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Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless, frequent relapses are common. In children with iNS, steroid-sparing agents are used to avoid prolonged steroid use and reduce steroid toxicity. Levamisole is one of these steroid-sparing drugs and although clinical effectiveness has been demonstrated, the molecular mechanisms of how levamisole exerts its beneficial effects remains poorly studied. Apart from immunomodulatory capacities, nonimmunological effects of levamisole on podocytes have also been suggested. We aimed to elaborate on the effects of levamisole on human podocytes in iNS. RNA sequencing data from a human podocyte cell line treated with levamisole showed that levamisole modulates the expression of various genes involved in actin cytoskeleton stabilization and remodeling. Functional experiments showed that podocytes exposed to puromycin aminonucleoside (PAN), lipopolysaccharides (LPS), and NS patient plasma resulted in significant actin cytoskeleton derangement, reduced cell motility, and impaired cellular adhesion when compared to controls, effects that could be restored by levamisole. Mechanistic studies revealed that levamisole exerts its beneficial effects on podocytes by signaling through the glucocorticoid receptor and by regulating the activity of Rho GTPases. In summary, our data show that levamisole exerts beneficial effects on podocytes by stabilizing the actin cytoskeleton in a glucocorticoid receptor-dependent manner.

Chemical exchange saturation transfer (CEST) has been explored for differentiation between tumour and benign tissue in prostate cancer (PCa) patients. With ultrahigh field strengths such as 7-T, the increase of spectral resolution and sensitivity could allow for selective detection of amide proton transfer (APT) at 3.5 ppm and a group of compounds that resonate at 2 ppm (i.e., [poly]amines and/or creatine). The potential of 7-T multipool CEST analysis of the prostate and the detection of PCa was studied in patients with proven localised PCa who were scheduled to undergo robot-assisted radical prostatectomy (RARP). Twelve patients were prospectively included (mean age 68.0 years, mean serum prostate-specific antigen 7.8ng/mL). A total of 24 lesions larger than 2 mm were analysed. Used were 7-T T2-weighted (T2W) imaging and 48 spectral CEST points. Patients received 1.5-T/3-T prostate magnetic resonance imaging and galium-68-prostate-specific membrane antigen-positron emission tomography/computerised tomography to determine the location of the single-slice CEST. Based on the histopathological results after RARP, three regions of interest were drawn on the T2W images from a known malignant zone and benign zone in the central and peripheral zones. These areas were transposed to the CEST data, from which the APT and 2-ppm CEST were calculated. The statistical significance of the CEST between the central zone, the peripheral zone, and tumour was calculated using a Kruskal-Wallis test. The z-spectra showed that APT and even a distinct pool that resonated at 2 ppm were detectable. This study showed a difference trend in the APT levels, but no difference in the 2-ppm levels when tested between the central zone, the peripheral zone, and tumour (H(2) = 4.8, p = 0.093 and H(2) = 0.86, p = 0.651, respectively). Thus, to conclude, we could most likely detect APT and amines and/or creatine levels noninvasively in prostate using the CEST effect. At group level, CEST showed a higher level of APT in the peripheral versus the central zone; however, no differences of APT and 2-ppm levels were observed in tumours., (© 2023 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Surgery for tongue cancer often results in a major loss in quality of life. While MRI may be used to minimise the volume of excised tissue, often the full tumour extent is missed. This tumour extent may be detected with metabolic imaging. One of the main reasons for the lack of metabolic information on tongue cancer would be the absence of an x-nuclear coil with the tongue as a focus target. Metabolic MRI through 31 P MRSI is known as a powerful tool to non-invasively study elevated cell proliferation and disturbed energy metabolism in tumours. Severe magnetic field non-uniformities are inherently caused by the substantial difference in magnetic susceptibilities of tissue and air in the mouth and its environs. Despite this, the wide chemical shift dispersion of 31 P could still facilitate precise detection of the cell proliferation biomarkers, phospomonoesters and diesters, as well as energy metabolites ATP, inorganic phosphate, and phosphocreatine potentially mapped over the tongue or tumour in vivo . In this study, we present the first 31 P MRSI data of the human tongue in vivo from healthy volunteers and a patient with a tongue tumour at 7 T MRI using a 1 H 8-channel transceiver setup placed inside a body 31 P transmitter, which is able to get a uniform excitation from the tongue while providing comfortable access to the patient. In addition, a user-friendly external 31 P receiver array is used to provide high sensitivity (80%) comparable to an uncomfortable inner mouth loop coil positioned on the tongue. The primary aim is the demonstration of 31 P metabolite profiles in the tongue and the differences between healthy and malignant tissue. Indeed, clear elevated cell proliferation expressed as enhanced phosphomonoesters is observed in the tumour vs. the healthy part of the tongue. This can be performed within a total scan duration of 30 min, comparable to clinical scans, with a spatial resolution of 1.5 cm for the 10-min 31 P MRSI scan., Competing Interests: RF was employed by company Ceresensa Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Forner, Nam, de Koning, van der Velden, van der Kemp, Raaijmakers and Klomp.)

