[Nutrition, lifestyle, physical activity, and supportive care during chemotherapeutic treatment]. / Ernährung, Lebensweise, Aktivitäten und supportive Massnahmen unter chemotherapeutischer Behandlung.

With improvements in cancer survival rates, more patients with cancer are living longer and the influence of nutrition, lifestyle, physical activity as well as supportive care during and after chemotherapy is of increasing interest. In several malignancies smoking cessation increases cancer survival. Similar effects are expected by healthy nutrition. Regular physical activity of cancer patients reduces drug interactions of chemotherapy, decreases the number of comorbid conditions, and helps patients maintain independence as long as possible. For supportive care during chemotherapy the 5-HT3 receptor antagonists are more effective for the prevention of chemotherapy-induced nausea and vomiting. There are several colony-stimulating factors (e.g. GCSF, erythropoietin) for hematopoietic recovery post-chemotherapy. Altogether supportive care of chemotherapy reduces toxicity and increases efficacy.

Differential transfection efficiency rates of the GM-CSF gene into human renal cell carcinoma lines by lipofection.

One of the major questions in any gene therapy approach is the selection of the appropriate vector system. Here, the optimization of a gene transfer protocol for renal cell carcinoma using lipofection as a nonviral gene transduction system was evaluated. To select the promoter which gives the highest expression, different plasmids which are able to express Escherichia coli beta-galactosidase gene as a reporter gene under the control of different promoters were tested: human cytomegalovirus promoter (pCMVbeta), simian virus 40 promoter (pSVbeta), adenovirus promoter (ADbeta), and herpes simplex virus thymidine kinase promoter (TKbeta). The pCMVbeta revealed the highest expression of the beta-gal gene in the renal cell carcinoma (RCC) lines. Thus this CMV promoter was selected for the expression of the granulocyte-macrophage colony stimulator factor (GM-CSF) gene. Three different lipids (LipofectAmine, LipofectAce, and Lipofectin) were compared for their transduction efficiency, and the optimal conditions for quantitatively high lipofection rates were established. The consistently best results regarding gene expression as well as viability of the RCC lines were obtained when Lipofectin was used. Gene expression was monitored by a specific enzyme-linked immunosorbent assay and functionally validated by a cell proliferation test. The GM-CSF expression profile showed a peak at 48 hours after transfection and was still detectable after 5 days. Here the feasibility of efficient lipofection of the GM-CSF gene into RCC lines is demonstrated. Most importantly, considerable differences in the relative quantity of GM-CSF gene transfer into the different RCC lines was observed here. This may be of critical relevance for the design of any clinical gene transduction protocol in tumor cell vaccination attempts.

Screening for renal carcinoma associated mutations in the von Hippel-Lindau tumor suppressor gene by temperature gradient gel electrophoresis.

Renal cell carcinoma is the most common neoplastic disease of the adult kidney and occurs in its sporadic and hereditary form. Approximately 57% of all renal carcinomas of the clear cell type analyzed revealed a mutation in the von Hippel-Lindau disease (VHL) gene. In the present work, temperature gradient gel electrophoresis (TGGE) is presented as a rapid and precise polymerase chain reaction (PCR)-employing methodology for the detection of mutations in the VHL gene. The theoretical efficiency of TGGE to detect mutations in every base pair of the gene was calculated. According to computer analysis, at least 92% of all known mutations in the VHL gene are detectable. This calculated figure appears to be in agreement with the experimental results. Primary difficulties in analyzing exon 1 of the VHL gene were overcome by the employment of psoralen-cross-linked PCR fragments. In addition, TGGE analysis was used in screening for possible mutations in thirteen renal carcinoma samples. With this protocol TGGE is successfully added to the array of methods for the screening of VHL mutations.

A new immunoassay for soluble fibrin enables a more sensitive detection of the activation state of blood coagulation in vivo.

