Early deformation mechanisms in the shear affected region underneath a copper sliding contact.

Dislocation mediated plastic deformation decisively influences the friction coefficient and the microstructural changes at many metal sliding interfaces during tribological loading. This work explores the initiation of a tribologically induced microstructure in the vicinity of a copper twin boundary. Two distinct horizontal dislocation traces lines (DTL) are observed in their interaction with the twin boundary beneath the sliding interface. DTL formation seems unaffected by the presence of the twin boundary but the twin boundary acts as an indicator of the occurring deformation mechanisms. Three concurrent elementary processes can be identified: simple shear of the subsurface area in sliding direction, localized shear at the primary DTL and crystal rotation in the layers above and between the DTLs around axes parallel to the transverse direction. Crystal orientation analysis demonstrates a strong compatibility of these proposed processes. Quantitatively separating these different deformation mechanisms is crucial for future predictive modeling of tribological contacts.

Mitosin and pHH3 predict poorer survival in astrocytomas WHO grades II and III.

AIMS: The limitations of the current WHO classification of astrocytomas call for a sustained effort to improve diagnostic and prognostic accuracy. The relationship between tumour growth and clinical outcome suggests that proliferative activity should be examined. The objective of this study was to evaluate the diagnostic and prognostic value of the proliferation markers mitosin and phosphohistone H3 (pHH3) in infiltrative astrocytomas WHO grades II and III and compare the findings with mitotic count and Ki-67/MiB-1 immunostaining. METHODS: Fifty-nine and thirty-three infiltrative astrocytomas WHO grades II and III, respectively, were immunostained with the proliferation markers mitosin and pHH3 using standard immunohistochemical procedures. The expression was quantified as a proliferative index (PI) and statistically evaluated with Spearman's rank correlation test, Wilcoxon-Mann-Whitney U test, and univariable and multivariable COX regression survival analyses. RESULTS: Significant positive correlations were found between these proliferation markers. The number of mitoses, pHH3 mitotic figures (MFs), the Ki-67/MiB-1 PI and the mitosin PI were greater in WHOgrade III anaplastic astrocytomas compared to WHO grade II diffuse astrocytomas, while pHH3 PI only showed a trend. All proliferation markers were associated with poorer prognosis, but mitotic count was not. Ki-67/MiB-1, mitosin and pHH3 MF achieved statistical significance in the univariable analyses of both time to relapse (TTR) and overall survival (OS). Only mitosin remained significant in both multivariable analyses. pHH3 was significant in the multivariable analysis of OS but not of TTR. Clinical factors including age, extent of surgical resection and WHO performance status were also significantly correlated with survival. CONCLUSIONS: In conclusion, mitosin and pHH3 immunostaining have prognostic and diagnostic value in the clinical assessment of patients with infiltrative astrocytomas. The inclusion of proliferation markers in a layered diagnosis should be considered in the upcoming revision of the WHO classification system.

Simple 3D images from fossil and recent micromaterial using light microscopy.

Abstract We present a technique for extracting 3D information from small-scale fossil and Recent material and give a summary of other contemporary techniques for 3D methods of investigation. The only hardware needed for the here-presented technique is a microscope that can perform dark field and/or differential interference contrast with a mounted digital camera and a computer. Serial images are taken while the focus is successively shifted from the uppermost end of the specimen to the lowermost end, resulting in about 200 photographs. The data are then processed almost completely automatically by successive use of three freely available programs. Firstly, the stack of images is aligned by the use of CombineZM, which is used to produce a combined image with a high depth of field. Secondly, the aligned images are cropped and sharp edges extracted with the aid of ImageJ. Thirdly, although ImageJ is also capable of producing 3D representations, we preferred to process the image stack further using osirix as it has the facility to export various formats. One of the interesting export formats is a virtual Quicktime movie file (QTVR), which can be used for documentation, and stereo images can also be produced from this Quicktime VR. This method is easy to apply and can be used for documenting specimens in 3D (at least some aspects) without having to prepare them. Therefore, it is particularly useful as a safe method for documenting limited material, before using methods that may destroy the specimen of interest, or to investigate type material that cannot be treated with any preparatory technique. As light microscopes are available in most labs and free computer programs are easily accessible, this method can be readily applied.