Shiga-toxin (Stx)-producing Escherichia coli hemolytic-uremic syndrome (STEC-HUS) is one of the most common causes of acute kidney injury in children. Stx-mediated endothelial injury initiates the cascade leading to thrombotic microangiopathy (TMA), still the exact pathogenesis remains elusive. Interestingly, there is wide variability in clinical presentation and outcome. One explanation for this could be the enhancement of TMA through other factors. We hypothesize that heme, as released during extensive hemolysis, contributes to the etiology of TMA. Plasma levels of heme and its scavenger hemopexin and degrading enzyme heme-oxygenase-1 (HO-1) were measured in 48 STEC-HUS patients. Subsequently, the effect of these disease-specific heme concentrations, in combination with Stx, was assessed on primary human glomerular microvascular endothelial cells (HGMVECs). Significantly elevated plasma heme levels up to 21.2 µM were found in STEC-HUS patients compared to controls and were inversely correlated with low or depleted plasma hemopexin levels (R 2 -0.74). Plasma levels of HO-1 are significantly elevated compared to controls. Interestingly, especially patients with high heme levels (n = 12, heme levels above 75 quartile range) had high plasma HO-1 levels with median of 332.5 (86-720) ng/ml (p = 0.008). Furthermore, heme is internalized leading to a significant increase in reactive oxygen species production and stimulated both nuclear translocation of NF-κB and increased levels of its target gene (tissue factor). In conclusion, we are the first to show elevated heme levels in patients with STEC-HUS. These increased heme levels mediate endothelial injury by promoting oxidative stress and a pro-inflammatory and pro-thrombotic state. Hence, heme may be a contributing and driving factor in the pathogenesis of STEC-HUS and could potentially amplify the cascade leading to TMA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Wijnsma, Veissi, de Wijs, van der Velden, Volokhina, Wagener, van de Kar and van den Heuvel.)

Background: Rising healthcare costs due to unnecessary referrals to secondary healthcare services underscore the need for optimizing current referral procedures. This study investigates whether the use of web-based consultation (WBC) in general practice is a feasible alternative to decrease referrals., Methods: Patients with lumbosacral radicular syndrome, knee complaints, or thyroid dysfunction, who visited the general practitioner (GP) between May 2015 and December 2016 were included for a WBC. We determined whether the GP would refer a patient to an outpatient clinic in the absence of a WBC and then compared this decision with the referral advice from a specialist. We further assessed the user-friendliness of the WBC service based on average recorded user time and feedback from the GPs., Results: Seventy eligible WBCs submitted by GPs were analyzed. Our data showed a 46% absolute reduction in in-persons referrals in our study population. These findings confirmed the feasibility of using WBC. The median time spent to submit a WBC was five and 10 min for GPs and specialists respectively. On average, the WBC service saved €286 per WBC. The results of a questionnaire showed that GPs found WBC to be a user-friendly option which could help reduce the number of in-person referrals., Conclusion: We demonstrated that WBC is not only feasible but has the potential to reduce nearly half of all in-person referrals to outpatient clinics. WBC decreased healthcare expenses and proved to be a user-friendly and safe alternative to the standard referral process. WBC may potentially have a profound impact on healthcare expenditure if applied in a wider medical setting. For follow-up research, we recommend including a control group for comparative analyses.

