Moderate increases in daily step count are associated with reduced IL6 and CRP in women with PCOS.

Aims Physical activity has been proposed to be an effective non-pharmacological method of reducing systemic inflammation and therefore may prove particularly efficacious for women with polycystic ovary syndrome (PCOS) who have been shown to have high levels of inflammation and an increased risk of type 2 diabetes (T2DM) and cardiovascular disease (CVD). Therefore, the aim of the present study was to assess whether modest changes in daily step count could significantly reduce levels of inflammatory markers in women with PCOS. Subjects and Methods Sixty-five women with PCOS were assessed at baseline and again at 6 months. All had been provided with an accelerometer and encouraged to increase activity levels. Multivariate linear regression analyses (adjusted for age, ethnicity, baseline step count, change in BMI and change in accelerometer wear-time) were used to assess changes in daily step count against clinical and research biomarkers of inflammation, CVD and T2DM. Results Mean step count/day at baseline was 6337 (±270). An increase in step count (by 1000 steps) was associated with a 13% reduction in IL6 (ß: -0.81 ng/L; 95% CI, -1.37, -0.25, P = 0.005) and a 13% reduction in CRP (ß: -0.68 mg/L; 95% CI, -1.30, -0.06, P = 0.033). Additionally, there was a modest decrease in BMI (ß: 0.20 kg/m2; 95% CI, -0.38, -0.01, P = 0.038). Clinical markers of T2DM and CVD were not affected by increased step count. Conclusions Modest increases in step count/day can reduce levels of inflammatory markers in women with PCOS, which may reduce the future risk of T2DM and CVD.

Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish.

Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 µM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1ß, it also induced the release of chemotactic chemokines, MCP-1, MIP-1ß, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host-pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination.

Mycobacterium tuberculosis Zinc Metalloprotease-1 Elicits Tuberculosis-Specific Humoral Immune Response Independent of Mycobacterial Load in Pulmonary and Extra-Pulmonary Tuberculosis Patients.

Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4(+) T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can be explored as antigen candidates for prime-boost vaccination strategies or extrapolated as markers for disease detection and progression.

Interpretation of coagulation test results under direct oral anticoagulants.

Diagnostic of global coagulation parameters is part of the daily clinical routine practice in conservative as well in operative disciplines. The correct interpretation of in vitro test results in context to the ex vivo influence of anticoagulant drugs and the in vivo hemostatic system of the individual patient is dependent on the doctors clinical and laboratory experience. This article shortly reviews the laboratory interference of oral anticoagulants including the target-specific inhibitors dabigatran, rivaroxaban and apixaban on coagulation parameters and discusses the potential of several methods for measuring the anticoagulant effect of the direct oral anticoagulants.

Clinical characteristics of people experiencing biochemical hypoglycaemia during an oral glucose tolerance test: cross-sectional analyses from a UK multi-ethnic population.

AIMS: People who experience biochemical hypoglycaemia during an oral glucose tolerance test (OGTT) may be insulin resistant, but this has not been investigated robustly, therefore we examined this in a population-based multi-ethnic UK study. METHODS: Cross-sectional data from 6478 diabetes-free participants (849 with fasting insulin data available) who had an OGTT in the ADDITION-Leicester screening study (2005-2009) were analysed. People with biochemical hypoglycaemia (2-h glucose <3.3mmol/l) were compared with people with normal glucose tolerance (NGT) or impaired glucose regulation (IGR) using regression methods. RESULTS: 359 participants (5.5%) had biochemical hypoglycaemia, 1079 (16.7%) IGR and 5040 (77.8%) NGT. Biochemical hypoglycaemia was associated with younger age (P<0.01), white European ethnicity (P<0.001), higher HDL cholesterol (P<0.01), higher insulin sensitivity (P<0.05), and lower body mass index (P<0.001), blood pressure (P<0.01), fasting glucose (P<0.001), HbA1C (P<0.01), and triglycerides (P<0.01) compared with NGT and IGR separately in both unadjusted and adjusted (age, sex, ethnicity, body mass index, smoking status) models. CONCLUSIONS: Biochemical hypoglycaemia during an OGTT in the absence of diabetes or IGR was not associated with insulin resistance, but instead appeared to be associated with more favourable glycaemic risk profiles than IGR and NGT. Thus, clinicians may not need to intervene due to biochemical hypoglycaemia on a 2-h OGTT.

