Effects of a nine-strain bacterial synbiotic compared to simethicone in colicky babies - an open-label randomised study.

The aim of the study was to determine effects of administration of simethicone and a multi-strain synbiotic on the crying behaviour of colicky babies. The study design consisted of an open-label, two parallel treatment group study involving 87 infants aged 3-6 weeks with infantile colic (defined as crying episodes lasting 3 or more hours per day and occurring at least 3 days per week within 3 weeks prior to enrolment) randomly, unequally [1:1.5] assigned to receive simethicone (n=33) or a multi-strain synbiotic (n=54) orally for 4 weeks. The multi-strain synbiotic contained Lactobacillus acidophilus LA-14, Lacticaseibacillus casei R0215, Lacticaseibacillus paracasei Lp-115, Lacticaseibacillus rhamnosus GG, Ligilactobacillus salivarius Ls-33, Bifidobacterium lactis Bl-04, Bifidobacterium bifidum R0071, Bifidobacterium longum R0175 and fructooligosaccharides). Primary outcome measures were the responder rates (effect ≥50% reduction from baseline) of the measures 'crying days last 3 weeks', 'average evening crying duration last 3 weeks' and 'reduction of average number of crying phases per day last three weeks' at the end of treatment. The study is registered at ClinicalTrials.gov under NCT04487834. Significantly higher responder rates (effect ≥50% reduction from baseline) of the multi-strain synbiotic compared to simethicone were found for the measures 'crying days last 3 weeks' (72% vs 18%, P<0.0001) and 'average evening crying duration last 3 weeks' (85% vs 39%, P=0.0001). No significant difference was found for the measure 'reduction of average number of crying phases per day last three weeks' (50% vs 42%, P=0.4852). No adverse effects were reported for the two treatment groups. Based on these results, the multi-strain synbiotic can be considered as an interesting therapeutic possibility for the treatment of infantile colic, worthwhile to be investigated further in non-clinical and clinical studies.

Orexin A but not orexin B regulates lipid metabolism and leptin secretion in isolated porcine adipocytes.

It is well known that orexins are involved in the metabolism and endocrine function of rodent adipocytes, but there are no data on other animal species, including pigs. Therefore, in this study, we tested the hypothesis that orexin A (OxA) and orexin B (OxB) modulate the metabolism and endocrine functions of isolated porcine adipocytes and adipose tissue explants. Moreover, we characterized the possible mechanism of OxA action in porcine adipocytes. According to the results, both orexin receptor 1 and orexin receptor 2 were expressed in the porcine adipose tissue. We found that OxA suppressed the release of glycerol from porcine adipocytes both in the absence (basal lipolysis; P < 0.05) and in the presence (stimulated lipolysis; P < 0.05) of isoproterenol. Orexin A increased basal and insulin-stimulated glucose uptake (P < 0.05), as well as it enhanced the rate of glucose incorporation into lipids with insulin (stimulated lipogenesis; P < 0.01) or without insulin (basal; P < 0.05). We have also shown that OxA stimulated the mRNA expression of glucose transporter 4 (P < 0.05) and its translocation into the plasma membrane (P < 0.01). Moreover, OxA upregulated the mRNA expression of leptin in isolated porcine adipocytes (P < 0.05) and increased the secretion of leptin (P < 0.05). We have also demonstrated one of the possible mechanisms of OxA action in adipocytes. In the presence of extracellular-signal-regulated kinase 1 and 2 (ERK1/2) inhibitor, the effect of OxA was not detectable in porcine adipocytes, which indicates that this peptide increased cell viability via ERK1/2 pathway (P < 0.05). However, OxB did not show any effect on the metabolism and endocrine functions of porcine adipocytes. In summary, we have shown for the first time that OxA has a significant impact on the intensity of lipolysis, glucose uptake, lipogenesis, as well as on the expression and secretion of leptin. Therefore, we conclude that OxA but not OxB regulates lipid metabolism in porcine adipose tissue and that this regulation is partly mediated via ERK1/2 pathway. The action of orexins should be further explored to better understand their role in the regulation of adiposity in pigs.

