Lipid Peroxidation and Vitamin E in Red Blood Cells and Plasma in Hemodialysis Patients Under rhEPO Treatment

Abstract: Lipid peroxidation, measured by malonyldial-dehyde (MDA) and vitamin E in red blood cells (RBC) and plasma, was investigated in 25 hemodialysis (HD) patients before and after 6 months rhEPO therapy. RBC-MDA was significantly elevated, but plasma MDA was in the reference range. After recombinant human erythro-poietin (rhEPO) treatment, the MDA level was significantly decreased in both compartments. Marked vitamin E deficiency was established in RBC as well as in plasma. rhEPO therapy restored vitamin E levels in both compartments. Our data suggest a possible positive rhEPO-antioxidant effect in HD patients.     

An optimal three-stage design for phase II clinical trials

A phase II clinical trial in cancer therapeutics is usually a single-arm study to determine whether an experimental treatment (E) holds sufficient promise to warrant further testing. When the criterion of treatment efficacy is a binary endpoint (response/no response) with probability of response p, we propose a three-stage optimal design for testing H₀: p ≤ p₀ versus H₁: p ≥ p₁, where p₁ and p₀ are response rates such that E does or does not merit further testing at given levels of statistical significance (α) and power (1 ? β). The proposed design is essentially a combination of earlier proposals by Gehan and Simon. The design stops with rejection of H₁ at stage 1 when there is an initial moderately long run of consecutive treatment failures; otherwise there is continuation to stage 2 and (possibly) stage 3 which have decision rules analogous to those in stages 1 and 2 of Simon's design. Thus, rejection of H₁ is possible at any stage, but acceptance only at the final stage. The design is optimal in the sense that expected sample size is minimized when p = p₀, subject to the practical constraint that the minimum stage 1 sample size is at least 5. The proposed design has greatest utility when the true response rate of E is small, it is desirable to stop early if there is a moderately long run of early treatment failures, and it is practical to implement a three-stage design. Compared to Simon's optimal two-stage design, the optimal three-stage design has the following features: stage 1 is the same size or smaller and has the possibility of stopping earlier when 0 successes are observed; the expected sample size under the null hypothesis is smaller; stages 1 and 2 generally have more patients than stage 1 of the two-stage design, but a higher probability of early termination under H₀; and the total sample size and criteria for rejection of H₁ at stage 3 are similar to the corresponding values at the end of stage 2 in the two-stage optimal design.     

Red blood cell methotrexate and folate levels in children with acute lymphoblastic leukemia undergoing therapy: a Pediatric Oncology Group pilot study

Summary We enrolled children with acute lymphoblastic leukemia (ALL) in a Pediatric Oncology Group (POG) pilot study to monitor erythrocyte (RBC) methotrexate (MTX) and folate (F) levels before and during treatment. The mean value for RBCF at diagnosis was 0.86±0.46 nmol/ml RBC in the 214 patients who achieved remission and 1.21±0.74 nmol/ml RBC in the 10 patients who did not (P=0.020). Folate levels tended to increase during remission induction, but they dropped following an intensive consolidation with methotrexate to levels that were sustained throughout chemotherapy treatment. Methotrexate levels reached mean values of approximately 0.15 nmol/ml RBC at the end of an intensive methotrexate consolidation, then fell to levels that were sustained throughout maintenance therapy. There was a weak correlation between improved event-free survival and higher RBCMTX levels after consolidation, but no correlation was found between improved survival and the level of RBCMTX or RBCF during maintenance therapy. A larger study with more complete data is needed to determine whether RBCMTX or RBCF might be useful in predicting event-free survival in patients with ALL.This work was supported in part by grants from the National Cancer Institute and the National Institute of Health (CA-30969, CA-28476, CA29139, CA-159-89, and CA-33587)     

Polyoma virus high molecular weight nuclear RNA codes for capsid protein VP2 in vitro.

RNA extracted from the nuclei of 3T6 mouse cells late after infection by pot yoma virus (Py) has been translated in vitro to give one predominant polypeptide, which was identified as virion protein VP2 by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide analysis. The active nuclear RNA species sediment between 15 and 50 S on sucrose gradients after denaturation, and resediment true after further denaturation and recentrifugation on 50% formamide-sucrose gradients. The bulk of the active viral nuclear RNA does not bind to oligo(dT)-cellulose and synthesis of Py VP2 is inhibited by m⁷Gp under conditions where translation of Py-cRNA and EMC RNA are not inhibited. These experiments suggest that large viral nuclear RNA molecules, which are probably capped but not polyadenylated, can serve as messengers for Py VP2.     

