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Biomedical subjects

M Wilhelm

Publications and source records attributed to M Wilhelm.

At least 37 records · Page 2Linked to original sources

[Induction of apoptosis by preoperative passive immunotherapy in resectable stomach carcinoma].

The antibody SC-1 is a human IGM molecule, which binds to a tumor specific receptor. This SC-1 receptor is detectable on biopsies, it is present in about 50% of gastric cancers. After binding of the antibody to the receptor the tumor cells go into apoptosis. 50 patients expressing the SC-1 receptor on their tumors have been treated with SC-1 prior to gastrectomy. In 80% of cases apoptosis induction could be demonstrated in the tumors. The only side effect of the SC-1 therapy was a reversible episode of fever during antibody infusion in 8% of our patients.

Antibodies, Monoclonal↗

Polypurine tract formation by Ty1 RNase H.

To better understand the mechanism by which Ty1 RNase H creates the polypurine tract (PPT) primer, we have demonstrated the polymerase-dependent hydrolytic activity of Ty1 reverse transcriptase (RT) during minus-strand synthesis. Using RNase H and polymerase mutants of the recombinant Ty1 RT protein, we show that the two domains of Ty1 RT can act independently of one another. Our results indicate that RNA/DNA substrates containing a short RNA PPT, which serve as primers for plus-strand DNA synthesis, are relatively resistant to RNase H cleavage. RNA substrates with a correct 5' end but with 3' end extending beyond the plus-strand initiation site were cleaved specifically to generate the correct 3' end of the PPT. Using long RNA/DNA duplexes containing the PPT, we show that Ty1 RT is able to make specific internal cleavages that could generate the plus-strand primer with correct 5' and 3' ends. Long RNA/DNA duplexes with mutations in the PPT or in a U-rich region upstream of the PPT, which abolish plus-strand initiation in vivo, were not cleaved specifically at the 5' end of the PPT. Our work demonstrates that the in vitro enzyme can recapitulate key processes that control proper replication in vivo.

Base Sequence↗

Human wig-1, a p53 target gene that encodes a growth inhibitory zinc finger protein.

We previously identified a novel p53-induced mouse gene, wig-1, that encodes a 290 amino acid zinc finger protein (Varmeh-Ziaie et al., 1997). Here we have identified and characterized the human homolog of mouse wig-1. The human wig-1 protein is 87% identical to the mouse protein and contains three zinc finger domains and a putative nuclear localization signal. Human wig-1 mRNA and protein is induced following activation of wild type p53 expression in our BL41-ts p53 Burkitt lymphoma cells. Wig-1 is also induced in MCF7 cells following treatment with the DNA-damaging agent mitomycin C. Northern blotting detected low levels of wig-1 mRNA in normal human tissues. Fluorescence in situ hybridization mapped wig-1 to human chromosome 3q26.3-27. FLAG-tagged human wig-1 localizes to the nucleus. Ectopic overexpression of human wig-1 inhibits tumor cell growth in a colony formation assay. These results suggest that human wig-1 has a role in the p53-dependent growth regulatory pathway.

Amino Acid Sequence↗

Rituximab therapy of patients with B-cell chronic lymphocytic leukemia.

Rituximab (IDEC-C2B8) is a chimeric antibody that binds to the B-cell surface antigen CD20. Rituximab has significant activity in follicular non-Hodgkin lymphomas. Much less is known about the effects in chronic lymphocytic leukemia (CLL). We have initiated a phase II trial to evaluate the efficacy and safety of rituximab in patients with CD20+ pretreated CLL. To avoid the rituximab-associated toxicity, we restricted the tumor cell load, as measured by the number of circulating lymphocytes and the spleen size, in the first 2 cohorts of patients included in the study. Patients received 4 intravenous infusions of 375 mg/m2 once a week over a period of 1 month. Of the 28 patients evaluable for response, 7 patients showed a partial remission (National Cancer Institute criteria) lasting for a median of 20 weeks, with 1 patient still in remission after 71 weeks. Based on lymphocyte counts only, we found at least a 50% reduction of lymphocyte counts lasting for at least 4 weeks in 13 (45%) of 29 patients. Fifteen patients from 3 institutions were monitored for the immunophenotype profile of lymphocyte subsets. The number of CD5+CD20+ cells decreased significantly and remained low until day 28 after therapy. T-cell counts were not affected. With the exception of one rituximab-related death, adverse events in the remaining patients were mild. The results suggest that rituximab has clinical activity in pretreated patients with B-CLL. Toxicity is tolerable. Response duration after withdrawal of rituximab is rather short. Therefore, other modes of application and the combination with other agents need to be tested.

