Reduce bladder cancer recurrence in patients treated for upper urinary tract urothelial carcinoma: The REBACARE-trial.

BACKGROUND: Following radical nephro-ureterectomy for urothelial carcinoma of the upper urinary tract (UUT), the reported bladder recurrence rate of urothelial carcinoma is 22-47%. A single intravesical instillation of chemotherapy within 10 days following nephro-ureterectomy has the potential to decrease the risk of a bladder recurrence significantly. Despite recommendation by the European Association of Urology guideline to administer a single instillation postoperatively, the compliance rate is low because the risk of extravasation of chemotherapy. AIM: To reduce the risk of bladder cancer recurrence by a single intravesical instillation of Mitomycin immediately (within 3 h) before radical nephro-ureterectomy or partial ureterectomy. METHODS: Adult patients (age ≥ 18 years) with a (suspicion of a) urothelial carcinoma of the UUT undergoing radical nephro-ureterectomy or partial ureterectomy will be eligible and will receive a single intravesical instillation of Mitomycin within 3 h before surgery. In total, 170 patients will be included in this prospective, observational study. Follow-up will be according to current guidelines. RESULTS: The primary endpoint is the bladder cancer recurrence rate up to two years after surgery. Secondary endpoints are: a) the compliance rate; b) oncological outcome; c) possible side-effects; d) the quality of life; e) the calculation of costs of a single neoadjuvant instillation with Mitomycin and f) molecular characterization of UUT tumors and intravesical recurrences. CONCLUSIONS: A single intravesical instillation of Mitomycin before radical nephro-ureterectomy or partial ureterectomy may reduce the risk of a bladder recurrence in patients treated for UUT urothelial carcinoma and will circumvent the disadvantages of current therapy.

A structural chemogenomics analysis of aminergic GPCRs: lessons for histamine receptor ligand design.

BACKGROUND AND PURPOSE: Chemogenomics focuses on the discovery of new connections between chemical and biological space leading to the discovery of new protein targets and biologically active molecules. G-protein coupled receptors (GPCRs) are a particularly interesting protein family for chemogenomics studies because there is an overwhelming amount of ligand binding affinity data available. The increasing number of aminergic GPCR crystal structures now for the first time allows the integration of chemogenomics studies with high-resolution structural analyses of GPCR-ligand complexes. EXPERIMENTAL APPROACH: In this study, we have combined ligand affinity data, receptor mutagenesis studies, and amino acid sequence analyses to high-resolution structural analyses of (hist)aminergic GPCR-ligand interactions. This integrated structural chemogenomics analysis is used to more accurately describe the molecular and structural determinants of ligand affinity and selectivity in different key binding regions of the crystallized aminergic GPCRs, and histamine receptors in particular. KEY RESULTS: Our investigations highlight interesting correlations and differences between ligand similarity and ligand binding site similarity of different aminergic receptors. Apparent discrepancies can be explained by combining detailed analysis of crystallized or predicted protein-ligand binding modes, receptor mutation studies, and ligand structure-selectivity relationships that identify local differences in essential pharmacophore features in the ligand binding sites of different receptors. CONCLUSIONS AND IMPLICATIONS: We have performed structural chemogenomics studies that identify links between (hist)aminergic receptor ligands and their binding sites and binding modes. This knowledge can be used to identify structure-selectivity relationships that increase our understanding of ligand binding to (hist)aminergic receptors and hence can be used in future GPCR ligand discovery and design.

Micturation related swelling of the scrotum.

Unlike small inguinal and femoral bladder hernias, massive bladder hernias into the scrotum, also named scrotal cystoceles, are rare. We describe and discuss the clinical appearance and management of a patient with a micturation related unilateral swelling of the scrotum.

Comparison of the Murex Hybrid Capture CMV DNA (v2.0) assay and the pp65 CMV antigenemia test for the detection and quantitation of CMV in blood samples from immunocompromised patients.

