Effects of arsenolipids on in vitro blood-brain barrier model.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids (AsLs) occurring in fish and edible algae, possess a substantial neurotoxic potential in fully differentiated human brain cells. Previous in vivo studies indicating that AsHCs cross the blood-brain barrier of the fruit fly Drosophila melanogaster raised the question whether AsLs could also cross the vertebrate blood-brain barrier (BBB). In the present study, we investigated the impact of several representatives of AsLs (AsHC 332, AsHC 360, AsHC 444, and two arsenic-containing fatty acids, AsFA 362 and AsFA 388) as well as of their metabolites (thio/oxo-dimethylpropionic acid, dimethylarsinic acid) on porcine brain capillary endothelial cells (PBCECs, in vitro model for the blood-brain barrier). AsHCs exerted the strongest cytotoxic effects of all investigated arsenicals as they were up to fivefold more potent than the toxic reference species arsenite (iAsIII). In our in vitro BBB-model, we observed a slight transfer of AsHC 332 across the BBB after 6 h at concentrations that do not affect the barrier integrity. Furthermore, incubation with AsHCs for 72 h led to a disruption of the barrier at sub-cytotoxic concentrations. The subsequent immunocytochemical staining of three tight junction proteins revealed a significant impact on the cell membrane. Because AsHCs enhance the permeability of the in vitro blood-brain barrier, a similar behavior in an in vivo system cannot be excluded. Consequently, AsHCs might facilitate the transfer of accompanying foodborne toxicants into the brain.

Arsenic-containing hydrocarbons: effects on gene expression, epigenetics, and biotransformation in HepG2 cells.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids found in fish and algae, elicit substantial toxic effects in various human cell lines and have a considerable impact on cellular energy levels. The underlying mode of action, however, is still unknown. The present study analyzes the effects of two AsHCs (AsHC 332 and AsHC 360) on the expression of 44 genes covering DNA repair, stress response, cell death, autophagy, and epigenetics via RT-qPCR in human liver (HepG2) cells. Both AsHCs affected the gene expression, but to different extents. After treatment with AsHC 360, flap structure-specific endonuclease 1 (FEN1) as well as xeroderma pigmentosum group A complementing protein (XPA) and (cytosine-5)-methyltransferase 3A (DNMT3A) showed time- and concentration-dependent alterations in gene expression, thereby indicating an impact on genomic stability. In the subsequent analysis of epigenetic markers, within 72 h, neither AsHC 332 nor AsHC 360 showed an impact on the global DNA methylation level, whereas incubation with AsHC 360 increased the global DNA hydroxymethylation level. Analysis of cell extracts and cell media by HPLC-mass spectrometry revealed that both AsHCs were considerably biotransformed. The identified metabolites include not only the respective thioxo-analogs of the two AsHCs, but also several arsenic-containing fatty acids and fatty alcohols, contributing to our knowledge of biotransformation mechanisms of arsenolipids.

[Do media reports and public brochures facilitate informed decision making about cervical cancer prevention?]. / Ermöglichen Medienberichte und Broschüren informierte Entscheidungen zur Gebärmutterhalskrebsprävention?

With the introduction and recommendation of the new HPV (human papillomavirus) vaccination in 2007, cervical cancer prevention has evoked large public interest. Is the public able to make informed decisions on the basis of media reports and brochures? To answer this question, an analysis of media coverage of HPV vaccination (Gardasil®) and Pap (Papanicolaou) screening was conducted from 2007-2009, which investigated the minimum requirement of completeness (pros and cons), transparency (absolute numbers), and correctness (references concerning outcome, uncertainty, magnitude) of the information. As a bench mark, facts boxes with concise data on epidemiology, etiology, benefits, harms, and costs were compiled in advance. Although all vaccination reports and brochures covered the impact of prevention, only 41% provided concrete numbers on effectiveness (90/220) and 2% on absolute risk reductions for the cancer surrogate dysplasia (5/220), whereby none of the latter numbers was correct. The prevention potential was correctly presented once. Only 48% (105/220) mentioned pros and cons. With regard to screening, 20% (4/20) provided explicit data on test quality and one expressed these in absolute numbers, while 25% (5/20) reported the prevention potential; all given numbers were correct. Finally, 25% (5/20) mentioned the possibility of false positive results. Minimum requirements were fulfilled by 1/220 vaccination and 1/20 screening reports. At present, informed decision making based on media coverage is hardly possible.

Membrane-bound CD95 ligand expressed on human antigen-presenting cells prevents alloantigen-specific T cell response without impairment of viral and third-party T cell immunity.