A novel sandwich immunoassay for measurement of soluble fibrin in plasma has been developed. For immunization we used the synthetic heptapeptide Gly-Pro-Arg-Val-Val-Glu-Arg representing the amino terminus of the alpha-chain of human fibrin. A monoclonal IgG1 antibody was obtained by conventional hybridoma technology. To increase convenience, the sandwich immunoassay was developed for the Enzymun-Test systems which are based on streptavidin pre-coated tubes. The new assay was designed with the same fibrin specific antibody both in biotinylated and in peroxidase-labelled form. Fibrin in native plasma samples could only be detected after pre-incubation of the plasma with chaotropic ions. Test results were calculated using a standard curve comprising six fibrin standards (0-50 micrograms/ml). Precision of the method was satisfactory; intra-assay CVs using plasma samples ranged between 5.0% (23.7 micrograms/ml) and 12.4% (0.2 microgram/ml). CVs of interassay precision measurements using standards as samples range between 7.3% (25.0 micrograms/ml) and 11.4% (1.0 micrograms/ml). The lower detection limit was 0.12 microgram/ml. Investigations of normal range in 70 age-matched healthy individuals resulted in a mean of 1.12 micrograms/ml. Linearity was excellent; recovery of high fibrin plasma after dilution with normal plasma was always between 100 and 107%. Fibrin specificity was due to the monoclonal antibody 2B5 used and no cross reactivity with fibrinogen, fibrinogen split products or fibrin D-dimer was observed. Fibrin fragment E1 (studied by Dempfle CE, et al. Blood Coag Fibrinol 1993; 4: 79-86) and fragments X and Y showed moderate cross reactivity in the assay and caused some overestimation of fibrin at high fibrin split product concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of 15 monoclonal antibodies against tumor-associated antigens of transitional cell carcinoma of the human bladder.

Quantitative urinary immunocytology with our monoclonal antibody (mab) 486p 3/12 proved to be valuable for diagnostic use in bladder-cancer patients' urine, especially in the followup of patients with superficial bladder carcinoma. To evaluate the use of other monoclonal antibodies in bladder cancer, we compared 15 mabs directed against bladder-tumor-associated antigens from seven research groups in a broad panel of cellular and tissue specimens (bladder tumor, prostatic adenoma, and kidney stone). Quantitative evaluation was done in cytocentrifuged preparations and tissue specimens. None of the 15 mabs was bladder-tumor-specific. All 15 stained normal urothelium to some extent and six stained granulocytes. Each of the 15 seemed to identify a different cellular antigen, as can be clearly demonstrated by the staining pattern of different regions in the normal kidney. The sensitivity of quantitative urinary immunocytology in bladder-tumor patients can be improved by using a panel, rather than one mab in bladder-tumor patients, but specificity decreases simultaneously. A main reason for the poor specificity of quantitative urinary immunocytology with all 15 mabs is that false-positive results are obtained with all mabs in kidney-stone patients. Our quantitative urinary immunocytology method is a general tool for the diagnostic use of all mabs in bladder-tumor patients. Mabs that have a high sensitivity might be useful in the followup of patients with superficial bladder carcinoma. None of the 15 mabs (because of their poor specificity) seems to be helpful in quantitative urinary immunocytology for screening a population for bladder carcinoma.

Primary structure of a precursor to the aspartic proteinase from Rhizomucor miehei shows that the enzyme is synthesized as a zymogen.

In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined. The deduced amino acid sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acid residues with a total molecular weight of 38,701. Clusters of identities around the active site cleft support the assumption that these proteinases have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified.

Precipitation methods for the determination of LDL-cholesterol.

The selective precipitation of low-density lipoproteins (LDL) with polyvinyl sulfate (PVS), and the immunoprecipitation of high-density lipoproteins (HDL) and very-low-density lipoproteins (VLDL) with an anti-HDL antibody, can both be used to establish simple methods for the determination of LDL cholesterol. Whereas the PVS method requires the calculation of LDL cholesterol as the difference of total and supernatant cholesterol, the immunoprecipitation method allows the direct measurement of LDL cholesterol in the supernatant. As a first step, both methods were optimized to yield accurate values for normolipemic and slightly hyperlipemic serum samples. Moreover, the determination of LDL-cholesterol in lipemic sera can be achieved by a combination of immunoprecipitation and polyanion precipitation.