Adolescent↗

DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1.

The fidelity of the yeast retrotransposon Ty1 reverse transcriptase (RT) was determined by an assay based on gel electrophoresis. Steady-state kinetics analyses of deoxyribonucleotide (dNTP) incorporation at a defined primer-template site indicate that Ty1 RT misincorporates dNTP at a frequency of 0.45 x 10(-5) for the A(t):A mispair in which dATP is misincorporated opposite a template A to 6.27 x 10(-5) for the C(t):A mispair. The G(t):G and T(t):T mispairs are formed with very low efficiency. The fidelity parameters of Ty1 RT do not depend on whether RNA or DNA are copied. Relative to lentiviral RTs (HIV-1, HIV-2 or EIAV) Ty1 RT is approximately 10-fold less error prone. Our data also show that the Ty1 RT is able to recapitulate two error-generating mechanisms: extension of mismatches and non-templated addition of nucleotides at the end of a blunt-end primer-template.

DNA, Fungal↗

Quality control of inactivated erysipelas vaccines: results of an international collaborative study to establish a new regulatory test.

According to the specifications of the European Pharmacopoeia (Ph. Eur.) monograph (Swine Erysipelas Vaccine (Inactivated), Monograph no. 64, European Pharmacopoeia, 3rd edn., 1997) on erysipelas vaccines for veterinary use, batch potency is estimated in a multi-dilution assay after immunisation and infection of mice. Recently, we described a serological assay system (ELISA) which has the potential to replace this challenge-based model (Beckmann R, Cussler K. Wirksamkeitsprüfung von Rotlaufimpfstoffen an der Labormaus. ELISA kontra Infektionsversuch. ALTEX 1994;Suppl. 1:39-45; Rosskopf-Streicher U, Johannes S, Hausleithner D, Gyra H, Cussler K. Suitability of an ELISA for the batch potency test in laboratory mice. Pharmeuropa BIO 1998;1:65-70). The humoral immune response is quantified in pooled sera of ten mice three weeks after immunisation. The results are expressed as relative potency (RP) in comparison to a reference serum. After a pre-validation study had been performed with success (Rosskopf-Streicher U, Johannes S, Wilhelm M, Gyra H, Cussler K. Potency testing of swine erysipelas vaccines by serology - results of a pre-validation study. ALTEX 1999;16:123-8), we initiated an international collaborative study with five European manufacturers and seven regulatory authorities to validate the assay and model. All participants were provided with blind-coded erysipelas vaccines of different potencies, the ELISA kit and test instructions. The participants had to immunise mice, to prepare serum samples and to perform the ELISA. Inter-laboratory reproducibility was reported by the pass/fail criteria of the vaccines under test. Intra-laboratory precision was assessed by comparing repeated measurements on three consecutive days. Day-to-day variation within the laboratories was statistically analysed by comparing pairs of RPs using Lin's concordance correlation coefficient. The results show that the ELISA is indeed a suitable alternative to replace the vaccination-challenge test. Furthermore, this new model reduces the number of animals required for the potency test by approximately 80%.

Analysis of Variance↗

Idiopathic CD4+ T cell lymphocytopenia evolving to monoclonal immunoglobulins and progressive renal damage responsive to IL-2 therapy.