Serum levels of spexin and kisspeptin negatively correlate with obesity and insulin resistance in women.

Spexin (SPX) and kisspeptin (KISS) are novel peptides relevant in the context of regulation of metabolism, food intake, puberty and reproduction. Here, we studied changes of serum SPX and KISS levels in female non-obese volunteers (BMI<25 kg/m(2)) and obese patients (BMI>35 kg/m(2)). Correlations between SPX or KISS with BMI, McAuley index, QUICKI, HOMA IR, serum levels of insulin, glucagon, leptin, adiponectin, orexin-A, obestatin, ghrelin and GLP-1 were assessed. Obese patients had lower SPX and KISS levels as compared to non-obese volunteers (SPX: 4.48+/-0.19 ng/ml vs. 6.63+/-0.29 ng/ml; p<0.001, KISS: 1.357+/-0.15 nmol/l vs. 2.165+/-0.174 nmol/l; p<0.01). SPX negatively correlated with BMI, HOMA-IR, insulin, glucagon, active ghrelin and leptin. Positive correlations were found between SPX and QUICKI index, McAuley index, serum levels of obestatin, GLP-1 and adiponectin and orexin-A Serum KISS negatively correlated with BMI, HOMA-IR, serum levels of insulin, glucagon, active ghrelin and leptin. KISS positively correlated with QUICKI index, McAuley index and adiponectin. In summary, SPX and KISS show negative correlations with obesity, insulin resistance indices, and hormones known to affect insulin sensitivity in females. Both, SPX and KISS could be therefore relevant in the pathophysiology of obesity and insulin resistance.

Chronic orexin-A (hypocretin-1) treatment of type 2 diabetic rats improves glucose control and beta-cell functions.

Orexin regulates food intake and energy expenditure. Here, we test the ability of orexin-A (OXA, hypocretin-1) at improving metabolic control in type 2 diabetic animals and elaborate potential mechanisms of action. Rats with experimentally induced type 2 diabetes by a combination of streptozotocin injection and high-fat diet feeding were chronically infused with OXA. In vitro experiments were conducted on isolated pancreatic islets, primary adipocytes and insulin secreting INS-1E cells. OXA improved glucose control, enhanced insulin sensitivity and attenuated pancreatic ß-cell loss in type 2 diabetic rats. Ex vivo, apoptotic death of pancreatic islets isolated from OXA-treated type 2 diabetic animals as well as the impairment of glucose-stimulated insulin secretion were attenuated, as compared to islets derived from vehicle-treated rats. OXA reduced plasma tumor necrosis factor-α (TNF-α) and non-esterified fatty acids (NEFA) levels in type 2 diabetic rats. OXA decreased palmitate- and TNF-α-induced apoptosis of INS-1E cells. OXA improves glucose control by enhancing insulin sensitivity and protecting ß-cells from apoptotic cell death in type 2 diabetic animals.

Resistin - from gene expression to development of diabetes.

Adipocyte-originated hormonal factors, playing a role of signaling particles, are widely engaged in energy control, feeding behavior and general glucose or lipid metabolism. One of them – resistin – has been suspected to initiate or develop insulin resistance and diabetes. From the moment of discovery of resistin, during last 13 years, numerous investigations put some light on a potential role of this hormone in mammals. In this review knowledge on resistin, including its structure, physiological role related to obesity and diabetes, as well as, gene sequence and phenotypic effects of the identified polymorphisms in human and domestic mammals is discussed.

Experimental demonstration of an OpenFlow based software-defined optical network employing packet, fixed and flexible DWDM grid technologies on an international multi-domain testbed.

Software defined networking (SDN) and flexible grid optical transport technology are two key technologies that allow network operators to customize their infrastructure based on application requirements and therefore minimizing the extra capital and operational costs required for hosting new applications. In this paper, for the first time we report on design, implementation & demonstration of a novel OpenFlow based SDN unified control plane allowing seamless operation across heterogeneous state-of-the-art optical and packet transport domains. We verify and experimentally evaluate OpenFlow protocol extensions for flexible DWDM grid transport technology along with its integration with fixed DWDM grid and layer-2 packet switching.