Background: In the majority of pediatric patients, the hemolytic-uremic syndrome (HUS) is caused by an infection with Shiga toxin-producing Escherichia coli (STEC), mostly serotype O157. It is important to discriminate between HUS caused by STEC and complement-mediated HUS (atypical HUS) due to differences in treatment and outcome. As STEC and its toxins can only be detected in the patient's stool for a short period of time after disease onset, the infectious agent may go undetected using only fecal diagnostic tests. Serum antibodies to lipopolysaccharide (LPS) of STEC persist for several weeks and may therefore be of added value in the diagnosis of STEC., Methods: All patients with clinical STEC-HUS who were treated at Radboud University Medical Center between 1990 and 2014 were included in this retrospective single-center study. Clinical and diagnostic microbiological data were collected. Immunoglobulin M (IgM) antibodies against LPS of STEC serotype O157 were detected by a serological assay (ELISA)., Results: Data from 65 patients weres available for analysis. Fecal diagnostic testing found evidence of an STEC infection in 34/63 patients (54 %). Serological evidence of STEC O157 was obtained in an additional 16 patients. This is an added value of 23 % (p < 0.0001) when the serological antibody assay is used in addition to standard fecal diagnostic tests to confirm the diagnosis STEC-HUS. This added value becomes especially apparent when the tests are performed more than 7 days after the initial manifestation of the gastrointestinal symptoms., Conclusions: The serological anti-O157 LPS assay clearly makes a positive contribution when used in combination with standard fecal diagnostic tests to diagnose STEC-HUS and should be incorporated in clinical practice., Competing Interests: Compliance with ethical standards Financial disclosure All authors declare they have no financial relationships relevant to this article to disclose. Funding source No external funding for this manuscript. Conflict of interest All the authors declare that they have no potential conflicts of interest to disclose.

Purpose: Fluctuations of the background magnetic field (B0 ) due to body and breathing motion can lead to significant artifacts in brain imaging at ultrahigh field. Corrections based on real-time sensing using external field probes show great potential. This study evaluates different aspects of field interpolation from these probes into the brain which is implicit in such methods. Measurements and simulations were performed to quantify how well B0 -fluctuations in the brain due to body and breathing motion are reflected in external field probe measurements., Methods: Field probe measurements were compared with scanner acquired B0 -maps from experiments with breathing and shoulder movements. A realistic simulation of B0 -fluctuations caused by breathing was performed, and used for testing different sets of field probe positions., Results: The B0 -fluctuations were well reflected in the field probe measurements in the shoulder experiments, while the breathing experiments showed only moderate correspondence. The simulations showed the importance of the probe positions, and that performing full 3(rd) order corrections based on 16 field probes is not recommended., Conclusion: Methods for quantitative assessment of the field interpolation problem were developed and demonstrated. Field corrections based on external field measurements show great potential, although potential pitfalls were identified., (© 2015 Wiley Periodicals, Inc.)

Spectral degradations as a result of temporal field variations are observed in MRSI of the human prostate. Moving organs generate substantial temporal and spatial field fluctuations as a result of susceptibility mismatch with the surrounding tissue (i.e. periodic breathing, cardiac motion or random bowel motion). Nine patients with prostate cancer were scanned with an endorectal coil (ERC) on a 7-T MR scanner. Temporal B0 field variations were observed with fast dynamic B0 mapping in these patients. Simulations of dynamic B0 corrections were performed using zero- to second-order shim terms. In addition, the temporal B0 variations were applied to simulated MR spectra causing, on average, 15% underestimation of the choline/citrate ratio. Linewidth distortions and frequency shifts (up to 30 and 8 Hz, respectively) were observed. To demonstrate the concept of observing local field fluctuations in real time during MRSI data acquisition, a field probe (FP) tuned and matched for the (19)  F frequency was incorporated into the housing of the ERC. The data acquired with the FP were compared with the B0 field map data and used to correct the MRSI datasets retrospectively. The dynamic B0 mapping data showed variations of up to 30 Hz (0.1 ppm) over 72 s at 7 T. The simulated zero-order corrections, calculated as the root mean square, reduced the standard deviation (SD) of the dynamic variations by an average of 41%. When using second-order corrections, the reduction in the SD was, on average, 56%. The FP data showed the same variation range as the dynamic B0 data and the variation patterns corresponded. After retrospective correction, the MRSI data showed artifact reduction and improved spectral resolution. B0 variations can degrade the MRSI substantially. The simple incorporation of an FP into an ERC can improve prostate cancer MRSI without prior knowledge of the origin of the dynamic field distortions., (Copyright © 2014 John Wiley & Sons, Ltd.)