BACKGROUND: Presently the semiquantitative pp65 cytomegalovirus (CMV) antigenemia test on white blood cells is often used for monitoring transplant patients for the appearance of active CMV infections. However, the need for immediate processing of the specimens and the lack of interlaboratory standardization of the test may sometimes be a disadvantage. OBJECTIVE: The aim of this study was to investigate the value of the recently developed second version of the Murex Hybrid Capture (MHC) CMV-DNA assay (v 2.0) in comparison with the CMV-pp65 test. The MHC CMV-DNA assay is a quantitative solution hybridization antibody capture assay, using alkaline phosphatase conjugated antibodies and a chemiluminescent substrate. STUDY DESIGN: 248 EDTA blood samples from 33 renal transplant patients and 32 samples from 22 other immunocompromised patients were tested by both the MHC CMV-DNA assay and the CMV-pp65 test. RESULTS: The qualitative ( + or -) results of both tests showed an overall concordance of 81.4%. Calculations on the basis of discordancy analyses showed that the sensitivity, the specificity, and the positive and negative predictive values were 87.7, 98.3, 98.6, 85.2% for the MHC CMV-DNA assay and 76.6, 100, 100, 75.5% for the CMV-pp65 test. Comparison of the quantitative results of both tests systems showed a correlation coefficient of 0.837. In addition, retesting of 50 samples with the MHC-CMV-DNA assay showed an excellent reproducibility with a correlation coefficient of 0.992. All patients which were tested regularly (at least five samples) became either positive with both tests or with none of them. Neither test system detected CMV significantly earlier than the other one. Both tests became strongly positive in all transplant patients with symptomatic CMV infections, and both tests showed a rapid decline of CMV during subsequent antiviral treatment. CONCLUSION: The quantitative Murex Hybrid Capture CMV-DNA assay (v 2.0) may become a valuable additional tool in CMV diagnosis. Further studies will be needed to support this preliminary judgement.

The prevention of anastomotic leakage after total gastrectomy with local decontamination. A prospective, randomized, double-blind, placebo-controlled multicenter trial.

OBJECTIVE: A prospective, randomized, double-blind, placebo controlled multicenter trial was undertaken in 205 patients treated with total gastrectomy for gastric malignancies to evaluate whether local antimicrobial measures reduce the incidence of esophagojejunal anastomotic leakage. SUMMARY BACKGROUND DATA: Anastomotic leakage of the esophagojejunostomy is always a septic complication of total gastrectomy for gastric malignancies, but it never has been attempted to prevent this complication with the administration of topical antimicrobial agents during the critical phase of anastomotic wound healing. METHODS: To evaluate the efficacy and safety of topical decontamination, the study was carried out as a prospective, randomized, double-blind and placebo-controlled clinical multicenter trial in patients with total gastrectomy for gastric cancer. Patients received either placebo or decontamination with polymyxin B (100 mg), tobramycin (80 mg), vancomycin (125 mg), and amphotericin B (500 mg) four times per day orally from the day before the operation until the seventh postoperative day. All patients received a perioperative intravenous prophylaxis with cefotaxime 2 x 2 g. Other interventions including the administration of antibiotics and fluids, were not affected by the study protocol. RESULTS: Of 260 patients who were randomized, total gastrectomy was not carried out in 55 patients. They dropped out of the study. Patients receiving an esophagojejunostomy were observed until day 42, when they were discharged from the clinic or died. An intention-to-treat analysis of the data was carried out. Among the 103 recipients of placebo, there were 11 (10.6%) with an anastomotic leakage of the esophagojejunostomy, and among the 102 recipients of decontamination, there were 3 (2.9%) with an anastomotic leakage of the esophagojejunostomy (p = 0.0492). Pulmonary infections were observed in 23 patients (22.3%) receiving placebo and in 9 patients (8.8%) who were decontaminated (p = 0.02). There were 11 deaths (10.6%) among the recipients of placebo and 5 deaths (4.9%) among the recipients of decontamination (p = 0.1). CONCLUSIONS: Decontamination with polymyxin, tobramycin, vancomycin, and amphotericin B during anastomotic wound healing is safe and effective in the prevention of esophagojejunal anastomotic leakage after total gastrectomy.