Obestatin inhibits lipogenesis and glucose uptake in isolated primary rat adipocytes.

Ghrelin and obestatin are encoded by the preproghrelin gene and originate from post-translational processing of the preproghrelin peptide. Obestatin is mainly present in the stomach, but its action is focused on appetite inhibition in opposition to ghrelin function. Recently, it has been presented that obestatin may regulate adipocyte metabolism and influence fat content. However, obestatin action is still poorly understood. Therefore, we aimed to investigate obestatin function on adipocyte metabolism in the rat. We studied changes in the mRNA expression of active and inactive isoforms of obestatin receptors. In addition, we analyzed influence of obestatin on lipogenesis, lipolysis and glucose transport in isolated adipocytes. Moreover, we also performed analysis of obestatin action on lipolysis in differentiated rat preadipocytes with silenced obestatin receptor. We found significantly higher expression of the obestatin receptor Gpr39-1a active form at an mRNA level following adipocytes incubation with obestatin. We did not observe expression changes in the inactive form of obestatin receptor Gpr39-1b. Additionally, we found significant changes in Gpr39-1a expression following obestatin receptor silencing in cells incubated with obestatin in comparison to control. Obestatin inhibited both, basal and insulin-stimulated lipogenesis and glucose transport in adipocytes. Furthermore, obestatin potentiated adrenalin-stimulated lipolysis. We also found reduced glycerol release following obestatin incubation in adipocytes with silenced Gpr39 gene. Our results indicate that obestatin acts via the GPR39 receptor in isolated adipocytes, and that through this mechanism obestatin influences lipid accumulation, glucose uptake and lipolysis.

Glucagon increases circulating fibroblast growth factor 21 independently of endogenous insulin levels: a novel mechanism of glucagon-stimulated lipolysis?

AIMS/HYPOTHESIS: Glucagon reduces body weight by modifying food intake, glucose/lipid metabolism and energy expenditure. All these physiological processes are also controlled by fibroblast growth factor 21 (FGF-21), a circulating hepatokine that improves the metabolic profile in obesity and type 2 diabetes. Animal experiments have suggested a possible interaction between glucagon and FGF-21 however, the metabolic consequences of this crosstalk are not understood. METHODS: The effects of exogenous glucagon on plasma FGF-21 levels and lipolysis were evaluated in healthy volunteers and humans with type 1 diabetes, as well as in rodents with streptozotocin (STZ)-induced insulinopenic diabetes. In vitro, the role of glucagon on FGF-21 secretion and lipolysis was studied using isolated primary rat hepatocytes and adipocytes. Fgf-21 expression in differentiated rat pre-adipocytes was suppressed by small interfering RNA and released FGF-21 was immunoneutralised by polyclonal antibodies. RESULTS: Glucagon induced lipolysis in healthy human volunteers, patients with type 1 diabetes, mice and rats with STZ-induced insulinopenic diabetes, and in adipocytes isolated from diabetic and non-diabetic animals. In addition, glucagon increased circulating FGF-21 in healthy humans and rodents, as well as in patients with type 1 diabetes, and insulinopenic rodents. Glucagon stimulated FGF-21 secretion from isolated primary hepatocytes and adipocytes derived from animals with insulinopenic diabetes. Furthermore, FGF-21 stimulated lipolysis in primary adipocytes isolated from non-diabetic and diabetic rats. Reduction of Fgf-21 expression (by approximately 66%) or immunoneutralisation of released FGF-21 markedly attenuated glucagon-stimulated lipolysis in adipocytes. CONCLUSIONS/INTERPRETATION: These results indicate that glucagon increases circulating FGF-21 independently of endogenous insulin levels. FGF-21 participates in glucagon-induced stimulation of lipolysis.

Effects of orexin A on proliferation, survival, apoptosis and differentiation of 3T3-L1 preadipocytes into mature adipocytes.