Background/aims: Diarrhea-associated hemolytic uremic syndrome is associated with the presence of Shiga toxin (Stx1, Stx2 and several variants) in the circulation. The aim of this study is to examine the possible triggering effect of Stx1 on the exocytosis of Weibel-Palade bodies (WPbs)., Methods: Cultured human umbilical venous endothelial cells (HUVECs) and glomerular microvascular endothelial cells (GMVECs) were stimulated by thrombin and Stx1 in both static and flowing conditions. The amount of secreted von Willebrand factor (VWF) in the supernatant as well as the remaining intracellular fraction was determined., Results: In HUVECs and in 2 out of 4 GMVECs, the stimulation of Stx1 in flow at 1 dyne/cm(2) resulted in a decrease of intracellular VWF. This is contrary to the results of Stx1 applied in static conditions. At a higher flow rate of 5 dyne/cm(2), no effect in GMVECs was observed., Conclusion: Stx1 can contribute, via an effect on WPbs, to the exocytosis of WPbs in flow conditions in HUVECs and probably in GMVECs. This results in the release of VWF, suggesting an initiating role of the coagulation system in the pathogenesis.

Driven by health care reform and the advent of the private sector in the late 1980s, and by commitments made to the Association of Southeast Asian Nations (ASEAN), Vietnam is faced with a need to increase the regulation and training of its health care professionals. Previously, a diploma from an accredited health professional school was sufficient to practice for a lifetime. Legislation has recently been passed that will institute a licensing system, will require continuing medical education (CME) to maintain the license, and will probably place a large burden on the health professional schools and training institutes to provide CME. Supported by international nongovernmental organizations and foreign universities, the medical universities in Vietnam are responding and are preparing for their new and expanded role.

This work describes the development of an animal-component free medium (IPT-AFM) that allows an optimal growth of Vero cells, an adherent cell line used for the production of viral vaccines. Statistical experimental design was applied to identify crucial nutrients that affect cell growth. Using Medium 199 or MEM as a basal medium, a serum-free medium (SFM) referred as IPT-SFM that only enclosed transferrin as a component of animal origin was developed at first. Then, the composition of IPT-SFM was further improved to obtain an animal-component free medium named IPT-AFM. IPT-AFM contains M199 as a basal medium, plant hydrolysates, epidermal growth factor, ethanolamine, ferric citrate, and vitamin C. Among various plant hydrolysates, specific combinations of soy (Hypep 1510) and wheat gluten (Hypeps 4601 and 4605) hydrolysates, were identified to promote cell growth; whereas individual Hypeps had a minor positive effect on cell growth. Nevertheless, the removal of serum did influence cell attachment. Coating tissue-culture flasks with teleostean, a product extracted from cold water fish skin, had not only enhanced cell attachment but also improved cell growth performance in static cultures. Different non-animal proteases were also assessed as an alternative to trypsin. TrypLE Select, a recombinant trypsin, gave the best cell growth performances. Kinetics of cell growth in IPT-AFM were investigated in T-flasks, cell growth was comparable with that obtained in MEM+10% fetal calf serum (FCS). A mean cell division number equal to 2.26 +/- 0.18 and a specific growth rate micro 0.019 +/- 0.003 h(-1) were achieved in IPT-AFM., ((c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009.)

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3+/-0.4 and a specific growth rate micro of 0.017+/-0.006 h(-1) were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM+10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10(7) FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5x10(6) cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5x10(6) cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5x10(7) FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.