Stromal inhibition of epithelial cell growth in the prostate; overview of an experimental study.

The paracrine influence of prostatic stroma on the proliferation of prostatic epithelial cells was investigated. Using a double-layer soft agar assay it was demonstrated that stromal cells from the human prostate inhibit the anchorage-independent growth of the prostatic tumor epithelial cell lines PC-3 and LNCaP. Anchorage-dependent growth was inhibited too as was shown in the semi-automated colorimetric MTT test performed on multiwell plates. Antiproliferative activity was mediated by a diffusible factor in the stromal cell conditioned medium and was found to be produced specifically by prostatic stromal cells. Although the putative inhibiting factor shared some properties with transforming growth factor beta (TGF-beta) evidence is presented that the factor is different from this well-known inhibitor of epithelial cell growth. Absence of TGF-beta activity was shown by the lack of inhibitory response of the TGF-beta-sensitive mink lung cell line CCL-64 to prostate stromal cell conditioned medium and to concentrated partially purified preparations of the inhibitor. Furthermore, neutralizing antibodies against TGF-beta 1 or TGF-beta 2 did not cause a decline in the level of PC-3 growth inhibition caused by partially purified inhibitor. It is concluded that the prostate stroma-derived factor may be a novel growth inhibitor different from any of the currently described inhibiting factors.

[Early endotoxin tolerance--a preventive concept against infection]. / Die frühe Endotoxintoleranz--ein prophylaktisches Konzept gegen Sepsis.

After induction of early-phase endotoxin tolerance in a porcine endotoxin shock model over a period of 3-4 days a highly significant survival-time extension (p < or = 0.0001) was achieved. The protective effect of the unspecific tolerant state led to a significant enhancement of cardiorespiratory parameters during the continuous endotoxin challenge. However, the level of tolerance is relative and can be overcome by increasing the endotoxin challenge dose.

Stromal inhibition of prostatic epithelial cell proliferation not mediated by transforming growth factor beta.

The paracrine influence of prostatic stroma on the proliferation of prostatic epithelial cells was investigated. Stromal cells from the human prostate have previously been shown to inhibit anchorage-dependent as well as anchorage-independent growth of the prostatic tumour epithelial cell lines PC-3 and LNCaP. Antiproliferative activity, mediated by a diffusible factor in the stromal cell conditioned medium, was found to be produced specifically by prostatic stromal cells. In the present study the characteristics of this factor were examined. It is demonstrated that prostate stroma-derived inhibiting factor is an acid- and heat-labile, dithiothreitol-sensitive protein. Although some similarities with type beta transforming growth factor (TGF-beta)-like inhibitors are apparent, evidence is presented that the factor is not identical to TGF-beta or to the TGF-beta-like factors activin and inhibin. Absence of TGF-beta activity was shown by the lack of inhibitory response of the TGF-beta-sensitive mink lung cell line CCL-64 to prostate stromal cell conditioned medium and to concentrated, partially purified preparations of the inhibitor. Furthermore, neutralising antibodies against TGF-beta 1 or TGF-beta 2 did not cause a decline in the level of PC-3 growth inhibition caused by partially purified inhibitor. Using Northern blot analyses, we excluded the involvement of inhibin or activin. It is concluded that the prostate stroma-derived factor may be a novel growth inhibitor different from any of the currently described inhibiting factors.

Immunocytochemical characterization of explant cultures of human prostatic stromal cells.

The study of stromal-epithelial interactions greatly depends on the ability to culture both cell types separately, in order to permit analysis of their interactions under defined conditions in reconstitution experiments. Here we report the establishment of explant cultures of human prostatic stromal cells and their immunocytochemical characterization. As determined by antibodies to keratin and prostate specific acid phosphatase, only small numbers (< 5%) of epithelial cells were present in primary cultures; subsequent passaging further reduced epithelial cell contamination. Antibodies against intermediate filament proteins (keratins, vimentin, and desmin) and smooth muscle actin microfilaments demonstrated that stromal cells from benign prostatic hyperplasia and prostate carcinoma differed in regard to their differentiation markers. Two contrasting phenotypes were identified in cultures derived from these two different lesions: One exhibiting fibroblastic features, was predominant in cultures derived from benign lesions and a second, showing varying degrees of smooth muscle differentiation, was more abundant in carcinoma-derived cultures. These findings are indicative of a remarkable divergence in the stromal-epithelial relationships associated with these pathological conditions and may provide us with a potential tool for studying these processes.