Metabolic activities of orexin A (OXA) in mature adipocytes are mediated via PI3K/PKB and PPARγ. However, the effects of OXA on preadipocytes are largely unknown. We report here that OXA stimulates the proliferation and viability of 3T3-L1 preadipocytes and protects them from apoptosis via ERK1/2, but not through PKB. OXA reduces proapoptotic activity of caspase-3 via ERK1/2. Inhibition of ERK1/2 prevents the differentiation of preadipocytes into adipocytes. Unlike insulin, neither short-term nor prolonged exposure of 3T3-L1 preadipocytes to OXA induces preadipocyte differentiation to adipocytes, despite increased ERK1/2 phosphorylation. Unlike insulin, OXA fails to activate PKB, which explains its inability to induce the differentiation of preadipocytes.

Orexin A stimulates glucose uptake, lipid accumulation and adiponectin secretion from 3T3-L1 adipocytes and isolated primary rat adipocytes.

AIMS/HYPOTHESIS: Orexin A (OXA) modulates body weight, food intake and energy expenditure. In vitro, OXA increases PPARγ (also known as PPARG) expression and inhibits lipolysis, suggesting direct regulation of lipid metabolism. Here, we characterise the metabolic effects and mechanisms of OXA action in adipocytes. METHODS: Isolated rat adipocytes and differentiated murine 3T3-L1 adipocytes were exposed to OXA in the presence or absence of phosphoinositide 3-kinase (PI3K) inhibitors. Pparγ expression was silenced using small interfering RNA. Glucose uptake, GLUT4 translocation, phosphatidylinositol (3,4,5)-trisphosphate production, lipogenesis, lipolysis, and adiponectin secretion were measured. Adiponectin plasma levels were determined in rats treated with OXA for 4 weeks. RESULTS: OXA PI3K-dependently stimulated active glucose uptake by translocating the glucose transporter GLUT4 from cytoplasm into the plasma membrane. OXA increased cellular triacylglycerol content via PI3K. Cellular triacylglycerol accumulation resulted from increased lipogenesis as well as from a decrease of lipolysis. Adiponectin levels in chow- and high-fat diet-fed rats treated chronically with OXA were increased. OXA stimulated adiponectin expression and secretion in adipocytes. Both pharmacological blockade of peroxisome proliferator-activated receptor γ (PPARγ) activity or silencing Pparγ expression prevented OXA from stimulating triacylglycerol accumulation and adiponectin production. CONCLUSIONS/INTERPRETATION: Our study demonstrates that OXA stimulates glucose uptake in adipocytes and that the evolved energy is stored as lipids. OXA increases lipogenesis, inhibits lipolysis and stimulates the secretion of adiponectin. These effects are conferred via PI3K and PPARγ2. Overall, OXA's effects on lipids and adiponectin secretion resemble that of insulin sensitisers, suggesting a potential relevance of this peptide in metabolic disorders.

Importance of ghrelin in hypothalamus-pituitary axis on growth hormone release during normal pregnancy in the rat.

Ghrelin is a hormone mainly produced in the stomach and its first discovered action was connected with regulating growth hormone secretion. It was found that ghrelin injection increases growth hormone release and that this action is dose-dependent. Ghrelin may influence growth hormone secretion both by central and peripheral action. Ghrelin acts via its receptors named growth hormone secretagogue receptors (GHSR). Ghrelin receptors were found in almost all tissues including the central nervous system. Besides influence on growth hormone secretion, ghrelin also regulates food intake and energy metabolism centrally as well as peripherally. In our study, active ghrelin and growth hormone levels in serum were measured. We also investigated gene expression of proghrelin, growth hormone releasing hormone (GHRH) and growth hormone receptor (GH-R) in the hypothalamus and the active form of ghrelin receptor (GHSR-1a) in hypothalamus and pituitary. Expression of growth hormone and growth hormone releasing hormone receptor (GHRH-R) in the pituitary were also measured. The results of our study indicate that active ghrelin and growth hormone levels in serum increased during pregnancy. Expression of ghrelin in hypothalamus and its receptor also increased in hypothalamus and pituitary during pregnancy. We also observed that growth hormone gene expression rose in pituitary, while its receptor mRNA level in hypothalamus decreased. Additionally, growth hormone expression in placenta decreased during pregnancy. Moreover, GHRH in hypothalamus and its receptor in pituitary showed reduced levels during pregnancy. Our results may indicate that ghrelin is a important factor influencing growth hormone release during pregnancy.