Rabies virus strain production in Vero cells grown on Cytodex 1 in a 2 L stirred tank bioreactor and in a medium free of components of human or animal origin (VP-SFM) is described. Cell banking procedure in VP-SFM supplemented with an animal components free mixture (10%DMSO+0.1%methylcellulose) was reported and cell growth after revitalization was assessed. Vero cells exhibited growth performances (specific growth rate and cell division number) similar to that obtained in serum containing medium. To design a scalable process that is totally free of animal-derived substances, two proteases: TrypLE Select and Accutase, were assessed as an alternative to trypsin which is routinely used for cell passage. Growth performance of Vero cells grown in VP-SFM and MEM+10% fetal calf serum (FCS) over four passages and subcultivated with either TrypLE Select or Accutase was evaluated. TrypLE Select showed the best performance in terms of specific growth rate and cell division number. Kinetics of cell growth and rabies virus production (LP2061/Vero strain) were investigated in spinner flask and in a 2 L bioreactor. In spinner flask, a maximal cell density level of 1.85x10(6) cells/mL was achieved when the cells were grown in VP-SFM on 2 g/L Cytodex 1. Cell infection experiments conducted at an MOI of 0.3 and without the medium exchange step, typically needed for serum containing rabies virus production, resulted in a maximal virus titer equal to 2x10(7) (Fluorescent Focus Unit) FFU/mL. In stirred tank bioreactor, Vero cell growth in VP-SFM on 3 g/L Cytodex 1 was shown to be sensitive to the aeration mode. Sparging the culture was detrimental for cell growth, whereas cell density level was greatly enhanced when only headspace aeration was used. A cell density level of 2.6x10(6) cells/mL was obtained when the cells were grown on 3g/L Cytodex 1 and in batch culture mode. Cell infection at an MOI of 0.1 without any medium exchange, yielded a maximal rabies virus titer of 2.4x10(7) FFU/mL. Furthermore, Vero cell growth in a 2 L bioreactor using recirculation culture mode during cell proliferation step and perfusion for virus multiplication phase was investigated. In comparison to batch culture, a higher cell density level that was equal to 5x10(6) cells/mL was reached. Cell infection under conditions similar to batch culture, resulted in a maximal virus titer equal to 1.38x10(8) FFU/mL. The potency of the pooled inactivated virus harvests showed an activity of 2.58 IU/mL which was comparable to that obtained in serum supplemented medium.

Acute renal failure hallmarks the pathogenesis of the epidemic form of hemolytic uremic syndrome (D+HUS), which is caused by E. coli strains that produce Shiga-like toxin (Stx). In this study, we investigated the influence of Stx-1 on nitric oxide (NO) production by human glomerular microvascular endothelial cells (GMVEC) and human mesangial cells. NO synthesis by human mesangial cells is in the micromolar range and that of GMVEC in the picomolar range. Stx-1 reduced NO production in non-stimulated GMVEC (5 nmol/l Stx-1 required) without inhibition of protein synthesis. In non-stimulated and TNFalpha-pretreated mesangial cells, NO production was reduced with a maximal reduction at 10 fmol/l shiga toxin. The cellular iNOS antigen content in mesangial cells was reduced in a concentration-dependent way (10 fmol/l-100 pmol/l), while partial inhibition of protein synthesis required 10 nmol/l Stx-1 in these cells. Our in vitro data suggest that Stx may reduce NO synthesis during the course of HUS development, contributing to the aggravation of the thrombotic microangiopathy and renal failure as observed in HUS.

The Netherlands Vaccine Institute (NVI) developed the micro-carrier technology for large-scale production of IPV in the late 1960s and has used this technology successfully to produce IPV as well as DTP-IPV for the national immunization program in the Netherlands. As a public sector organization, and as one of the Millennium Development Goals, NVI has supported over the years access to vaccine technology like DTP and Hib for vaccine manufacturers in developing countries. In line with this role as a resource institute, NVI has recently been approached by a number of vaccine manufacturers, predominantly from developing countries, for transfer of IPV technology to meet the anticipated increase in demand for IPV following OPV cessation. Since WHO encourages new manufacturers to use the attenuated Sabin virus instead of wild polio strains in the production of IPV, NVI decided to respond positively to this WHO policy. The existing NVI experience in large-scale production of IPV and OPV using Vero cell based micro-carrier technology and its experience with experimental Sabin-IPV is an attractive start for the development of Sabin-IPV. This paper discusses the approach followed and the experience already gained in the project, as well as factors critical to its success.