Idiopathic CD4+ T cell lymphocytopenia was unexpectedly detected in a 33-year-old, otherwise healthy young woman with no HIV or other viral infection, autoimmune, or neoplastic disease or increased susceptibility to infection. CD4+ T cell levels were 60-140/microl over a 3.5-year period. Following an uneventful pregnancy, the patient developed anemia and interstitial nephritis associated with a plasma cell dyscrasia with a monoclonal IgA gammopathy and a shifting immunoglobulin pattern that included IgG and IgA monoclonal proteins and increased urinary light chains. Osteolytic lesions were never detected and bone marrow aspirations revealed up to 10% atypical plasma cells. Various therapies often used in treating multiple myeloma only temporarily controlled the increasing renal damage. IL-2 therapy of 600,000 to 1 million units subcutaneously daily resulted in increased CD4+ T cells to normal levels, a decrease in the gammopathy, a return of renal function, energy, and weight gain, and apparently normal health status sustained for 2 years. The findings are compatible with a potentially fatal but nonmalignant immunoregulatory disorder that can be controlled by IL-2 administration.

Adult↗

Reverse transcription of retroviruses and LTR retrotransposons.

Retroelements are mobile genetic entities that replicate via reverse transcription of a template RNA. A key component to the life cycle of these elements is the enzyme reverse transcriptase (RT), which copies the single-stranded genomic RNA of the element into a linear double-stranded DNA that is ultimately integrated into the host genome by the element-encoded integrase. RT is a multifunctionnal enzyme which possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity that specifically degrades the RNA strand of RNA-DNA duplexes. At some stages of the replication a strand-displacement activity of RT is also necessary. All activities are essential for the conversion of single-stranded genomic RNA into the double-stranded preintegrative DNA. This review focuses on the role of RT in the different steps of the replication process of retroelements. The features of retrotransposon replication which differ from the retroviral ones will be emphasized. In a second part of the review, the biochemical and enzymatic properties of two newly characterized retrotransposon RTs will be described. The role of the integrase domain in reverse transcriptase activity of some retroviral and retrotransposon RTs will be discussed.

Amino Acid Sequence↗

Structure and function of photoreceptor and second-order cell mosaics in the retina of Xenopus.

The structure, physiology, synaptology, and neurochemistry of photoreceptors and second-order (horizontal and bipolar) cells of Xenopus laevis retina is reviewed. Rods represent 53% of the photoreceptors; the majority (97%) are green light-sensitive. Cones belong to large long-wavelength-sensitive (86%), large short-wavelength-sensitive (10%), and miniature ultraviolet wavelength-sensitive (4%) groups. Photoreceptors release glutamate tonically in darkness, hyperpolarize upon light stimulation and their transmitter release decreases. Photoreceptors form ribbon synapses with second-order cells where postsynaptic elements are organized into triads. Their overall adaptational status is regulated by ambient light conditions and set by the extracellular dopamine concentration. The activity of photoreceptors is under circadian control and is independent of the central body clock. Bipolar cell density is about 6000 cells/mm2 They receive mixed inputs from rods and cones. Some bipolar cell types violate the rule of ON-OFF segregation, giving off terminal branches in both sublayers of the inner plexiform layer. The majority of them contain glutamate, a small fraction is GABA-positive and accumulates serotonin. Luminosity-type horizontal cells are more frequent (approximately 1,000 cells/mm2) than chromaticity cells (approximately 450 cells/mm2). The dendritic field size of the latter type was threefold bigger than that of the former. Luminosity cells contact all photoreceptor types, whereas chromatic cells receive their inputs from the short-wavelength-sensitive cones and rods. Luminosity cells are involved in generating depolarizing responses in chromatic horizontal cells by red light stimulation which form multiple synapses with blue-light-sensitive cones. Calculations indicate that convergence ratios in Xenopus are similar to those in central retinal regions of mammals, predicting comparable spatial resolution.

Animals↗

Aluminum balance in intensive care patients.