Plasma obestatin and autonomic function are altered in orexin-deficient narcolepsy, but ghrelin is unchanged.

Narcolepsy-cataplexy is characterised by orexin deficiency, sleep disturbance, obesity and dysautonomia. Ghrelin and obestatin affect both energy intake and sleep. Our aim was to investigate ghrelin, obestatin and metabolic/autonomic function in narcolepsy-cataplexy. Eight narcolepsy-cataplexy patients (seven CSF orexin-deficient) and eight matched controls were studied. The subjects had a fixed energy meal with serial blood samples and measurement of heart rate variability (HRV). Fasting plasma obestatin was more than threefold higher in narcolepsy subjects (narcolepsy 89.6 ± 16 pg/ml vs. control 24.9 ± 3 pg/ml, p < 0.001). There was no change in HRV total power, but post-prandial low-frequency (LF) power and high-frequency (HF) power were lower in the narcolepsy group [area under the curve (AUC): HF power narcolepsy 1.4 × 10(5) ± 0.2 × 10(5) vs. control 3.3 × 10(5) ± 0.6 × 10(5 )ms(2)/h, p < 0.001]. On multiple regression analyses, the only significant predictor of plasma obestatin was HF power, which was inversely correlated with obestatin (ß = -0.65 R (2) = 38 %, p = 0.009). Fasting and post-prandial plasma ghrelin were similar in both groups (narcolepsy 589.5 ± 88 pg/ml vs. control 686.9 ± 81 pg/ml, p = 0.5; post-prandial AUC-narcolepsy 161.3 ± 22 ng/ml/min vs. control 188.6 ± 62 ng/ml/min, p = 0.4). Only the narcolepsy group had significant suppression of plasma ghrelin after the meal (ANOVA, p = 0.004). In orexin-deficient narcolepsy, fasting plasma ghrelin is unaltered, and post-prandial suppression is preserved. Fasting plasma obestatin is increased and correlates with autonomic dysfunction. As obestatin affects NREM sleep, we suggest that increased plasma levels contribute to the disrupted sleep-state control in narcolepsy.

[Progress in diagnostic evaluation of platelet function disorders]. / Fortschritte in der Thrombozytenfunktionsdiagnostik.

Both for diagnosis of congenital and acquired platelet dysfunction as well as for therapy monitoring after application of platelet function inhibitors various methods have been established for evaluation of platelet function. In contrast to the gold standard of platelet function testing, the light transmission aggregometry in platelet rich plasma the Point-of-care (POC) analyzers allow fast analysis of platelet function without extensive laboratory work up. The conditions of the pre-analytical phase, however, are still of enormous importance in the prevention of medical errors. There is increasing clinical data in monitoring the effect of platelet aggregation inhibitors, showing that quantitative determination of the platelet function degree correlates with risk of increased bleeding or stent thrombosis. However, it is still unclear, which is the optimal test system, to predict the clinical outcome of these patients.

Ghrelin inhibits autonomic function in healthy controls, but has no effect on obese and vagotomized subjects.