[Registration of clinical trials: a statement from the International Committee of Medical Journal Editors]. / Registratie van klinische trials: een verklaring van het International Committee of Medical Journal Editors.

Altruistic motives and trust are central to scientific investigations involving people. These prompt volunteers to participate in clinical trials. However, publication bias and other causes of the failure to report trial results may lead to an overly positive view of medical interventions in the published evidence available. Registration of randomised controlled trials right from the start is therefore warranted. The International Committee of Medical Journal Editors has issued a statement to the effect that the 11 journals represented in the Committee will not consider publication of the results of trials that have not been registered in a publicly accessible register such as www.clinicaltrials.gov. Patients who voluntarily participate in clinical trials need to know that their contribution to better human healthcare is available for decision making in clinical practice.

Influence of hepatocyte growth factor, epidermal growth factor, and mycophenolic acid on endothelin-1 synthesis in human endothelial cells.

BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which plays an important pathophysiological role in ischaemic renal failure and drug-induced renal injury such as cyclosporin A (CsA)- and tacrolimus-associated nephrotoxicity. In contrast, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) seem to accelerate renal regeneration after ischaemic and drug-induced renal injury. This study aimed to investigate the influence of HGF and EGF on ET-1 synthesis in cultured human umbilical vein endothelial cells (HUVEC) and renal artery endothelial cells (RAEC). In addition, we have investigated whether mycophenolic acid (MPA), a new immunosuppressive drug, which in contrast to CsA and tacrolimus lacks nephrotoxic side effects, modulates ET-1 synthesis in endothelial cells. METHODS: ET-1 release was measured with a specific enzyme-linked immunosorbent assay. ET-1 mRNA expression was investigated by reverse transcription polymerase chain reaction. RESULTS: HGF and EGF (0.001-10 nM) exerted a significant concentration-dependent inhibitory effect on ET-1 release by HUVEC and RAEC (minimum 56.1+/-4.3% of control, n=6, mean+/-SE). The suppressive effect of HGF and EGF on ET-1 synthesis was dose-dependently antagonized by the tyrosine kinase inhibitors tyrphostin AG1478, lavendustin A and methyl 2,5-dihydroxycinnamate. Incubation of HUVEC and RAEC with MPA (2.5, 10, 25, and 50 microg/ml) for 3-5 h induced a significant reduction of ET-1 mRNA expression. After 48 h incubation with MPA (1-50 microg/ml) a significant decrease of ET-1 release and DNA content per culture well was observed, whereas ET-1 release referred to the DNA content in the corresponding culture well did not differ significantly from controls. CONCLUSIONS: The present findings demonstrate that HGF and EGF reduce ET-1 synthesis in endothelial cells via their receptor tyrosine kinase activity and suggest that the renoprotective effects of HGF and EGF might be linked to their inhibitory action on ET-1 synthesis. This study also provides evidence that, in contrast to CsA and tacrolimus, MPA does not stimulate ET-1 synthesis. This might explain the clinical observation that renal function often improves when CsA or tacrolimus is replaced by mycophenolate mofetil.