The diarrhea-associated form of the hemolytic uremic syndrome (D+ HUS) is characterized by a triad of symptoms, namely thrombocytopenia, hemolytic anemia, and acute renal failure. Histopathological studies of patients with D+ HUS show microthrombi in arterioles and glomeruli of the kidney. Recently, it was suggested that antiphospholipid antibodies might play a pathogenic role in D+ HUS. However, an epiphenomenon could not be excluded. In this study we investigated the relationship between antiphospholipid antibodies and clinical symptoms in 22 patients with the classical form of HUS (D+ HUS). The first sample was obtained on the day of admission. The next samples were taken on day 7 and 14. We measured anticardiolipin (aCL) antibodies (IgM, IgA, and IgG) in the samples using an ELISA. A significant increase in IgM (60%) and IgG (41%) aCL antibodies was seen in patients versus controls. No relationship between aCL antibody levels and severity of renal failure could be demonstrated. These data suggest that antiphospholipid antibodies are increased, but have not been shown to have a role in the pathogenesis of the microangiopathy seen in D+HUS.

The objectives of this study are to determine whether Norplant would be an acceptable contraceptive method for Cambodian women, given its technology and the socio-cultural context, and whether it can be delivered by a private sector clinic with good quality care. This is a prospective cohort study of the first 966 acceptors. It was found the one-year continuation rate was 90.5%, there were no pregnancies and client satisfaction was high. In general, Norplant was delivered with high quality of care. Findings indicate that Norplant suits the contraceptive needs of many Cambodian women and is appropriate for their socio-cultural context. Norplant can be introduced, with high quality care, in a private clinic in a developing country.

The hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children. The role of a verocytotoxin (VT)-producing Escherichia coli has been strongly implicated in the epidemic form of HUS. Although direct toxicity of VT on glomerular endothelial cells has been demonstrated, it remained still unclear how the VT is transported from the intestine to the target organs. In this study we demonstrate that VT, when incubated in whole blood, binds rapidly and completely to human polymorphonuclear leukocytes (PMNs) and not to other components of blood. Binding studies with (125)I-VT-1 showed a single class of binding sites on freshly isolated, nonstimulated human PMNs. The K(d) of VT-binding to PMNs was 10(-8) mol/L, 100-fold less than that of the VT-receptor globotriaosylceramide. On incubation of VT-preloaded PMNs with human glomerular microvascular endothelial cells (GMVECs), transfer of VT-1 to the endothelial cells occurred. Incubation of nonstimulated GMVECs with VT-preloaded PMNs, but not with PMNs or VT-1 alone, caused inhibition of protein synthesis and cell death. Our data are in concert with a role of PMNs in the transfer of VT from the intestine to the kidney endothelium. This transfer occurs by selective binding to a specific receptor on PMNs and subsequent passing of the ligand VT to the VT-receptor on GMVECs, which causes cell damage. This new mechanism further underpins the important role of PMNs in HUS.

Background: Strains of verocytotoxin-producing Escherichia coli (VTEC) belonging to serogroup O157 (O157 VTEC) can cause a spectrum of disease that includes nonspecific diarrhea, hemorrhagic colitis and the diarrhea-associated form of the hemolytic uremic syndrome (D+ HUS)., Methods: We conducted a retrospective study of 34 children with D+ HUS caused by O157 VTEC to determine the frequency of VTEC infection in their household members., Results: Gastrointestinal tract symptoms were reported in 1 or more household contacts of 17 (50%) of the 34 index cases. Of the 26 household members with gastrointestinal tract symptoms, 15 were parents and 11 were siblings. Evidence of VTEC infection was reported in 1 or more household contacts in 23 (68%) of the 34 families (in 46% of the siblings and in 28% of the parents). Nineteen (48%) siblings had a positive stool sample and in only 5 (12%) of the siblings IgM class serum antibodies to O157-lipopolysaccharide (LPS) were detected. Nineteen (31%) parents had a positive stool sample. Antibodies to O157-LPS were not detected in any of the parents. The occurrence of (bloody) diarrhea significantly correlated with the occurrence of IgM class serum antibodies to O157-LPS., Conclusions: It was concluded that household members of children with D+ HUS are often asymptomatically infected with O157 VTEC. Differences in the pathogenesis of the infection between infected individuals may be related to differences in the number of ingested O157 VTEC bacteria and to differences in susceptibility.