Genetically modified antigen-presenting cells (APC) represent an attractive strategy for in vitro immunomodulation. In the human system, APC expressing HLA-A1 and a membrane-bound form of CD95L (m-CD95L) were used for selective depletion of HLA-A1-specific T cells. In short-term assays, m-CD95L-expressing APC-induced apoptosis in activated T cells and the constitutive presence of m-CD95L and HLA-A1 expressing APC in long-term T cell cultures prevented the expansion of CD4(+) and CD8(+) HLA-A1-specific T cells and the development of HLA-A1-specific cytotoxicity. However, immunity towards third party, viral and bacterial antigens was maintained and T cells spared from depletion could be induced to develop cytotoxicity towards unrelated antigens. Interestingly, inhibition of HLA-A1-specific T cell response absolutely requires the coexpression of m-CD95L and HLA-A1 antigen on the same APC. Thus, m-CD95L expressing APC might be used in clinical settings to obtain tolerance induction in allogeneic transplantation systems or autoimmune diseases.

Arsenic-containing hydrocarbons disrupt a model in vitro blood-cerebrospinal fluid barrier.

Lipid-soluble arsenicals, so-called arsenolipids, have gained a lot of attention in the last few years because of their presence in many seafoods and reports showing substantial cytotoxicity emanating from arsenic-containing hydrocarbons (AsHCs), a prominent subgroup of the arsenolipids. More recent in vivo and in vitro studies indicate that some arsenolipids might have adverse effects on brain health. In the present study, we focused on the effects of selected arsenolipids and three representative metabolites on the blood-cerebrospinal fluid barrier (B-CSF-B), a brain-regulating interface. For this purpose, we incubated an in vitro model of the B-CSF-B composed of porcine choroid plexus epithelial cells (PCPECs) with three AsHCs, two arsenic-containing fatty acids (AsFAs) and three representative arsenolipid metabolites (dimethylarsinic acid, thio/oxo-dimethylpropanoic acid) to examine their cytotoxic potential and impact on barrier integrity. The toxic arsenic species arsenite was also tested in this way and served as a reference substance. While AsFAs and the metabolites showed no cytotoxic effects in the conducted assays, AsHCs showed a strong cytotoxicity, being up to 1.5-fold more cytotoxic than arsenite. Analysis of the in vitro B-CSF-B integrity showed a concentration-dependent disruption of the barrier within 72 h. The correlation with the decreased plasma membrane surface area (measured as capacitance) indicates cytotoxic effects. These findings suggest exposure to elevated levels of certain arsenolipids may have detrimental consequences for the central nervous system.

Prospective randomized comparison of mezlocillin therapy alone with combined ampicillin and gentamicin therapy for patients with cholangitis.

Forty-six patients with cholangitis were randomized to receive therapy with mezlocillin sodium (24 patients) or a combination of ampicillin sodium--gentamicin sulfate (22 patients). The biliary concentration of mezlocillin was 112 times higher than that of ampicillin and 778 times higher than that of gentamicin. The ratio of the concentration in serum or bile over the minimum inhibitory concentration against aerobic gram-negative bacilli (therapeutic index) was higher for mezlocillin than for either ampicillin or gentamicin. Twenty (83%) of 24 patients were cured following mezlocillin therapy compared with 9 (41%) of 22 patients after ampicillin-gentamicin therapy. The 3 patients with superinfection were in the ampicillin-gentamicin arm of the study. Fewer toxic or adverse effects occurred in association with mezlocillin treatment than with ampicillin-gentamicin treatment. Mezlocillin therapy was more effective, less toxic, and less expensive than treatment with ampicillin and gentamicin for patients with cholangitis.

In vitro toxicological characterisation of arsenic-containing fatty acids and three of their metabolites.

Arsenic-containing fatty acids are a group of fat-soluble arsenic species (arsenolipids) which are present in marine fish and other seafood. Recently, it has been shown that arsenic-containing hydrocarbons, another group of arsenolipids, exert toxicity in similar concentrations comparable to arsenite although the toxic modes of action differ. Hence, a risk assessment of arsenolipids is urgently needed. In this study the cellular toxicity of a saturated (AsFA 362) and an unsaturated (AsFA 388) arsenic-containing fatty acid and three of their proposed metabolites (DMAV, DMAPr and thio-DMAPr) were investigated in human liver cells (HepG2). Even though both arsenic-containing fatty acids were less toxic as compared to arsenic-containing hydrocarbons and arsenite, significant effects were observable at µM concentrations. DMAV causes effects in a similar concentration range and it could be seen that it is metabolised to its highly toxic thio analogue thio-DMAV in HepG2 cells. Nevertheless, DMAPr and thio-DMAPr did not exert any cytotoxicity. In summary, our data indicate that risks to human health related to the presence of arsenic-containing fatty acids in marine food cannot be excluded. This stresses the need for a full in vitro and in vivo toxicological characterisation of these arsenolipids.