Aluminum (Al) is a well known contaminant of intravenous solutions. The aim of the present study was the estimation of the aluminum load in patients of an intensive care unit (ICU). 15 patients with normal renal function took part. The study period was 15 days. Al was measured in serum, 24 h-urine and 132 samples of parenterals. Daily Al doses were recorded. Al balance was calculated on the basis of the iatrogenic Al dose and renal Al excretion. Al analysis was performed by graphite furnace atomic absorption spectrometry (AAS) with Zeeman background correction under careful quality control. Solutions with Al levels >100 microg/l were: calcium salts, additives for parenteral nutrition solutions, antibiotics, acetylcysteine, triflupromazine, catecholamines and colloids. The Al content of solutions for parenteral nutrition ranged from 4.3 to 69 microg/l. Al doses amounted to 46-456 (median 119) microg/d, equivalent to 0.7 to 6.5 (median 1.7) microg/kg b.w. Renal Al excretion ranged from 10.5 to 723.1 microg/d (median 53 microg/d). These amounts partly exceeded the maximal dose (2 microg/kg b.w. per day), recommended by ASPEN/ASCN. Despite of the highly elevated renal Al excretion the median serum concentration of Al was only moderately increased (6.1 microg/l; range: <1.5 to 23.6 microg/l). However, calculations on the basis of the iatrogenic Al dose and renal Al excretion resulted in a net Al uptake (median) of 61 microg/d (maximum: 291 microg/d). Al amounts of this magnitude must be considered potentially harmful in ICU patients, especially with impaired renal function. Parenteral therapy resulted in a considerable Al dose with a positive Al balance in ICU patients. Threshold values for Al contamination of parenterally administered drugs and solutions should be established.

Adolescent↗

Fludarabine plus cyclophosphamide is an efficient treatment for advanced chronic lymphocytic leukaemia (CLL): results of a phase II study of the German CLL Study Group.

The efficacy and toxicity of a combination of fludarabine and cyclophosphamide (FC) was evaluated in patients with B-cell chronic lymphocytic leukaemia (CLL). Between April 1997 and July 1998, 36 patients with CLL (median age 59 years) received a regimen that consisted of fludarabine 30 mg/m(2) in a 30-min IV infusion, d 1-3, and cyclophosphamide 250 mg/m(2) in a 30-min IV infusion on d 1-3. Cycles were repeated every 28 d. Twenty-one patients had received between one and three different treatment regimens prior to the study, while 15 patients had received no prior therapy. The median Eastern Cooperative Oncology Group performance score was 1. One patient was at Binet stage A, 18 were stage B and 17 patients were stage C. Objective responses, assessed according to the revised guidelines of the National Cancer Institute-sponsored Working Group, were recorded in 29 out of 32 assessable patients (90.6%). Twenty-four partial remissions and five complete remissions were observed. Two patients showed no change and one patient showed disease progression. At February 2000, three of the responders had relapsed. Severe neutropenia, anaemia and thrombocytopenia (Common Toxicity Criteria grade 3 and 4) were observed in 25, six and six patients (69.4%, 16.7% and 16.7%) respectively. Other side-effects were uncommon. No treatment-related deaths and no grade 3 or 4 infections occurred. We conclude that the combination of fludarabine and cyclophosphamide showed significant activity in patients with CLL. Myelosuppression was the major side-effect. These results warrant further study on the FC combination in randomized trials.

Adult↗

Noncardiac surgery in patients with left ventricular assist devices.

BACKGROUND: Noncardiac surgery, especially abdominal surgical procedures in patients with long-term mechanical circulatory support and strong anticoagulation, is difficult. METHODS: We report on 14 patients (aged 44 +/- 15 years) with a portable Novacor or HeartMate system, who underwent noncardiac surgical procedures while being supported by the device. RESULTS: The patients underwent 20 procedures for noncardiac reasons; most had an intestinal operation or cholecystectomy. Half of the procedures were performed within 30 days after placement of the device (mean interval, 53 +/- 57 days), only 6 interventions were necessary after 100 days of mechanical support. Complications occurred in 8 patients (57%), 5 of whom had undergone cholecystectomy and had unacceptably high sanguineous drainage losses. CONCLUSIONS: An elective surgical procedure can be performed with an acceptable risk if the operation is carefully managed. Postponing resumption of full anticoagulation is advisable as it may reduce bleeding complications without apparently increasing the risk for thromboembolism. Emergency interventions remain a difficult task.