Endothelin-1 synthesis and endothelin B receptor expression in human coronary artery smooth muscle cells and monocyte-derived macrophages is up-regulated by low density lipoproteins.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide exerting its effects predominantly by paracrine and autocrine mechanisms. ET-1 acts as a mitogen and co-mitogen on vascular smooth muscle cells, and accumulating evidence suggests that ET-1 is involved in the pathogenesis of atherosclerosis. Deposition of low density lipoproteins (LDL) in the vessel wall is known to play a crucial role in the development of atherosclerotic lesions. In the present study, we have investigated the effect of native LDL (nLDL) and oxidatively modified LDL (oxLDL) on ET-1 synthesis and endothelin receptor expression in cultured human coronary artery smooth muscle cells and human monocyte-derived macrophages. Native LDL and oxLDL induced a significant stimulation of ET-1 release and ET-1 mRNA expression in human coronary artery smooth muscle cells and monocyte-derived macrophages. Antibodies against the scavenger receptor CD36 significantly reduced the oxLDL-induced stimulation of ET-1 synthesis. The antioxidants trolox and probucol did not significantly inhibit the LDL-induced rise of ET-1 release. Endothelin B receptor expression was up-regulated in both cell types after incubation with nLDL and oxLDL. In coronary smooth muscle cells, endothelin A receptor expression was slightly increased by LDL, whereas endothelin A receptor was not detectable in monocyte-derived macrophages. Coronary artery smooth muscle cells secreted a more than 150-fold higher amount of immunoreactive ET-1 into the cell culture medium than monocyte-derived macrophages. In summary, the present data, demonstrating a LDL-induced up-regulation of the endothelin system in coronary smooth muscle cells and in monocyte-derived macrophages, provide further support for a pathophysiological role of endothelin in coronary atherosclerosis and suggest that ET-1 might be involved in mediating the atherogenic effects of LDL.

A novel method for continuous online glucose monitoring in humans: the comparative microdialysis technique.

The aim of this study was to prove the feasibility of continuous subcutaneous glucose monitoring in humans using the comparative microdialysis technique (CMT). The performance of the CMT was determined by comparing tissue glucose values with venous or capillary blood glucose values in healthy volunteers and type 1 diabetic subjects. The CMT is a microdialysis-based system for continuous online glucose monitoring in humans. This technique does not require calibration by the patient. Physiological saline with glucose (5.5 mM) is pumped in a stop-flow mode through a microdialysis probe inserted into the abdominal s.c. tissue. Tissue glucose concentration is calculated by comparing the dialysate and perfusate glucose concentrations. The time delay due to the measurement process is 9 min. We tested the CMT on six healthy volunteers and six type 1 diabetic patients for 24 h in our clinical setting. Comparisons were made to HemoCue analyzer (Angelholm, Sweden) capillary blood glucose measurements (healthy volunteers) and to venous blood glucose concentration determined with a Hitachi analyzer (diabetic patients). The mean absolute relative error of the CMT glucose values from the blood glucose values was 17.8+/-15.5% (n = 167) for the healthy volunteers and 11.0+/-10.8% (n = 425) for the diabetic patients. The mean difference was 0.42+/-1.06 mM (healthy volunteers) and -0.17+/-1.22 mM (diabetic patients). Error grid analysis for the values obtained in diabetic patients demonstrated that 99% of CMT glucose values were within clinically acceptable regions (regions A and B of the Clarke Error Grid). The study results show that the CMT is an accurate technique for continuous online glucose monitoring.

Recent advances in continuous glucose monitoring.

Continuous glucose monitoring, providing more detailed information on glucose excursions than single spot measurements, should help to improve the therapy in diabetic patients and is also required for feedback-controlled insulin delivery. At the Institute for Diabetes-Technology in Ulm, founded by EF Pfeiffer, a portable glucose sensor for continuous tissue glucose monitoring has been developed. The combination of microdialysis and enzymatic amperometric glucose measurement implemented in this device marked a break-through in achieving reliable and precise continuous tissue glucose monitoring. In several studies, we have demonstrated that continuous subcutaneous glucose monitoring for up to 72 hours is feasible under 'in-house' and 'daily life' conditions in diabetic patients. The measured tissue glucose concentrations correlated closely to glucose control measurements in venous and capillary blood. A reliable continuous glucose monitoring device is a prerequisite for the development of an artificial pancreas. Our group developed an algorithm for subcutaneous application of the fast acting insulin analogon lispro. In experiments performed over 7 and 24 hours good metabolic control was achieved by algorithm-based insulin application. In addition, the algorithm was able to maintain acceptable metabolic control during and after moderate physical exercise. Further work is needed to optimize continuous tissue glucose monitoring systems and to develop a closed loop system for insulin application based on continuously measured tissue glucose concentrations.

Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis.

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.

Hepatocyte growth factor is upregulated by low-density lipoproteins and inhibits endothelin-1 release.

Low-density lipoproteins (LDL) are known to cause endothelial injury and to promote the development of atherosclerotic lesions. This study demonstrates a significant concentration-dependent stimulatory effect of LDL on hepatocyte growth factor (HGF) synthesis (maximum release: 423 +/- 16% of control) and HGF receptor mRNA expression in cultured human coronary artery endothelial cells (HCAEC). HGF is a potent mitogen for endothelial cells but does not affect smooth muscle cell proliferation. In contrast, endothelin-1 (ET-1) acts as a mitogen on vascular smooth muscle cells and seems to be upregulated in coronary atherosclerosis. In this study, the basal ET-1 synthesis in HCAEC was concentration-dependently reduced by HGF (minimum: 54 +/- 3% of control). This inhibitory effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met, since it was antagonized by the tyrosine kinase inhibitor lavendustin A. In addition, HGF also significantly reduced the LDL-stimulated ET-1 release. The LDL-induced upregulation of HGF synthesis in HCAEC and the inhibitory effect of HGF on ET-1 synthesis suggest a protective role of HGF in coronary atherosclerosis.

Inhibitory effect of epidermal growth factor and hepatocyte growth factor on endothelin-1 release by rabbit proximal tubule cells.

Several studies have demonstrated an upregulation of endothelin-1 (ET-1) synthesis in acute and chronic renal failure. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) have been shown to stimulate renal tubular cell proliferation and to accelerate renal regeneration after drug-induced and ischemia-induced renal injury. This study aimed to investigate the effect of EGF and HGF on ET-1 release, and whether the effect of EGF and HGF is antagonized by the tyrosine kinase inhibitor lavendustin A. Rabbit proximal tubule cells were incubated for 48 h with EGF or HGF (0.1-10.0 nM), lavendustin A (0.1-10.0 microM) or co-incubated with EGF or HGF (1 nM) and lavendustin A. ET-1 concentrations in the culture medium were measured with a specific enzyme-linked immunosorbent assay (ELISA). EGF and HGF exerted a significant (p < 0.001) dose-dependent inhibitory effect on ET-1 release. Lavendustin A induced a dose-dependent stimulation of ET-1 release and antagonized the inhibitory effect of EGF and HGF on ET-1 release. The inhibition of EGF and HGF receptor tyrosine kinase activity by lavendustin A was confirmed by Western blotting. These data suggest that EGF and HGF reduce ET-1 release via EGF and HGF receptor tyrosine kinase activity. The inhibitory action of EGF and HGF on ET-1 release might be involved in mediating the protective effects of EGF and HGF in renal injury.

Depending on their concentration oxidized low density lipoproteins stimulate extracellular matrix synthesis or induce apoptosis in human coronary artery smooth muscle cells.