Inhibition of prostatic epithelial cell proliferation by a factor secreted specifically by prostatic stromal cells.

Stromal cells from the prostate were recently shown to inhibit clonal growth of the prostatic carcinoma cell lines PC-3 (hormone-independent) and LNCaP (hormone-sensitive) in coculture. Our study revealed that stromal cell-conditioned medium strongly inhibited proliferation of PC-3 and LNCaP cells when grown in monolayer culture. Antiproliferative activity was found to be reversible, and was produced specifically by prostatic stromal cells and not by stromal cells derived from skin, foreskin, uterus, kidney, and Wilms' tumor. Inhibition was not species-specific, since the cell lines AT-2.1 and MATLyLu, derived from the Dunning rat prostate tumor, were also sensitive. No inhibition, however, occurred on breast and renal carcinoma cell lines, suggesting a prostate-specific action. The putative inhibiting factor(s) could be concentrated and partially purified by ammonium sulfate precipitation. The possible role in stromal control of epithelial cell proliferation is discussed.

[Surgical therapy of high grade stenoses of the carotid artery. Choice of procedure and results]. / Die operative Therapie hochgradiger Stenosen der Arteria carotis. Verfahrenswahl und Ergebnisse.

During the last 10 years 1514 endarterectomies of the carotid artery were performed by 9 surgeons. Complication rate was 3.6%, perioperative stroke incidence 3%, and lethality rate 1.1%. The results show, that conventional endarterectomy with obligate use of intraluminal shunt is a save management in therapy of severe stenosis of the carotid artery.

Topoisomerase II as a target of VM-26 and 4'-(9-acridinylamino)methanesulfon-m-aniside in atypical multidrug resistant human small cell lung carcinoma cells.

The Adriamycin-resistant small cell lung carcinoma cell line, GLC4/ADR, showed large differences in cross-resistance to drugs such as Adriamycin, etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and mitoxantrone, which stimulate the formation of topoisomerase (Topo) II-DNA complexes. GLC4/ADR cells demonstrated a reduced Topo II activity and no detectable levels of the P-glycoprotein compared to the parental GLC4 cells (S. De Jong et al., Cancer Res., 50: 304-309, 1990). In the present study, the resistance to VM-26 (59.5-fold) and to m-AMSA (4-fold) of GLC4/ADR after a 1-h incubation was further analyzed. Using the K(+)-sodium dodecyl sulfate precipitation assay, a reduction in VM-26- and m-AMSA-induced cleavable complex formation was found in GLC4/ADR cells compared to GLC4 cells that was related to the degree of resistance to each drug. Cellular accumulation of the VM-26 analogues VP-16 was 3- to 8-fold less and the accumulation of m-AMSA 1- to 2-fold less in GLC4/ADR cells than in the parental cells. Following the removal of VM-26, the cleavable complexes in GLC4/ADR cells disappeared at least 2-fold faster than in GLC4 cells, while the efflux of VP-16 was also enhanced in the resistant cells. On the contrary, no differences in cleavable complex disappearance or drug efflux between these cell lines were observed with m-AMSA. Efflux of both drugs, however, occurred at a much higher rate than cleavable complex disappearance. Using isolated nuclei, a reduction in cleavable complexes in GLC4/ADR was still observed with VM-26 as well as m-AMSA compared to GLC4. The resistant nuclei and nuclear extracts showed a 3-fold decrease in M(r) 170,000 Topo II by immunoblotting. No differences in cleavable complex formation were found between nuclear extracts of both cell lines, when the Topo II activities were equalized. These findings suggest that the cross-resistance to m-AMSA is due to a decreased amount of Topo II and decreased drug accumulation, while in addition to these mechanisms an increased rate of cleavable complex disappearance is involved in the cross-resistance to VM-26 of the GLC4/ADR cell line.