OBJECTIVE: Ghrelin inhibits sympathetic nervous system (SNS) activity in rodents. We studied the effect of ghrelin on healthy humans, in obesity and in vagotomized subjects. DESIGN: Randomized, double-blinded, placebo-controlled crossover. SUBJECTS: Seven lean [mean body mass index (BMI) 23·6 ± 0·9 kg/m(2) ], seven morbidly obese (mean BMI 50·9 ± 4·4 kg/m(2) ) and seven post-gastrectomy subjects (mean BMI 22·0 ± 1·1 kg/m(2) ). MEASUREMENTS: Subjects were randomized to intravenous ghrelin (5 pmol/kg/min) or saline over 270 min. Subjects had a fixed calorie meal and a free choice buffet during the infusion. Heart rate variability (HRV) was measured. Total power (TP) represents overall autonomic function, low-frequency (LF) power represents sympathetic and parasympathetic activity, and high-frequency (HF) power represents parasympathetic activity. Very low (VLO) frequency represents the frequency band associated with thermogenesis. RESULTS: Preliminary anova analysis, looking at all three subject groups together, showed that ghrelin had an overall highly significant inhibitory effect on TP (P = 0·001), HF power (P = 0·04), VLO power (P = 0·03) and no effect on LF (P = 0·07). Further subset analysis revealed that ghrelin had a significant effect on TP (P = 0·03), borderline effect on LF power (P = 0·06) and no effect on HF power (P = 0·1) in healthy controls. By contrast in obese subjects, ghrelin had no effect on TP (P = 0·3), LF (P = 0·5) and HF (P = 0·06) and also no effect in the vagotomized subjects on TP (P = 0·7), LF (P = 0·7) and HF (P = 0·9). Ghrelin had no effect on the LF/HF ratio. CONCLUSIONS: Ghrelin inhibits SNS activity in healthy controls with a moderate effect on parasympathetic nervous system activity but had no effect on obese subjects. Vagotomized subjects also did not respond to ghrelin, suggesting the vagus nerve is important for the effects of peripheral ghrelin on the SNS.

[Significance of platelet function tests]. / Stellenwert der Thrombozytenfunktionsdiagnostik.

Numerous laboratory tests are in use to detect congenital or acquired platelet function disorders. Platelet aggregometry, using ADP, collagen, arachidonic acid or ristocetin as inductor is the standard test system for diagnosis. It is also used to detect platelet non-response to antiplatelet therapy. Studies have demonstrated that laboratory assessment of platelet non response to aspirin or clopidogrel is associated with adverse outcomes, and they indicate the importance of adjusting antiplatelet therapy in patients with a low degree of platelet inhibition. Nevertheless, a standardized method for identifying these patients is still missing.

'Microalbuminuric anaemia'--the relationship between haemoglobin levels and albuminuria in diabetes.

BACKGROUND: There is an increasing incidence of anaemia in diabetes despite the absence of significant renal impairment. AIMS: This study set out to determine the prevalence of anaemia in a diabetic population and to explore the relationship between anaemia and urinary albumin excretion in diabetes mellitus. Also, to determine the difference between those with overt nephropathy, microalbuminuria and those without evidence of renal disease. METHODS: Five hundred and two consecutive diabetes patients were screened for anaemia. Anaemia was defined by World Health Organization criteria (<13 g/dl for men and <12 g/dl for women). Urinary albumin excretion was determined by urinary albumin creatinine ratio (ACR) from a single urine sample. RESULTS: Anaemia was present in 118 (23.5%) patients. There was a rise in the prevalence of anaemia from 19% in patients with a normal ACR to 29% in those with microalbuminuria and to 41% in macroalbuminuria. This increase in the prevalence of anaemia in microalbuminuria compared to normoalbuminuria was not explained by declining renal function as there was no significant difference in eGFR between the two groups. CONCLUSION: Anaemia was common in the study population. Early detection and correction of anaemia in diabetes is important for patients at risk of impaired quality of life and increased cardiovascular risk.

Matched wideband low-noise amplifiers for radio astronomy.

Two packaged low noise amplifiers for the 0.3-4 GHz frequency range are described. The amplifiers can be operated at temperatures of 300-4 K and achieve noise temperatures in the 5 K range (<0.1 dB noise figure) at 15 K physical temperature. One amplifier utilizes commercially available, plastic-packaged SiGe transistors for first and second stages; the second amplifier is identical except it utilizes an experimental chip transistor as the first stage. Both amplifiers use resistive feedback to provide input reflection coefficient S11<-10 dB over a decade bandwidth with gain over 30 dB. The amplifiers can be used as rf amplifiers in very low noise radio astronomy systems or as i.f. amplifiers following superconducting mixers operating in the millimeter and submillimeter frequency range.