Renal toxicity during therapy with gentamicin or tobramycin.

Twenty-seven patients who had normal pretherapy renal clearance by the [125I]iothalamate test were randomly assigned either gentamicin or tobramycin for therapy of infections due to susceptible bacteria. No patients were critically ill or had evidence of bacteremia. Mean age and duration of therapy were 51 years and 14 days, respectively, for 15 patients treated with gentamicin, and 45 years and 13 days for 12 patients treated with tobramycin. At the completion of therapy, six (40%) gentamicin and seven (58%) tobramycin patients had a decrease in renal clearance of at least 14% below baseline. The mean decrease was 26% in the gentamicin group and 23% in the tobramycin group. Simultaneous increases in serum creatinine concentrations (greater than or equal to 0.2 mg/dl) occurred in only 4 (31%) of the 13 patients. Of four patients who had renal clearance studies repeated 3 weeks to 6 months after therapy, two had stable function, but at 16 to 19% below baseline. Mean urinary concentration of N-acetyl glucosaminidase and alanine aminopeptidase increased faster and to higher levels with gentamicin than with tobramycin. However, on an individual patient basis, they were not predictive of a decrease in renal clearance in either therapy group.

Ciprofloxacin versus trimethoprim-sulfamethoxazole: treatment of community-acquired urinary tract infections in a prospective, controlled, double-blind comparison.

In this study, we determined the safety and efficacy of the treatment of adults with urinary tract infection with ciprofloxacin hydrochloride (250 mg twice daily for 10 days) in comparison with trimethoprim-sulfamethoxazole (160 mg of trimethoprim and 800 mg of sulfamethoxazole twice daily for 10 days). Patients with signs and symptoms of urinary tract infection were randomized to receive ciprofloxacin (98 women and 5 men) or trimethoprim-sulfamethoxazole (92 women and 8 men). The success rate of therapy was 91% for both treatment arms of the study. Among seven failures after ciprofloxacin therapy, three were due to relapse of infection and two to side effects that necessitated a change in medication; in addition, two patients had persistent symptoms and required hospitalization. Among the six failures associated with trimethoprim-sulfamethoxazole therapy, four were due to relapse, one to persistence of infection, and one to a side effect that necessitated a change in medication. Among the patients treated with trimethoprim-sulfamethoxazole, 32% had mild or moderate adverse reactions; in comparison, 17% of the ciprofloxacin-treated patients had adverse reactions (P = 0.026). For the treatment of urinary tract infection in adult patients in this study, ciprofloxacin and trimethoprim-sulfamethoxazole were equally effective, but ciprofloxacin was associated with fewer adverse reactions.

Empiric therapy with moxalactam alone in patients with bacteremia.

Moxalactam was administered (20 mg/kg intravenously every 8 hours) as single-drug empiric antimicrobial therapy to 63 patients with bacteremia who were neither neutropenic nor immunosuppressed. Six patients (10%) had microorganisms that were susceptible to moxalactam and resistant to all other antimicrobial agents tested; two patients (3%) had microorganisms that were resistant to moxalactam and other agents tested. Of these 63 patients, 47 (75%) were cured with moxalactam therapy. Nine patients (14%) had breakthrough bacteremia while receiving other antimicrobial therapy and were cured subsequently with moxalactam therapy alone. The two major risk factors for failure of moxalactam therapy were polymicrobial bacteremia and an extrahepatic intra-abdominal source of infection; these two conditions frequently coexisted. Six of nine patients with polymicrobial bacteremia died. Superinfection (one pseudomonal, five enterococcal) was responsible for 6 of the 16 treatment failures. Enterococcal superinfection occurred exclusively among patients who had received relatively prolonged therapy with moxalactam for extrahepatic intra-abdominal infection, especially intraabdominal abscess. These five patients died, and postmortem examination showed that enterococcal superinfection was the major cause of death in all. Mild, reversible adverse reactions associated with use of moxalactam occurred in 14 of the 63 patients (22%). None had clinically overt bleeding. The use of moxalactam alone seems to be safe and effective and a cost-effective alternative empiric antimicrobial therapy for most patients with bacteremia who are not immunosuppressed or neutropenic and who are not at high risk of having Pseudomonas or polymicrobial bacteremia.

Reticular dysgenesis: HLA non-identical bone marrow transplants in a series of 10 patients.