Adult↗

Comparison of different digestive tract models for estimating bioaccessibility of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) from red slag 'Kieselrot'.

'Kieselrot' (red slag), a highly PCDD/F-contaminated leaching residue from a copper production process, has been used as surface layer for more than 1,000 sports fields, playgrounds and pavements in Germany and neighbouring countries. Children can ingest this material directly by hand-to-mouth activities or soil-pica behaviour. Furthermore secondary contamination of farm land or kitchen gardens by drift of red slag dust may lead to an enrichment of PCDD/F within the food-chain. PCDD/F can be mobilized from contaminated materials by digestive juices and thus become bioaccessible for intestinal absorption. Two different digestive tract models were used to estimate the bioaccessibility of PCDD/F from red slag and to study the influence of food material on the mobilization of the contaminants. The bioaccessibility of PCDD/F from red slag depends on the charge of red slag material used, the bile content of the intestinal juice and on the presence of lipophilic foodstuffs. A low bioaccessibility of less than 5% was found when using a digestive tract model with a low bile content and in absence of food material. The bioaccessibility was estimated to be more than 60% when using a model with a higher bile content and in the presence of whole milk powder. A low bioaccessibility of PCDD/F from red slag in general--as assumed until now and mentioned in legal provision--was not confirmed by our study. Considering observations for the different homologue groups it is obvious that bioaccessibility is the first of several important steps to estimate human health risks arising from contaminated materials. In case red slag contaminated with PCDD/F their absorption rate in the digestive tract and/or metabolism might be at least just like important.

Benzofurans↗

Selective and sensitive assay for the determination of benzodiazepines by high-performance liquid chromatography with simultaneous ultraviolet and reductive electrochemical detection at the hanging mercury drop electrode.

An isocratic chromatographic method for the simultaneous determination of 10 benzodiazepines is presented. The selectivity of the assay was optimized by variation of stationary phase, temperature, as well as ionic strength, composition and pH of the mobile phase and the dependence of the detector response on the applied potential was investigated. The best results with respect to resolution at moderate retention times were obtained with a mixture of 0.02 mol/l phosphate buffer (pH 6) and acetonitrile in a volume ratio of 55:45 (v/v) on a LiChrospher-100 RP-8ec column (150x4.6 mm I.D.). Considerable improvement of selectivity was achieved if the column temperature was kept constant at 12 degrees C. Two detection modes were applied, UV detection at 250 nm, inserted upstream to the electrochemical detector, and reductive electrochemical detection at the hanging mercury drop electrode at -1.4 V (vs. Ag/AgCl), which proved to be especially sensitive in case of nitrophenyl-containing benzodiazepine species. The elaborated assay showed to be linear up to at least 2 mg/l for each compound. Detection limits generally were in the range of 6.5-123 ng/ml (130 pg-2.46 ng on-column, using a 20-microl loop) with relative standard deviations between 1.1 and 8.6% depending on the actual benzodiazepine investigated.

Benzodiazepines↗

Amacrine cells of the anuran retina: morphology, chemical neuroanatomy, and physiology.

