Increased binding of beta-2-microglobulin to blood cells in dialysis patients treated with high-flux dialyzers compared with low-flux membranes contributed to reduced beta-2-microglobulin concentrations. Results of a cross-over study.

BACKGROUND: Patients on long-term dialysis eventually develop amyloid deposits with beta2-microglobulin as a predominant component. Although several studies have suggested that high-flux membranes reduce beta2-microglobulin in plasma compared with low-flux dialyzers, the mechanisms underlying this observation are still discussed. METHODS: We revisited this important subject and measured beta2-microglobulin in the plasma of healthy individuals (n = 8), and patients undergoing hemodialysis (n = 20) who for assigned periods of time were either treated with a low-flux membrane (cuprophan) or high-flux (polyamide) dialyzer with an ELISA. The number of blood cells was determined by FACS. Beta2-microglobulin was also measured on the surface of granulocytes, lymphocytes, and monocytes before, directly after, and 4 h after hemodialysis. Expression of beta2-microglobulin, c-fos, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 mRNA was determined in whole blood samples with quantitative RT-PCR using an internal standard in parallel. In the second part of the study, patients were assigned in a two-group cross-over design either to low- or high-flux dialyzers (n = 9 in each group), and dialyzer membranes were changed every 4 weeks for two consecutive periods. Serum beta2-microglobulin concentrations were measured at the end of each period. RESULTS: Healthy controls had a low plasma beta2-microglobulin level of 1.2 +/- 0.3 mg/l. Before hemodialysis, patients on low-flux dialyzers had a plasma beta2-microglobulin level of 42.0 +/- 14.0 mg/l, patients treated with high-flux dialyzers 21.5 +/- 10.8 mg/l (p < 0.05 vs. low-flux dialyzers). In contrast, there was no significant difference in plasma concentrations of active transforming growth factor-beta1 with the two different membrane types. The difference in serum beta2-microglobulin between low- and high-flux membranes was more prominent directly after hemodialysis as well as 4 h after hemodialysis compared with the values directly before the start of treatment. At all studied time-points, leukocytes and platelets were significantly higher in patients on low-flux membranes. Healthy control persons exhibited a significantly higher amount of beta2-microglobulin bound to granulocytes, lymphocytes, and monocytes compared with dialysis patients. Interestingly, beta2-microglobulin bound to granulocytes, lymphocytes, and monocytes was significantly increased in patients treated with high-flux membranes compared with low-flux filters. Quantitative RT-PCR revealed no significant difference in beta2-microglobulin expression in whole blood before hemodialysis, directly after hemodialysis, and 4 h after hemodialysis. However, TNF-alpha and c-fos transcripts were significantly higher in whole blood obtained from patients treated with low-flux membranes compared to high-flux dialyzers. The two-group cross-over study over three periods of 4 weeks revealed that switching from low-flux to high-flux dialyzers significantly reduced serum beta2-microglobulin levels. CONCLUSION: Patients treated with a polyamide high-flux membrane had lower beta2-microglobulin concentrations compared with those patients on low-flux dialyzers. This difference might not be mediated by an increase in beta2-microglobulin mRNA, but may be caused by less beta2-microglobulin released from the blood cells in patients treated with high-flux dialyzers, in addition to a better beta2-microglobulin clearance.

Correlations of mRNA expression and in vitro chemosensitivity to enzastaurin in freshly explanted human tumor cells.

PURPOSE: Enzastaurin (LY317615) is a novel serine/threonine kinase inhibitor, targeting Protein Kinase C-beta (PKC-beta), and PI3K/AKT pathways to inhibit angiogenesis and tumor cell proliferation. The aims of this study were to determine whether Enzastaurin has direct antitumor activity against freshly explanted tumor cells and to correlate mRNA expression of genes related to the proposed mechanism of action of enzastaurin with in vitro chemosensitivity. EXPERIMENTAL DESIGN: Freshly biopsied tumor cells were studied using soft-agar cell cloning experiments (SACCE) to determine the in vitro chemosensitivity to enzastaurin. An aliquot of the same tumor specimens was shock-frozen and total RNA was isolated for standardized multiplex rt-PCR experiments for gene expression of PKC-beta1, PKC-beta2, IL-8, IL-8RA, IL-8RB, Glycogen Synthase Kinase 3 beta (GSK-3beta) and TGF-beta1. Correlations, threshold optimization, sensitivity, specificity, and efficiency were analyzed using the appropriate statistical methodologies. RESULTS: Seventy-two tumor samples were collected and 63 were fully evaluable. Low levels of mRNA expression of GSK-3beta and high levels of mRNA expression of IL-8 were highly significantly correlated with chemosensitivity to enzastaurin. Optimization analyses demonstrated threshold values of 4,000 copies for IL-8 and three copies for GSK-3beta relative to 10(4) copies of beta-actin. However, no correlation between mRNA expression of PKC-beta1, PKC-beta2, IL-8RA, IL-8RB and chemosensitivity to enzastaurin was observed. Expression of TGF-beta1 mRNA was not detectable in the specimens investigated. CONCLUSIONS: mRNA expression levels of IL-8 and GSK-3beta correlate with antitumor activity of enzastaurin. These results form a rational basis for clinical trials to evaluate the expression of these genes as potential predictors for treatment outcome after enzastaurin chemotherapy.