Negative control of epithelial cell proliferation by prostatic stroma.

The influence of prostatic fibroblasts on the growth of the prostatic carcinoma cell lines PC-3 and LNCaP was examined. In a double layer soft agar system, clonal growth of both cell lines was inhibited by all prostatic fibroblasts tested, irrespective of whether they were derived from malignant or non malignant prostate tissue. Irradiated fibroblasts and quiescent fibroblasts showed similar effects. The finding that growth was also inhibited in the presence of fibroblasts conditioned medium suggests that the observed effects were mediated by a diffusable growth inhibiting factor. Organ-specific production was suggested by the observation that skin fibroblasts stimulated rather than inhibited PC-3 cell growth. These findings indicate a negative control of epithelial cell proliferation by prostatic stroma.

K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung.

BACKGROUND: The capability of activated oncogenes to induce malignant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocarcinomas. METHODS: We studied the clinical importance of this oncogene-activation in 69 patients with lung adenocarcinoma in whom complete resection of the tumor was possible. The polymerase chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation-specific oligonucleotide probes. RESULTS: Nineteen of the tumors harbored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon-12 point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-ras point-mutation-positive tumors died during the follow-up period, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P less than 0.001). CONCLUSIONS: The presence of K-ras point mutations defines a subgroup of patients with lung adenocarcinoma in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.

Can hemofiltration increase survival time in acute endotoxemia--a porcine shock model.

In a porcine endotoxin shock model employing a continuous intravenous administration of Salmonella abortus equi endotoxin the cardiorespiratory and metabolic parameters were studied with main emphasis on the effect of hemofiltration (HF) as the only therapeutical measurement on the enhancement of survival time. Arachidonic acid (AA) metabolites Thromboxan B2 and 6-Keto-PGF 1-alpha could be lowered significantly by hemofiltration. Measuring the inadequacy of the supply and delivery systems in terms of O2-uptake, CO2 production, lung mechanics, TPR, CO, heart rate and MAP the control group seemed to be more severely compromised than the hemofiltrated groups, although the final outcome as for survival time could not be increased significantly. HF can nonselectively counteract some toxic effects of shock mediators without depriving the organism of beneficial components of a protective system being stimulated at the same time. Once the AA cascade is initiated, pharmacologic inhibition is of limited value as long as a direct specific therapeutic manipulation is still not available. Elimination of mediators by HF helps to combat the overstimulation of host defense mechanisms in ET shock which represents the ultimate threat to the host.

Fluctuations in the enzymatic activity of the human endometrium.

Cyclic fluctuations were studied in the activity of oxidoreductases playing a role in the major energy metabolic pathways, lysosomal and non-lysosomal hydrolases and some non-enzymatic cytochemical components demonstrable in different developmental physiological or pathophysiological phases of human endometrium. The total scope of the study involved 170 tissues and cytological specimens. The cytological material included microcurettings, aspirates, brush preparations and tissue prints. An evaluation of the usefulness of the application of enzyme cytochemistry to cytological material is included. The most important results were a cyclic fluctuation and a progestagenic controlled increase in the activity of many oxidoreductases, especially the NADPH regenerating enzymes of the pentose phosphate pathway, and of the NADP+ dependent isocitrate dehydrogenase. The histochemical evaluation of the activity of these NADP+ linked enzymes can therefore be recommended for the evaluation of the physiological status of the endometrial cells, especially in patients with infertility problems.

Concentration and purification of poliovirus by immune adsorption on immobilized antibodies.

Antibodies to poliovirus type 1, 2 and 3 have been coupled to Sepharose 4 B. The immune sorbentia have high binding capacities for both live and inactivated virus. Bound antigen is eluted with a high concentration of ammoniumisothiocyanate. Immediately after elution the chaotropic salt is removed by gelfiltration. The antigen recovery with inactivated virus was low; however, with live virus a good recovery was obtained. Therefore, immune adsorption seems to be applicable for concentration and purification of large amounts of live poliovirus.