgery.

New surface biopolymers for oxygenators: an in vitro hemocompatibility test of poly(2-methoxyethylacrylate).

BACKGROUND/OBJECTIVE: The continuous interaction of blood with artificial contact surfaces under cardiopulmonary bypass can lead to a substantial damage of blood cells and plasma factors. Surface biopolymers in oxygenation systems can help increasing the hemocompatibility, often combined with anticoagulative agents such as heparin. The poly(2-methoxyethylacrylate) [PMEA] is a new heparin-free polymer. The objective of this experimental study was to evaluate the hemocompatibility of a PMEA-coated oxygenator (Terumo Capiox RX-25) (PTX) under standardized in vitro conditions compared to two ionic-bound and one covalent-bound heparin-coated models. METHOD: Each oxygenator was mounted in a separate standardized closed circulation system. Heparinized (5 IE/mL) fresh human blood from the same donor (hemodilution: Ringer's solution) was used. Circulation time: 120 - 180 min with a flow rate of 4.0 L/min. Blood samplings: at the beginning, 5. min and every 30 min of the circulation. PARAMETERS: platelets, granulocytes, plasma factors (p-selectin, alpha-granulomeres expression, and TAT(III)-complex). After the experiment, oxygenators were dismantled and examined by scanning electron microscopy. RESULTS: All of the oxygenators led to an initial reduction of platelets and granulocytes. PTX had the lowest platelet and granulocyte reduction rates. With a lower p-selectin release compared to covalent-bound heparin-coated oxygenator and higher expression of alpha-granulomeres compared to ionic-bound heparin-coated oxygenators, the results of PTX indicated that a high number of circulating platelets were intact on the PMEA surface. TAT(III)-complex showed a steady increase in all of the oxygenators during the tests, more remarkably in PTX. In contrast to ionic-bound heparin-coated oxygenators, the electron microscopy displayed virtually no cellular accumulation on hollow fiber and housing surfaces of PTX and covalent-bound heparin-coated oxygenator. CONCLUSIONS: 1. The hemocompatibility characteristics of PTX were remarkably better than ionic-bound heparin-coated oxygenators and slightly better than the covalent-bound heparin-coated model under in vitro conditions. 2. The PMEA coating can be a useful alternative for patients with heparin-associated disorders. 3. The clinical feasibility of PTX should be evaluated under in vivo conditions.