The epidemic form of the hemolytic uremic syndrome (HUS) in children is hallmarked by endothelial cell damage, most predominantly displayed by the glomerular capillaries. The influx of mononuclear (MO) and polymorphonuclear cells (PMNs) into the glomeruli may be an important event in the initiation, prolongation, and progression of glomerular endothelial cell damage in HUS patients. The molecular mechanisms for the recruitment of these leukocytes into the kidney are unclear, but monocyte chemoattractant protein-1 (MCP-1) and IL-8 are suggested to be prime candidates. In this study, we analyzed the presence of both chemokines in 24-h urinary (n = 15) and serum (n = 14) samples of HUS children by specific ELISAs. Furthermore, kidney biopsies of three different HUS children were examined for MO and PMN cell infiltration by histochemical techniques and electron microscopy. Whereas the chemokines MCP-1 and IL-8 were present in only very limited amounts in urine of 17 normal control subjects, serial samples of HUS patients demonstrated significantly elevated levels of both chemokines. HUS children with anuria showed higher initial and maximum chemokine levels than their counterparts without anuria. A strong positive correlation was observed between urinary MCP-1 and IL-8 levels. Whereas initial serum IL-8 levels were significantly increased in HUS children, serum MCP-1 levels were only slightly elevated compared with serum MCP-1 in control children. No correlation was found between urinary and serum chemokine concentrations. Histologic and EM studies of HUS biopsy specimens clearly showed the presence of MOs and to a lesser extent of PMNs in the glomeruli. The present data suggest an important local role for MOs and PMNs in the process of glomerular endothelial-cell damage. The chemokines MCP-1 and IL-8 may possibly be implicated in the pathogenesis of HUS through the recruitment and activation of MOs and PMNs, respectively.

In the pathogenesis of the hemolytic uremic syndrome (HUS), endothelial damage of glomeruli and arterioles of the kidney appears to play a central role. Previous studies have shown that verocytotoxin-1 (VT-1) cytotoxicity on human vein endothelial cells require additional stimuli, in particular the inflammatory mediator tumor necrosis factor alpha (TNF alpha). In this study the effects of VT on human glomerular microvascular endothelial cells (GMVEC) were examined. A reproducible method was developed for the isolation and purification of large numbers of highly purified GMVEC. The obtained GMVEC were over 99% pure; their endothelial origin was demonstrated by the expression of the endothelial antigens von Willebrand factor, EN-4, PECAM-1 and V,E-cadherin. Upon stimulation with TNF alpha the cells expressed the endothelial-specific adhesion molecule E-selectin. A limited number of fenestral structures was observed by scanning electron microscopy (SEM), suggesting glomerular origin of the endothelial cells. Cytotoxicity of VT-1 to GMVEC was evaluated by determination of the number of viable adherent cells and by assay of overall protein synthesis after exposure to varying concentrations of VT-1. In non-stimulated GMVEC, cytotoxicity of VT-1 was inversely related to the degree and duration of confluence, subconfluent cells being the most sensitive. In highly confluent GMVEC, VT cytotoxicity required pre-exposure of the cells to the inflammatory mediator TNF alpha, which induced an increase in the number of VT receptors on GMVEC. Thin layer chromatography of extracted glycolipids from the GMVEC showed binding of VT-1 to globotriaosylceramide (Gb3), known to be the functional receptor for VT. There were no major differences in protein synthesis inhibition with equal concentrations VT-1 and VT-2. In conclusion, in this study we provide a reproducible method to isolate, purify and culture well characterized human GMVEC on a routine basis. In vitro studies with these GMVEC demonstrate that VT cytotoxicity depends on the degree of confluence and the additional preexposure to the inflammatory mediator TNF alpha. These observations provide further insight into the complex events that may occur in glomeruli in the pathogenesis of HUS.