Various lines of evidence indicate that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of atherosclerosis and coronary heart disease. We have investigated the effect of modified (oxidized) low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis in cultured human coronary artery smooth muscle cells (HCA-SMC). As shown by immunofluorescence microscopy and time-resolved fluorescence immunoassay, oxLDL dose-dependently stimulated collagen type I and fibronectin synthesis in cultured HCA-SMC. The effect on matrix synthesis was biphasic, with a maximum effect at concentrations between 1 and 10 microg/ml oxLDL. Higher oxLDL concentrations (>25 microg/ml) were cytotoxic. Beside oxLDL, malondialdehyde-modified LDL also stimulated extracellular matrix synthesis. In the presence of 100 microg/ml ascorbic acid, 25, 50 and 100 microg/ml oxLDL induced apoptosis within 6-8 hours (demonstrated by TUNEL-reaction, annexin-V binding and APO-2.7-expression). Apoptosis was not induced by normal (unmodified) LDL and malondialdehyde-modified LDL. The radical scavengers and antioxidants TROLOX and probucol and the hydrogen peroxide eliminator catalase significantly reduced oxLDL-induced apoptosis. Our results demonstrate that low concentrations of oxLDL are profibrogenic by stimulating extracellular matrix synthesis, whereas higher oxLDL concentrations induce oxidative stress and apoptosis in coronary artery smooth muscle cells. The profibrogenic effect might be relevant in the formation of atherosclerotic plaques, and the proapoptotic effect might contribute to an increased plaque vulnerability.

Decreased bone formative and enhanced resorptive markers in human immunodeficiency virus infection: indication of normalization of the bone-remodeling process during highly active antiretroviral therapy.

As cytokines and 1,25-dihydroxyvitamin D [1,25-(OH)2D] appear to have an important role in bone homeostasis, we examined the possibility that human immunodeficiency virus (HIV)-infected patients, characterized by enhanced levels of proinflammatory cytokines and 1,25-(OH)2D deficiency, have disturbed bone metabolism by analyzing serum markers of bone formation (osteocalcin) and bone resorption (C-telopeptide) in 73 HIV-infected patients. HIV-infected patients with advanced clinical and immunological disease and high viral load were characterized by increased C-telopeptide and particularly by markedly depressed osteocalcin levels. HIV-infected patients had enhanced activation of the TNF system. Serum concentrations of p55 and p75-TNF receptors were negatively correlated with osteocalcin, and p75-TNF receptor was positively correlated with C-telopeptide. HIV-infected patients with advanced disease also had decreased serum concentrations of 1,25-(OH)2D, but this parameter was not correlated with osteocalcin or C-telopeptide. During 24 months with highly active antiretroviral therapy there was a marked rise in serum osteolcalcin levels together with a profound fall in viral load and TNF components and a marked rise in CD4+ T cell counts. Also, there was a shift from no correlation to a significant correlation between osteocalcin and C-telopeptide levels during such therapy. The present study suggests disturbed bone formation and resorption during HIV infection. Our findings indicating synchronization of bone remodeling during highly active antiretroviral therapy may represent a previously unrecognized beneficial effect of such therapy and expand our knowledge of the interactions between cytokines and bone in the bone-remodeling process.

Endothelin release by rabbit proximal tubule cells: modulatory effects of cyclosporine A, tacrolimus, HGF and EGF.

BACKGROUND: Previous studies have suggested that endothelins, a family of 21 amino acid peptides with potent vasoconstrictive and mitogenic properties, are involved in the pathogenesis of acute and chronic renal failure. In addition, endothelin seems to play an important role in mediating the nephrotoxic side effects of cyclosporine A (CsA) and tacrolimus. The present study investigated the production of endothelin-1 (ET-1) and endothelin-3 (ET-3) by bipolar differentiated rabbit proximal tubule cells (PT-1 cells), and the modulatory effect of CsA, tacrolimus, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on ET-1 and ET-3 release. METHODS: ET-1 and ET-3 mRNA was detected by RT-PCR, immunoreactive endothelin was localized to PT-1 cells by immunofluorescence staining with antibodies against ET-1 and ET-3. ET-1 and ET-3 release into the culture medium was determined by specific radioimmunoassays after solid phase extraction. RESULTS: PT-1 cells exhibited a time-dependent increase of ET-1 release up to an incubation period of 36 hours, whereas ET-3 release already reached a steady state level after four hours. PT-1 cells, cultured on filter membranes, released a significantly higher amount of immunoreactive ET-1 into the basolateral compartment than into the apical compartment. ET-3 release did not differ significantly between the basolateral and the apical compartment. Supplementation of the cell culture medium with 10% fetal calf serum induced a marked increase of the basolateral and apical ET-1 release, whereas ET-3 release was only slightly increased. CsA and tacrolimus (0.5 to 5000 microgram/liter) induced a significant, dose-dependent increase of ET-1 and ET-3 release by PT-1 cells with a maximum stimulation at a CsA concentration of 500 microgram/liter (P < 0.001) and a tacrolimus concentration of 50 microgram/liter (P < 0.001). HGF and EGF (10-10 to 10-8 mol/liter) exerted a significant (P < 0.001) dose-dependent inhibitory effect on ET-1 release, whereas ET-3 release was not significantly reduced. Coincubation of PT-1 cells with CsA or tacrolimus and HGF or EGF also resulted in a marked reduction of ET-1 release. CONCLUSIONS: The present data suggest that ET-1 and ET-3 release by cultured rabbit proximal tubule cells are regulated differently, and that the stimulatory effect of CsA and tacrolimus on ET-1 release is antagonized by HGF and EGF.

Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeostasis.

The serum level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D], the biologically most potent metabolite of vitamin D, is tightly regulated within narrow limits in human healthy adults. 1,25-(OH)2D deficiency is rare and is associated with disturbances in calcium and bone metabolism. We have previously reported a marked decrease in serum levels of 1,25-(OH)2D in human immunodeficiency virus (HIV)-infected patients. The present study was designed to further examine the causes and consequences of severe 1,25-(OH)2D deficiency in these patients. The design was a prospective cohort study. Fifty-four HIV-infected patients clinically classified according to the revised criteria from Centers for Disease Control and Prevention and healthy controls were studied. Parameters related to vitamin D and calcium metabolism as well as immunological and nutritional status were determined. Twenty-nine of the patients (54%) had serum levels of 1,25-(OH)2D below the lower reference limit, and 18 of these had undetectable levels. In contrast, HIV-infected patients had normal serum levels of 25-hydroxyvitamin D and vitamin D-binding protein. HIV-infected patients as a group had modestly depressed serum calcium and PTH levels. There were, however, no correlations between these parameters and serum levels of 1,25-(OH)2D. There were no differences in serum calcium or PTH levels or nutritional status when patients with severe 1,25-(OH)2D deficiency were compared to other patients, but patients with undetectable 1,25-(OH)2D had significantly elevated serum phosphate levels. Furthermore, patients with undetectable 1,25-(OH)2D levels were characterized by advanced clinical HIV infection, low CD4+ lymphocyte counts, and high serum levels of tumor necrosis factor-alpha (TNFalpha). We conclude that inadequate 1alpha-hydroxylation of 25-hydroxyvitamin D seems to be the most likely cause of 1,25-(OH)2D deficiency in HIV-infected patients, possibly induced by an inhibitory effect of TNFalpha. The low 1,25-(OH)2D and high TNFalpha levels observed may impair the immune response in HIV-infected patients both independently and in combination and may represent an important feature of the pathogenesis of HIV-related immunodeficiency. Markedly depressed 1,25-(OH)2D serum levels are also present in certain other disorders characterized by immunological hyperactivity. Thus, the findings in the present study may not only represent a previously unrecognized immune-mediated mechanism for induction of 1,25-(OH)2D deficiency in human disease, but may also reflect the importance of adequate serum levels of 1,25-(OH)2D for satisfactory performance of the immune system in man.

Radiometric quantification of Mycobacterium avium complex.

The purpose of this study was to establish a system that would allow rapid and reliable quantification of Mycobacterium avium complex infection with a method that was as sensitive as counting of colony-forming units but less time-consuming and safer in the laboratory. The radiometric system for quantification of mycobacteria (Bactec, Becton Dickinson, USA) which calculates growth curves, was found to be faster, safer, and as sensitive as the established method of counting colony-forming units, with a low intra-assay variation and a wide assay range. Furthermore, the calculated growth curves provided important additional information about replication characteristics of the mycobacteria.