Reticular dysgenesis is a very rare congenital immunodeficiency classified within the severe combined immunodeficiencies (SCID) and characterized by impairment of both lymphoid and myeloid cell development. We report our experience in 10 patients with RD, treated between 1979 and 1999 with HLA-haploidentical hematopoietic stem cell transplantation (HSCT). All children but one were symptomatic within the first days of their lives. Five patients required two HSCT. Five patients received conditioning therapy with busulfan (16 mg/kg) and cyclophosphamide. Three of them are alive and well with myeloid and T and B cell lymphoid reconstitution, whereas two patients died (one chronic graft-versus-host disease, one pneumonitis). Transplantation without or with other conditioning regimens in the other five cases led to absent or incomplete engraftment and none of these cases survived. These results demonstrate the mandatory need for intensive conditioning before haploidentical HSCT in RD to achieve full lymphoid and myeloid engraftment.

In vitro toxicological characterisation of three arsenic-containing hydrocarbons.

Arsenic-containing hydrocarbons are one group of fat-soluble organic arsenic compounds (arsenolipids) found in marine fish and other seafood. A risk assessment of arsenolipids is urgently needed, but has not been possible because of the total lack of toxicological data. In this study the cellular toxicity of three arsenic-containing hydrocarbons was investigated in cultured human bladder (UROtsa) and liver (HepG2) cells. Cytotoxicity of the arsenic-containing hydrocarbons was comparable to that of arsenite, which was applied as the toxic reference arsenical. A large cellular accumulation of arsenic, as measured by ICP-MS/MS, was observed after incubation of both cell lines with the arsenolipids. Moreover, the toxic mode of action shown by the three arsenic-containing hydrocarbons seemed to differ from that observed for arsenite. Evidence suggests that the high cytotoxic potential of the lipophilic arsenicals results from a decrease in the cellular energy level. This first in vitro based risk assessment cannot exclude a risk to human health related to the presence of arsenolipids in seafood, and indicates the urgent need for further toxicity studies in experimental animals to fully assess this possible risk.

The impact of domain-specific beliefs on decisions and causal judgments.

Extensive evidence suggests that people often rely on their causal beliefs in their decisions and causal judgments. To date, however, there is a dearth of research comparing the impact of causal beliefs in different domains. We conducted two experiments to map the influence of domain-specific causal beliefs on the evaluation of empirical evidence when making decisions and subsequent causal judgments. Participants made 120 decisions in a two-alternative forced-choice task, framed in either a medical or a financial domain. Before each decision, participants could actively search for information about the outcome ("occurrence of a disease" or "decrease in a company's share price") on the basis of four cues. To analyze the strength of causal beliefs, we set two cues to have a generative relation to the outcome and two to have a preventive relation to the outcome. To examine the influence of empirical evidence, we manipulated the predictive power (i.e., cue validities) of the cues. Both experiments included a validity switch, where the four selectable cues switched from high to low validity or vice versa. Participants had to make a causal judgment about each cue before and after the validity switch. In the medical domain, participants stuck to the causal information in causal judgments, even when evidence was contradictory, while decisions showed an effect of both empirical and causal information. In contrast, in the financial domain, participants mainly adapted their decisions and judgments to the cue validities. We conclude that the strength of causal beliefs (1) is shaped by the domain, and (2) has a differential influence on the degree to which empirical evidence is taken into account in causal judgments and decision making.

Regulation of urokinase receptor transcription by Ras- and Rho-family GTPases.

How cell adhesion is coordinated with extracellular proteolysis is a key question in understanding cell migration. Potentially, the small GTP-binding proteins that affect actin organisation and signal transduction may also regulate the expression of genes associated with extracellular proteolysis. We investigated the ability of Ras, Rac-1, Cdc42Hs, and RhoA to regulate transcription from the1.55-kb promoter region of the human urokinase plasminogen activator receptor (uPAR) gene. Constitutively active V12 H-Ras and Rho-A stimulated uPAR transcription while Cdc42Hs and Rac-1 did not. The use of Ras effector-loop mutants indicated that signalling via multiple Ras-effectors is necessary for the maximum activation of transcription.

Expression of retinoid-X receptors (-alpha,-beta,-gamma) and retinoic acid receptors (-alpha,-beta,-gamma) in normal human skin: an immunohistological evaluation.