Somatostatin receptor subtypes 2 and 5 mediate inhibition of egg yolk-induced gall bladder emptying in mice.

BACKGROUND: Somatostatin inhibits gall bladder contraction. Impaired gall bladder emptying is associated with gall bladder stone formation. The incidence of cholecystolithiasis is high in patients treated with a somatostatin agonist octreotide, which predominantly interacts with somatostatin receptor subtype 2 (SSTR2). Therefore, it is believed that SSTR2 regulates gall bladder contraction; however, evidence has not been provided. Here, we evaluate the effects of SSTR1-SSTR5-selective agonists on egg yolk-induced gall bladder contraction in mice. METHODS: Homozygous deletion of SSTR2 and SSTR5 was generated by cross-mating of SSTR2(-/-) with SSTR5(-/-) mice. Mice of different genotypes were injected with SSTR1-5-selective agonists or octreotide 15 min before induction of gall bladder emptying by egg yolk. One hour later, gall bladders were removed and weighed. KEY RESULTS: Egg yolk-reduced gall bladder weights in all mice, irrespective of their genotype. Octreotide was the most potent inhibitor of gall bladder emptying in wild-type mice. In contrast, agonists with high selectivity for SSTR2 or SSTR5 inhibited gall bladder emptying by approximately 50-60%, whereas SSTR1-, SSTR3- and SSTR4-selective agonists failed to influence gall bladder contraction. In SSTR2(-/-) mice, octreotide and an SSTR5-selective agonist inhibited gall bladder emptying by approximately 50%, whereas SSTR2-selective agonists were inactive. Octreotide inhibited gall bladder emptying in SSTR5(-/-) mice by approximately 50%, without any effect in SSTR2(-/-)/SSTR5(-/-) mice. CONCLUSIONS & INFERENCES: Our study provides evidence for the role of SSTR2 and SSTR5 in regulating gall bladder emptying in mice.

Proton pump inhibitors reduce mycophenolate exposure in heart transplant recipients-a prospective case-controlled study.

This prospective study investigates the impact of proton pump inhibitors (PPI) on mycophenolic acid (MPA) pharmacokinetics in heart transplant recipients receiving mycophenolate mofetil (MMF) and tacrolimus. MPA plasma concentrations at baseline (C(0 h)), 30 min (C(0.5 h)), 1(C(1 h)) and 2 h (C(2 h)) were obtained by high-performance liquid chromatography (HPLC) in 22 patients treated with pantoprazole 40 mg and MMF 2000 mg. Measurements were repeated 1 month after pantoprazole withdrawal. A four-point limited-sampling strategy was applied to calculate the MPA area under the curve (MPA-AUC). Predose MPA concentrations with PPI were 2.6 +/- 1.6 mg/L versus 3.4 +/- 2.7 mg/L without PPI (p = ns). Postdose MPA concentrations were lower with PPI at C(0.5 h) (8.3 +/- 5.7 mg/L vs. 18.3 +/- 11.3 mg/L, p = 0.001) and C(1 h) (10.0 +/- 5.6 mg/L vs. 15.8 +/- 8.4 mg/L, p = 0.004), without significant differences at C(2 h) (8.3 +/- 6.5 mg/L vs. 7.6 +/- 3.9 mg/L). The MPA-AUC was significantly lower with PPI medication (51.2 +/- 26.6 mg x h/L vs. 68.7 +/- 30.3 mg x h/L; p = 0.003). The maximum concentration of MPA (MPA-C(max)) was lower (12.2 +/- 7.5 mg/L vs. 20.6 +/- 9.3 mg/L; p = 0.001) and the time to reach MPA-C(max) (t(max)) was longer with PPI (60.0 +/- 27.8 min vs. 46.4 +/- 22.2 min; p = 0.05). This is the first study to document an important drug interaction between a widely used immunosuppressive agent and a class of drugs frequently used in transplant patients. This interaction results in a decreased MMF drug exposure which may lead to patients having a higher risk for acute rejection and transplant vasculopathy.