Expression of the large T protein of polyoma virus promotes the establishment in culture of "normal" rodent fibroblast cell lines.

Transfer into mouse and rat embryo fibroblasts in primary culture of cloned polyoma virus genes encoding only the large T protein led to the establishment of flat colonies in sparse subcultures at a frequency equal to that of transformation by wild-type virus. Cell lines could be derived from such colonies and maintained in culture for large numbers of generations without entering crisis. They exhibited a normal phenotype, by the criteria of growth on plastic to a low saturation density and of anchorage dependency. However, they required a lower serum concentration for growth than spontaneously established 3T3 cells. Similar results were obtained after transfer of recombinant DNA molecules encoding only the amino-terminal 40% of the large T protein, suggesting that this "immortalization" function corresponds to the activity of an amino-terminal domain of the protein. Immunoprecipitation analysis of T antigens in cell lines established after transfer of the full-size and of the truncated large T genes demonstrated the expression of the full-size large T protein and of a Mr 40,000 antigen expressed from the amino-terminal part of the gene, respectively. After transfer of a "large T only" plasmid that carries a tsa mutation, cell lines were established at 33 degrees C with the same efficiency as with the wild-type large T gene, but their growth was arrested after a shift to 40 degrees C, with a progressive loss in cell viability. This result indicates a continuous requirement for a large T function in the maintenance of "immortality."     

Phase II trial of oral aminopterin for adults and children with refractory acute leukemia.

PURPOSE: To determine the antileukemic activity of weekly oral aminopterin in patients with refractory acute leukemia; to describe the pharmacodynamic properties of aminopterin; and to contrast the intracellular metabolism of aminopterin and methotrexate by patients' blasts in vitro. EXPERIMENTAL DESIGN: Forty-six patients were enrolled in three strata: children with acute lymphoblastic leukemia (ALL), adults with ALL, and patients with acute myeloid leukemia (AML). Aminopterin was given weekly, in two doses of 2 mg/m(2), 12 hours apart. Limited sampling pharmacokinetic analysis was done during the first week of therapy. Accumulation of [(3)H]aminopterin and [(3)H]methotrexate by leukemic blasts was studied in vitro. RESULTS: Six of 22 children with ALL (27%; 95% confidence interval, 8-47%) had clinically significant responses. None of those with AML and only two of 11 adults with ALL had responses meeting protocol definitions, although peripheral blast counts tended to decrease with therapy in all groups. Mucosal toxicity was minimal, even with limited use of leucovorin rescue. Complete bioavailability of aminopterin was confirmed, with a mean area under the curve of 0.52 +/- 0.03 micromol hour/L after oral dosing. No relationship between aminopterin pharmacokinetics and response was seen. In vitro, aminopterin showed more consistent metabolism by leukemic blasts to polyglutamates than methotrexate. Lineage-specific differences in the pattern of intracellular antifolylpolyglutamates were observed. CONCLUSIONS: Weekly oral aminopterin has significant activity among children with refractory ALL. With greater cellular accumulation and metabolism, more reliable bioavailability than methotrexate, and tolerable toxicity at this dose and schedule, aminopterin deserves further study as a potent alternative to methotrexate.     

RADIOACTIVE IRON AND ITS EXCRETION IN URINE,BILE, AND FECES

Radioactive iron as ferrous gluconate given by vein enables us to study iron excretion in urine, bile, and feces. There is an initial extra output in urine and feces during a few days (3 to 15 days) following the iron injection and this may total 2 to 8 per cent of the injected iron. Following this initial reaction the urinary excretion of radio-iron drops to traces or even to zero. The feces always contain measurable amounts of radio-iron—in five dogs receiving 100 to 250 mg. of radio-iron the fecal excretion per day settled down to 0.05 to 0.4 mg. per day. Blood destruction (acetyl-phenylhydrazine) causes a definite increase in radio-iron eliminated in the feces (0.1 to 1.0 mg. per day). Probably most of this excess iron comes through the biliary tract (bile fistula). The bile under usual conditions contributes very little iron to the intestine (0.01 mg. radio-iron per day or less). The body controls its iron stores by absorption or lack of it rather than by its capacity to eliminate it. The evidence is overwhelming that the dog excretes iron with difficulty and in small amounts (even in the plethoric state) by means of the biliary and gastro-intestinal tracts.