Amacrine cells are third-order retinal interneurons, projecting their processes into the inner plexiform layer. Historically, they were not considered as neurons first. By the middle of the 20th century, their neuronal nature was confirmed, and their enormous diversity established. Amacrine cells have been most successfully subdivided into morphological categories based on two parameters: diameter of the dendritic field and ramification pattern in the inner plexiform layer. Works combining anatomy, physiology, and neurochemistry are scarce and in the case of the anuran retina, the situation is even worse. Correlation between morphology, neurochemistry, and physiology is little studied. Here we try to build up a database and pinpoint some of the missing data. Obtaining those could help to better understand retinal function. Sporadic attempts did not make it possible to develop a comprehensive catalog of morphologically distinct amacrine cell types in the anuran retina. The number of morphologically identified amacrine cells currently stands at 16. The list of neurochemically identified distinct cell types can be given as follows: five types GABA-containing cell types with secondary markers and at least one without; two glycinergic cell types and one interplexiform cell where glycine colocalizes with somatostatin; one dopaminergic amacrine cell and also a variant of this with interplexiform morphology; two types of serotoninergic cells; three NADPHdiaphorase-positive cells, one substance P-positive cell type without identified second marker; one CCK-positive cell type without identified second marker and the calbindin positive cells (at least one but potentially more types). This adds up to 19 cell types, out of which two are interplexiform in character. This is more than that could be identified by purely morphological means. Out of Cajal's original 13 amacrine cell types described in the frog retina, 5 parallel unequivocally with neurons defined by neurochemistry. Three others have one close match each, but their exact identity is uncertain. The remaining amacrine cells have more than one potential matches. At the same time, on one hand the amacrine cell named two-layered by Cajal so far has no match among the neurochemically identified amacrine cells. On the other hand, the interplexiform subtype of the dopaminergic cell, the somatostatin-containing glycinergic interplexiform cell, the starburst cell, and the bistratified neuropeptide Y-immunoreactive cell have no match among Cajal's cells. All in all, the number of known amacrine and interplexiform cells now stands at at least 21 in the anuran retina. Physiological characterization of amacrine cells shows that their general features seem to be rather similar to those described in tiger salamander retina. In Xenopus retina, morphologically and physiologically identified amacrine cells responded to light stimulation most frequently with ON-OFF characteristics. Immunhistochemical identification of the recorded and dye injected cells showed that amacrine cells of the "same physiological type" might have different morphology. In other words, amacrine cells with different morphology can respond similarly to illumination. Even so, small differences between almost identical responses may reflect that the cell they stem from indeed belongs to different cell types.

Animals↗

Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma.

Bisphosphonates are well-known inhibitors of osteoclastic bone resorption, but recent clinical reports support the possibility of direct or indirect antitumor effects by these compounds. Because bisphosphonates share structural homologies with recently identified gamma delta T-cell ligands, we examined the stimulatory capacity of bisphosphonates to gamma delta T cells and determined whether gamma delta T-cell stimulation by bisphosphonates could be exploited to generate antiplasma cell activity in multiple myeloma (MM). All tested aminobisphosphonates (alendronate, ibandronate, and pamidronate) induced significant expansion of gamma delta T cells (V gamma 9V delta 2++ subset) in peripheral blood mononuclear cell cultures of healthy donors at clinically relevant concentrations (half-maximal activity, 0.9-4 mcmol/L). The proliferative response of gamma delta T cells to aminobisphosphonates was IL-2 dependent, whereas activation of gamma delta T cells (up-regulation of CD25 and CD69) occurred in the absence of exogenous cytokines. Pamidronate-activated gamma delta T cells produced cytokines (ie, interferon [IFN]-gamma) and exhibited specific cytotoxicity against lymphoma (Daudi) and myeloma cell lines (RPMI 8226, U266). Pamidronate-treated bone marrow (BM) cultures of 24 patients with MM showed significantly reduced plasma cell survival compared with untreated cultures, especially in cultures in which activation of BM-gamma delta T cells was evident (14 of 24 patients with MM). gamma delta T-cell depletion from BM cultures completely abrogated the cytoreductive effect on myeloma cells in 2 of 3 tested patients with MM. These results show that aminobisphosphonates stimulating gamma delta T cells have pronounced effects on the immune system, which might contribute to the antitumor effects of these drugs. (Blood. 2000;96:384-392)

Alendronate↗

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities.

Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.

Amino Acid Sequence↗