Novel mutation in the NHLRC1 gene in a Malian family with a severe phenotype of Lafora disease.

We studied a Malian family with parental consanguinity and two of eight siblings affected with late-childhood-onset progressive myoclonus epilepsy and cognitive decline, consistent with the diagnosis of Lafora disease. Genetic analysis showed a novel homozygous single-nucleotide variant in the NHLRC1 gene, c.560A>C, producing the missense change H187P. The changed amino acid is highly conserved, and the mutation impairs malin's ability to degrade laforin in vitro. Pathological evaluation showed manifestations of Lafora disease in the entire brain, with particularly severe involvement of the pallidum, thalamus, and cerebellum. Our findings document Lafora disease with severe manifestations in the West African population.

Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary.

BACKGROUND: Light transmittance aggregometry (LTA) is considered to be the 'gold standard' of platelet function testing. As LTA has been poorly standardized, we analyzed the results of LTA in healthy subjects and patients with antiplatelet therapy using different concentrations of agonists and performing tests in non-adjusted and platelet count-adjusted platelet-rich plasma (PRP). METHODS: LTA was performed in 20 healthy subjects and in patients treated with aspirin (n = 30) or clopidogrel (n = 30) monotherapy, as well as in patients on combination therapy (n = 20), using arachidonic acid (ARA 0.25 and 0.5 mg mL(-1)) and adenosine diphosphate (ADP 2 and 5 microm) as agonists and performing platelet function tests in non-adjusted and platelet count (250 nL(-1) +/- 10%)-adjusted PRP. RESULTS: The overall platelet aggregation response is decreased after adjusting the PRP for platelet count compared with measurements in unadjusted PRP. The variability of aggregation results is high in adjusted PRP in the subgroup of healthy subjects, ranging from 9.2-95.3% (5th-95th percentile) relative to 77.6-95.5% in non-adjusted PRP when determining maximum aggregation to ARA 0.5 mg mL(-1). Late aggregation using ADP 2 microm ranges from 3.8-89.9% in adjusted PRP compared with 42.9-92.5% in non-adjusted PRP. Maximum aggregation using ARA 0.5 mg mL(-1) in non-adjusted PRP differentiates between aspirin-treated patients and healthy controls well, whereas late aggregation using ADP 2 microm in non-adjusted PRP offers the best discrimination between clopidogrel-treated patients and healthy controls. CONCLUSION: Adjustment of PRP for platelet count does not provide any advantage and therefore the time-consuming process of platelet count adjustment is not necessary.

[Resistance to acetylsalicylic acid and clopidogrel: current status]. / Resistenz gegen Azetylsalizylsäure und Clopidogrel. Aktuelles Wissen.

Resistance to acetylsalicylic acid (ASA) or clopidogrel is understood from the clinical point of view as failure of the drugs to prevent recurrent vascular occlusions. Non-response to ASA and clopidogrel is defined from the laboratory aspect as an inability to cause in vitro detectable platelet function inhibition. It would be beneficial to monitor non-response to ASA or clopidogrel with platelet function methods, which detect the specific effect of these drugs, and thus prevent clinical events caused by failure of therapy. Non-response to ASA and clopidogrel are detected with different platelet function methods, which are not always clinically standardized and are assessing only the global platelet function and not the specific drug effect. Although various studies reporting 5 to 59% non-response for both drugs, support a clinical relevance of ASA and clopidogrel non-response, well-designed clinical prospective trials are required to identify patients with antiplatelet drug resistance. Furthermore, mechanisms explaining this phenomenon of drug resistance are still unknown.

Development and clinical evaluation of two chromogenic substrate methods for monitoring fondaparinux sodium.