[3H]t-Butylbicycloorthobenzoate ([3H]TBOB) binds to specific sites on crude synaptic rat brain membranes. The dissociation constant, Kd, determined from saturation experiments is near 8 nM and the receptor density Bmax is about 20 pmol/g wet tissue. Non-specific binding constitutes about 35% of the total binding at 4 nM [3H]TBOB. The association of [3H]TBOB is monophasic but its dissociation is biphasic. Kd values of 8 nM (70% of the binding sites) and 20 nM (30% of the binding sites) were estimated from the kinetic data. These values differ from those previously reported. Specifically bound [3H]TBOB is displaced by picrotoxin and by t-butylbicyclophosphorothionate (TBPS). No simple competitive interaction of picrotoxin with [3H]TBOB binding was found. Micromolar quantities of the GABAergic facilitating compounds, GABA, muscimol and diazepam inhibited [3H]TBOB binding in an allosteric manner.

The biochemical mechanism responsible for the convulsive effects of folates was investigated. The epileptogenic effects of folates were determined in vivo by quantification of the seizures following intracortical application in rats. The rank order of epileptogenic effects is: folic acid greater than or equal to 5-HCO-H4 folate greater than H2 folate greater than 5-CH3-H4 folate. This sequence of epileptogenicity in vivo is compared to the rank order of the effects of folates on radioligand binding to the GABAA-receptor complex in vitro. The inhibitory potencies of folates on [3H]muscimol and [3H]diazepam bindings did not correlate with their epileptogenic effects. However, folates reverse the inhibiting effect of GABA on the binding of the cage convulsant [3H]TBOB [( 3H]t-butylbicycloorthobenzoate). The rank order of this in vitro effect (folic acid greater than 5-HCO-H4 folate greater than H2 folate = 5-CH3-H4 folate) resembles the rank order of epileptogenicity determined in vivo. A relationship between the in vivo and in vitro effects is therefore suggested.

The rate of ATP synthesis (JP) in isolated rat-liver mitochondria was strongly dependent on the magnitude of the protonmotive force (delta mu H+) across the mitochondrial inner membrane. Addition of different concentrations of various uncouplers or malonate to mitochondrial incubations in State 3 led to a depression of delta mu H+ and a concomitant decrease in JP. A unique relationship between JP and delta mu H+ was obtained, which was independent of the way in which delta mu H+ was varied. This unique relationship was observed when K+ (in the presence of valinomycin) was used as a probe for delta psi. Different relationships between JP and delta mu H+ were observed when K+ was used as a probe for delta psi and when K+ was measured after separation of the mitochondria by centrifugation without silicone oil. This led to a serious underestimation of delta psi, specifically when uncouplers were present, and non-unique flow-force relationships were thus obtained. Anomalous relationships between JP and delta mu H+ were also found when TPMP+ was used as a probe for delta psi. However, in uncoupler incubations the presence of TBP- strongly affected the TPMP+ accumulation ratio without any effect on the K+ accumulation or on JP and in the presence of TBP- unique relationships between JP and delta mu H+ were again obtained. This indicates that the accumulation of TPMP+ inside the mitochondria is not a straightforward function of delta psi but also depends on conditions like the presence of TBP- or uncouplers. We conclude that there is a unique relationship between the rate of phosphorylation and the protonmotive force in mitochondria and that under some conditions the behaviour of TPMP+ is anomalous.

The site of action responsible for the convulsive effect of folic acid was investigated in vitro. Folic acid (ECmax 5 x 10(-4) M) enhances the binding of the cage convulsant [3H]t-butylbicycloorthobenzoate ([3H]TBOB) to rat brain membranes, namely to 130% of control in the absence of GABA and to over 300% of control in the presence of physiological concentrations of GABA. Analysis of the binding parameters reveals that folic acid increases the apparent number of [3H]TBOB binding sites.