Increasing evidence suggests that the retinoid-X receptors (RXR-alpha,-beta,-gamma) play a crucial role in regulating the transcriptional activity of several steroid hormone receptors, including the receptors for retinoic acid (RAR-alpha,-beta,-gamma), 1,25-dihydroxyvitamin D3 and thyroid hormone. We investigated the localization of the different types of RXR-alpha,-beta,-gamma and RAR-alpha,-beta,-gamma proteins in frozen sections of normal human skin (n = 12) in situ, applying recently raised corresponding specific monoclonal antibodies and an immunohistochemical technique that we established for the detection of these nuclear receptors. Our findings indicate that RXR-alpha,-beta,-gamma and RAR-alpha,-beta,-gamma proteins can be detected by immunohistochemistry in normal human skin. In contrast to RXR-alpha,-beta,-gamma as well as RAR-alpha and RAR-gamma proteins that were consistently detected in cell layers of the viable epidermis, RAR-beta was only focally demonstrated in single epidermal cells in three out of 12 biopsies analysed. Immunohistochemical labelling of RAR-alpha,-beta,-gamma and RXR-alpha,-beta,-gamma proteins in epidermal nuclei was also pronounced in the stratum granulosum, suggesting a function of RXR and RAR proteins in the transition from proliferation to differatiation in epidermal keratinocytes. Expression of RXRs and RARs in hair follicles, sebaceous glands and endothelial cell points to a biological function from these nuclear receptors to hair growth as well as to the physiology of sebaceous glands and endothelial cells.

[The relationship between fovea capitis femoris and weight bearing area in the normal and dysplastic hip in adults: a radiologic study]. / Die radiologische Beziehung der Fovea capitis femoris zur azetabulären Belastungszone bei der normalen und dysplastischen Hüfte des Erwachsenen.

AIM: In residual hip dysplasia the fovea capitis femoris lays often more cranial than in the normal hip morphology, the ligamentum capitis femoris thereby articulating with the weight-bearing area of the acetabular cartilage. The aim of this study was to quantitate this aspect with regard to its potential negative effect for the degeneration of the dysplastic acetabulum. METHOD: The relation between the fovea capitis femoris and the weight-bearing area were studied using the a.p.-pelvis view in normal and dysplastic hips. The measurements were made by digital image analysis. RESULTS: The hypothesis that the "fovea alta" is characteristic for the dysplastic hip was confirmed. In dysplastic hips the typically wider fovea lays on average 30 degrees more cranial, touching the weight-bearing area over 11 degrees. The fovea in normal hips has on average a distance of 26 degrees to the acetabular roof. In dysplastic hips the steeper roof, the tendency for decentering of the femoral head as well as a higher CCD angle explain this phenomenon to a certain degree. In a theoretical model to correct the acetabular position over the femoral head alone one quarter of the foveae would still touch the weight-bearing area. Nevertheless, in our series after periacetabular osteotomy this was never the case due to better centering. CONCLUSION: lt is our hypothesis that a "fovea alta", which further reduces the already smaller loaded cartilage surface, is one part leading to the early degeneration of the dysplastic hip. Therefore, it should be considered in biomechanical models and in the planning of corrective procedures.

Similar pattern of thymic-dependent T-cell reconstitution in infants with severe combined immunodeficiency after human leukocyte antigen (HLA)-identical and HLA-nonidentical stem cell transplantation.

Donor T cells after stem cell transplantation reconstitute by 2 different pathways: by expansion from grafted, mature T cells and by intrathymic maturation from progenitor cells. This study characterized thymic-dependent reconstitution of CD4(+) T cells following different transplant modalities in patients with severe combined immunodeficiency (SCID). Three groups of patients were studied: one group after transplantation from human leukocyte antigen (HLA)-identical siblings with unmanipulated grafts without conditioning, a second group after transplantation from HLA-nonidentical parents with T-cell-depleted grafts without preconditioning, and a third group with prior conditioning. Reconstitution of the T-cell compartment was monitored by determining the expression of CD45 isoforms by developing CD4(+) cells in the peripheral blood and in discriminating expanded (CD45RO(+)) and newly generated (CD45RA(+)) T cells. Concomitantly, changes in the size of the thymus were evaluated sequentially by ultrasonography. Reconstitution of CD4(+)CD45RA(+) cells was delayed in all patients for several months, including patients after HLA-identical transplantation, and was always paralleled by normalization of the size of the thymus. No engraftment of donor progenitor cells was observed, as studied in one patient transplanted without conditioning. CD4(+)CD45RO(+) cells were detected early after transplantation only in patients given unmanipulated grafts. The study showed that thymic-dependent T-cell maturation in these patients with SCID runs an autonomous course, independent of graft manipulation, of major HLA disparities, and of whether conditioning is used or not. In addition, thymic maturation may not require engraftment of donor-derived CD34(+) cells in the marrow. (Blood. 2000;96:4344-4349)