The image of motor units architecture in the mechanomyographic signal during the single motor unit contraction: in vivo and simulation study.

The mechanomyographic (MMG) signal analysis has been performed during single motor unit (MU) contractions of the rat medial gastrocnemius muscle. The MMG has been recorded as a muscle surface displacement by using a laser distance sensor. The profiles of the MMG signal let to categorize these signals for particular MUs into three classes. Class MMG-P (positive) comprises MUs with the MMG signal similar to the force signal profile, where the distance between the muscle surface and the laser sensor increases with the force increase. The class MMG-N (negative) has also the MMG profile similar to the force profile, however the MMG is inverted in comparison to the force signal and the distance measured by using laser sensor decreases with the force increase. The third class MMG-M (mixed) characterize the MMG which initially increases with the force increases and when the force exceeds some level it starts to decrease towards the negative values. The semi-pennate muscle model has been proposed, enabling estimation of the MMG generated by a single MU depending on its localization. The analysis have shown that in the semi-pennate muscle the localization of the MU and the relative position of the laser distance sensor determine the MMG profile and amplitude. Thus, proposed classification of the MMG recordings is not related to the physiological types of MUs, but only to the MU localization and mentioned sensor position. When the distance sensor is located over the middle of the muscle belly, a part of the muscle fibers have endings near the location of the sensor beam. For the MU MMG of class MMG-N the deflection of the muscle surface proximal to the sensor mainly influences the MMG recording, whereas for the MU MMG class MMG-P, it is mainly the distal muscle surface deformation. For the MU MMG of MMG-M type the effects of deformation within the proximal and distal muscle surfaces overlap. The model has been verified with experimental recordings, and its responses are consistent and adequate in comparison to the experimental data.

Polymorphism of the porcine leptin gene promoter and analysis of its association with gene expression and fatness traits.

We describe for the first time a 245 bp fragment of the porcine leptin gene promoter in the proximity of the transcription start site. Altogether, 720 pigs were screened with the PCR-SSCP technique for polymorphism in this region. Four SNPs, segregating as two haplotypes, have been identified, one of them (C113G) in the putative consensus site for the AP-2 transcription factor. This polymorphism was evenly distributed in the Duroc breed (n=21) and was absent in the Polish Landrace (n=248) and Pietrain breed (n=12). In the Polish Large White (n=191) and synthetic line 990 (n=243), allele G occurred with a very low frequency. The investigation was performed to test if the C113G SNP affects leptin mRNA level in subcutaneous fat and leptin protein concentration in serum. Additionally, the effect of this polymorphism on fatness traits was statistically analyzed. Although there was a trend toward decreased expression in GG animals, the differences were not significant between genotypes. We also found no evidence for an association of the LEP promoter genotype with the analyzed fatness traits.

Increased expression of vascular endothelial growth factor associated with accumulation of lipids in Bruch's membrane of LDL receptor knockout mice.

AIM: To investigate the pathogenesis of age related macular degeneration (ARM) with respect to lipid accumulation within Bruch's membrane (BrM) in a knockout model with low density lipoprotein (LDL) receptor deficiency. METHODS: LDL receptor deficient mice and C57BL/6 controls were fed a standard diet or a high fat (HF) diet. Plasma total cholesterol (pTC) was determined. Eyes were examined by transmission electron microscopy. Immunohistochemical staining for VEGF was performed. RESULTS: pTC were highest in LDL receptor deficient mice after HF diet and elevated after standard diet compared to controls with and without HF diet. While BrM of controls did not exhibit any visible changes, membrane bound translucent particles were seen in all BrM of knockout mice. The amount of these particles was substantially increased and membranes were thickened after HF diet. VEGF staining was positive in knockout mice only and was located in retinal pigment epithelial cells, the outer plexiform layer, and photoreceptor inner segments. Most intensive VEGF expression was documented after HF diet. CONCLUSION: LDL receptor deficient mice exhibit an accumulation of lipid particles in BrM which is further increased after fat intake. VEGF expression is found in the outer retinal layers of LDL receptor deficient mice and appears to correlate with the amount of lipid particles present in BrM.