This study was designed to develop methods for monitoring of the selective factor Xa inhibitor fondaparinux sodium (ARIXTRA) based on standard laboratory methods for the chromogenic determination of the anti-factor Xa activity of low molecular weight heparin. To examine the biologic activity of fondaparinux in comparison to its plasma concentration, 2 methods were investigated: 1 working with the addition of antithrombin (AT), the other without exogenous AT. Both methods showed a linear relationship of fondaparinux concentration and OD/min on a log-lin scale in the range from 0.1 to 2 microg/mL. Inter- and intra-assay variability was <6% in all cases. The results of spiked samples from patients on vitamin K antagonists (VKA) were in good agreement with both methods, and the determination of the fondaparinux concentration was not influenced by reduced levels of factor X in plasma caused by VKA-intake. Ex vivo samples from orthopedic patients (n=18) on prophylactic treatment with fondaparinux showed concentrations between 0.2 to 0.7 microg/mL 3 hours after s.c. injection. No significant differences were detected between both methods with these samples. The presented methods are suitable tools for monitoring of fondaparinux. The linear calibration curve in the range 0.1 to 2 microg/mL is suitable for determination of prophylactic and therapeutic application of fondaparinux. Both methods, with and without addition of AT, can be performed fully automated in clinical routine on an automated coagulation analyzer (STA coagulation analyzer). No significant differences were detected between both methods with these samples.

Use of native or platelet count adjusted platelet rich plasma for platelet aggregation measurements.

BACKGROUND: It is still not clear whether native or platelet count adjusted platelet rich plasma (PRP) should be used for platelet aggregation measurements. AIM: To evaluate the necessity of using adjusted PRP in platelet function testing. METHODS: Platelet aggregation with native PRP and adjusted PRP (platelet count: 250/nl, obtained by diluting native PRP with platelet poor plasma) was performed on the Behring Coagulation Timer (BCT(R)) using ADP, collagen, and arachidonic acid as agonists. Healthy subjects, patients on antiplatelet treatment, and patients with thrombocytosis (platelet counts in PRP > 1250/nl) were investigated. RESULTS: No significant differences in the maximum aggregation response were seen when using either native or adjusted PRP from healthy subjects and patients on antiplatelet treatment. Nevertheless, some patients taking aspirin or clopidogrel showed reduced inhibition of ADP and arachidonic acid induced aggregation in adjusted PRP but not in native PRP. The maximum velocity of healthy subjects and patients on antiplatelet treatment varied significantly as a result of the degree of dilution of the adjusted PRP. Surprisingly, the BCT provided good results when measuring platelet aggregation of native PRP from patients with thrombocytosis, whereas commonly used aggregometers could not analyse platelet aggregation of native PRP in these patients. CONCLUSION: The time consuming process of PRP adjustment may not be necessary for platelet aggregation measurements. Moreover, using adjusted PRP for monitoring aspirin or clopidogrel treatment may falsify results. Therefore, it may be better to use native PRP for platelet aggregation measurements, even in patients with thrombocytosis.

Influence of blood collection techniques on platelet function.

For investigations of platelet function it is recommended that venipuncture should be performed using ordinary needle systems instead of butterfly cannulae systems. Platelets might be activated in the long plastic tubes of butterfly systems. The aim of this study was to investigate the dependency of platelet function results on blood sampling using different collection systems. Therefore, blood of 25 healthy volunteers was collected from both arms using at the same time on one side a 21-gauge needle and on the other side a 21-gauge butterfly cannula system. Both samples of each volunteer were analyzed on the PFA-100. Platelet aggregation was performed on the Behring Coagulation Timer (BCT) and the optical aggregometer PAP-4 using ADP, collagen and arachidonic acid to induce platelet aggregation in platelet-rich plasma. No significant prolongation of the closure times on the PFA-100 with the COL/EPI cartridge and the COL/ADP cartridge was observed when using butterfly cannulae. The results of optical aggregometry were not significantly different. The maximum aggregation response did not differ significantly for both collection systems. Aggregometry and the PFA-100 system are not affected by different blood collection systems. Therefore butterfly cannulae can be used for sample collection to investigate platelet function.