Antitumor activity of enzastaurin (LY317615.HCl) against human cancer cell lines and freshly explanted tumors investigated in in-vitro [corrected] soft-agar cloning experiments.

Enzastaurin (LY317615.HCl) is an antiproliferative agent targeted specifically against PKC-beta. We have investigated the antitumoral effects of Enzastaurin against human cancer cell lines and freshly explanted human tumor tissue. Ten human cancer cell lines (NSCLC, colon, and thyroid) and human tumor specimens from 72 patients were used for in vitro studies in a cloning assay (HTCA). Cell lines and primary tumor cells were exposed to Enzastaurin for either 1 h or 7 days, or for 1 h or 21 days. At clinically achievable concentrations of Enzastaurin, inhibition of cell growth was observed for lung, colorectal, and thyroid cancer cell lines in a concentration dependent manner. Patient specimens exposed 1 h or 21 days to 1,400 nM Enzastaurin demonstrated inhibition rates of 24 and 32%, respectively. Marked inhibitory effects were observed in breast, thyroid, head/neck, non-small cell lung cancer, pancreatic cancer, and melanoma. In addition to its established antiangiogenic effects, Enzastaurin has direct antitumor activity against established human cancer cell lines and primary tumor specimens. This warrants further clinical development in tumors which have been identified to be potentially sensitive to Enzastaurin.

In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression.

AIM OF THE STUDY: mRNA expression of genes involved in the mechanism of action of pemetrexed was correlated with in vitro chemosensitivity of freshly explanted human tumor specimens. EXPERIMENTAL DESIGN: Chemosensitivity to pemetrexed was studied in soft-agar. Multiplex rtPCR experiments for reduced folate carrier (RFC), folate receptor-alpha (FR-alpha), folylpolyglutamate synthetase (FPGS), thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyl transferase (GARFT), mrp4, and mrp5 were performed in parallel. Correlations, threshold optimization, sensitivity, specificity, and efficiency were analyzed using the appropriate statistical methodologies. RESULTS: In 61 samples, low levels of TS, GARFT, DHFR, and mrp4 gene expression significantly correlated with chemosensitivity to pemetrexed. Optimization analyses demonstrated threshold values of 144 copies for TS and six copies for mrp4 relative to 10(4) copies of beta-actin. CONCLUSIONS: These results form a rational basis for the design of clinical trials to evaluate the expression of these enzymes as predictors for treatment outcome.

Fibronectin splice variants--prognostic markers for the stage of renal interstitial fibrosis in the rat.

BACKGROUND/AIMS: Renal interstitial fibrosis (RIF) is the main cause for progressive renal failure, but its pathogenic factors are not well known. In animal models of renal fibrogenesis done thus far an increase of total fibronectin (FN) mRNA has been proved. Recent studies have pointed to a key role of the splice variant EIIIA(+)-FN and EIIIB(+)-FN for the development of organ fibrosis. However, a broader knowledge of the distribution of these different FN mRNA isoforms is still lacking. Our aim was to study the particular expression of the EIIIA(+)-FN and EIIIB(+)-FN during the process of fibrogenesis in two rat models and to evaluate the FN isoforms as diagnostic/prognostic marker for the stage of interstitial damage in rat kidneys. METHODS: Kidneys of unilateral ureteral obstruction (UUO) and control rats were removed in intervals of 5, 14 or 21 days after surgery. For the investigation of kidney damage due to uranyl nitrate (UN), rats obtained a single i.p. dose of 5 mg/kg body weight UN and were killed 2, 10 and 20 weeks thereafter. The quantitative RT-PCR method was used to estimate the total FN, EIIIA(+)-FN and EIIIB(+)-FN transcription rate. RESULTS: In the UUO model, a significant augmentation of both isoforms was obtained in the kidneys in the first 5-day interval, which was more pronounced at the 21-day interval. In the UN-treated kidneys there appeared only a continuous increase of EIIIA(+)-FN and the splice variant EIIIB(+)-FN failed to show a shift in these animals as compared to the controls. CONCLUSION: Both animal models generated fibrogenic damages of the tubulointerstitium, whereas only the UUO resulted in progressive fibrosis. Absence of EIIIB(+)-FN seems to enhance the progression of fibrogenesis.