Experimentally verified model of mechanomyograms recorded during single motor unit contractions.

The aim of the paper is to create a model which enables to observe the mechanomyographic (MMG) wave generated during single motor unit contractions in a muscle, while the muscle is immersed in paraffin oil. The muscle model is described as a rheological membrane. Both the muscle and the medium models have been built by using Stiff-Finite-Element-Method (SFEM), which allows one to simulate the muscle surface displacement and the acoustic propagation of this effect in the oil. Such a modelling enables one to determine the impact of the rheological properties of the liquid environment on the shape of the MMG wave. In order to verify the model, the MMG signals and the contraction forces have been recorded in vivo from the medial gastrocnemius muscle of a rat. In these experiments single motor units were stimulated with various stimulation frequencies. A piezotransducer, immersed in paraffin oil, has been used to record the MMG signal recording. The signals recorded during individual twitches of the motor units have been used to estimate the parameters of the model. Subsequently, the model has been experimentally verified. The signals recorded in experiments during unfused and fused tetani have been compared with the simulated model responses in the analogous stimulation program. It has been observed that the MMG signals obtained with the proposed linear model have been consistent with the results of in vivo experiments.

T cell receptor-mediated signalling in T cells from multiple myeloma patients.

PURPOSE: Several abnormalities of the T-cell receptor (TCR) CD3 transduction pathway in cancer patients including B-cell neoplasms have been reported. Adequate activation of the receptor is important to provide stimulus for the T-cell to start its effector functions. Activation of TCR/ CD3 results in recruitment of tyrosine kinases and changes of tyrosine phosphorylation levels. We investigated phosphorylation levels in T-cells from multiple myeloma patients at diagnosis and compared them to normal individuals. PATIENTS AND METHODS: We quantitatively analyzed protein phosphorylation of resting and anti-CD3 stimulated Tcells in 10 previously untreated multiple myeloma patients and 6 healthy donors using two-colour flow cytometry. The results were shown as a median fluorescence intensity values. A specific monoclonal antibody directed against phosphotyrosine was used. RESULTS: No significant differences between helper Tcells and cytotoxic T-cells and no significant differences between multiple myeloma patients and controls were found. CONCLUSION: The flow cytometry technique used in our experiment allows a quick insight into phosphorylation pattern following activation of T-cells. Further experiments combining this method with measuring the activity of particular tyrosine kinases of the activation cascade may bring the necessary information explaining the nature of T-cell dysfunction.

The influence of coumestrol, zearalenone, and genistein administration on insulin receptors and insulin secretion in ovariectomized rats.

The influence of phytoestrogens (genistein and coumestrol) and mycoestrogen (zearalenone) on insulin secretion, liver insulin receptors and some aspects of lipid and carbohydrate metabolism were investigated in this study. Ovariectomized rats were injected s.c. with the above mentioned compounds in the amount of 1 mg for three days. Coumestrol and zearalenone caused a significant increase in uterus weight, similar to the effects observed after estrone action, while this effect was not observed after the genistein injection. Blood insulin level was not changed after phyto- or mycoestrogen treatment. However, coumestrol and genistein significantly decreased the binding capacity of liver insulin receptors. These changes corresponded with alterations in glucose and free fatty acids profiles in blood, as well as with glycogen content in liver. The effects observed after genistein and coumestrol injections differed from those noticed in rats treated with zearalenone or estrone. On the basis of these results we conclude that metabolic effects of high doses of coumestrol and genistein in ovariectomized rats are partly mediated by changes in insulin sensitivity of the liver and that the action of plant estrogens on metabolism is, at least to the some degree, independent of their estrogen activity.