Cyclosporin A and PSC 833 prevent up-regulation of MDR1 expression by anthracyclines in a human multidrug-resistant cell line.

We have previously demonstrated that within 24 h of exposure of the CEM/A7R cell line to epirubicin (EPI), MDR1 gene expression is induced. The aim of the current study was to investigate the role of cyclosporin A (CyA) and PSC 833, two biochemical modulators of the classical multidrug-resistant phenotype, in this model. CEM/A7R cells were exposed to EPI in the presence or absence of various concentrations of CyA or PSC 833. MDR1 expression was assessed using Northern blot analysis and quantitated using a phosphorimager. P-glycoprotein (P-gp) expression was analyzed by the determination of MRK16 binding using flow cytometry. P-gp function was measured in an assay of [3H]daunomycin accumulation. The coincubation of CyA or PSC 833 with EPI prevented the increase in MDR1 gene expression induced by EPI alone. This effect of the two modulators was dose dependent. Neither modulator alone had any significant effect on the expression of MDR1. In these experiments, changes in MDR1 expression correlated with changes in P-gp levels (based on MRK16 binding) and P-gp function. Thus, both PSC 833 and CyA appear to prevent the induction of MDR1 gene expression caused by the short-term exposure of CEM/A7R cells to EPI.

Altered methylation of the human MDR1 promoter is associated with acquired multidrug resistance.

One of the most important forms of drug resistance in acute myeloid leukemia is the multidrug resistance (MDR) phenotype, which is characterized by the expression of the MDR1 gene product, P-glycoprotein. Although a number of factors affect MDR1 gene expression, the genetic events that "switch on" the human MDR1 gene in tumor cells that were previously P-glycoprotein negative have remained elusive. Here, we report evidence that the methylation status of the human MDR1 promoter may serve as a basis for this "switch." Based on Southern analysis using methylation-sensitive and methylation-insensitive restriction enzymes, a tight correlation was found between MDR phenotype and demethylation of the 5' region of the MDR1 gene in a human T cell leukemia cell line. Similar results were obtained from the analysis of P-glycoprotein-positive and P-glycoprotein-negative samples of chronic lymphocytic leukemia. Treatment of the cell lines with the demethylating agent 5'-azadeoxycytidine altered the methylation pattern of the MDR1 promoter in P-glycoprotein-negative cells to resemble that of P-glycoprotein-positive cells and activated the promoter such that MDR1 mRNA was now detectable. Treatment also resulted in an increased resistance to epirubicin and decreased daunomycin accumulation, both of which were reversible by verapamil, a characteristic of the classical MDR phenotype in cells expressing P-glycoprotein. These results suggest that the MDR phenotype may be acquired as a result of changes in methylation of the MDR1 promoter.

Clinical application of a rapid, functional assay for multidrug resistance based on accumulation of the fluorescent dye, fluo-3.

A rapid and simple functional assay for P-glycoprotein (Pgp) using flow cytometry to measure the accumulation of the flurophore fluo-3 has been applied to samples from patients with B-cell chronic lymphocytic leukaemia (B-CLL). Peripheral blood lymphocytes from 37 patients with B-CLL were studied for Pgp. Pgp expression, using MRK-16, a monoclonal antibody recognising an external surface epitope of Pgp, was detected in 92% of patients with B-CLL. The functional assays for Pgp expression were positive in 78 and 59% of patients using the fluo-3 and doxorubicin (dox) assays, respectively. When compared with the MRK-16 assay, the fluo-3 assay had a sensitivity of 82% compared to a sensitivity of 56% for the dox assay (P = 0.004). The specificity of the fluo-3 and dox assays could not be evaluated because of the low number of MRK-16 negative CLL cells.

Purification and characterization of the sand crab (Ovalipes bipustulatus) coagulogen (fibrinogen).

From the coagulocytes (amoebocytes) coagulogen (fibrinogen) was isolated, and purified on Sephacryl S-200. The cell homogenate contained one major protein species with a minimum molecular weight of 70,000. This protein clotted in the presence of human thrombin, human factor XIII and Ca++. The coagulogen contained no free thiol groups, however these were detectable in the reduced protein. Using the cyanoethylation procedure, it was estimated that one coagulogen molecule contained two lysine residues which participated in the cross-linking reaction. The total amino acid composition of the crab coagulogen and coagulin (fibrin) was estimated and compared with the amino acid composition of the Limulus polyphemus, lobster, (Panulirus interruptus) and porcine fibrinogen.

Coagulation in the sand crab (Ovalipes bipustulatus).

The coagulation mechanism of the sand crab (O. bipustulatus) has been investigated. From the coagulocytes (amoebocytes) present in the crab haemolymph (blood), fibrinogen (coagulogen) was isolated. It was shown to be homogeneous by electrophoresis on S.D.S. polyacrylamide gel and had a molecular weight similar to the A alpha-chain of human fibrinogen. Unlike human fibrinogen. Unlike human fibrinogen it cannot be dissociated by reduction. In fibrin polymerization, a crosslinking process takes place and this process was inhibited by glycine ethyl ester. A fibrin stabilizing factor is present in the crab haemolymph and this protein was able to cross-link human fibrin in the same manner as human factor XIII.

Confronting cisternae presenting as intracytoplasmic inclusions in monocytes.

Small gray-blue intracytoplasmic inclusions were noted in the peripheral monocytes and neutrophils from a patient with renal failure. Ultrastructural analysis revealed confronting cisternae within the cytoplasm. The authors submit a case report to introduce a previously undescribed morphologic feature of leukocytes that may be mistaken at the light microscopic level for Döhle bodies or other inclusions.

DNA ploidy patterns and cytokinetics of non-Hodgkin's lymphoma.

Flow cytometry studies for cellular DNA analysis were performed in 115 cases of non-Hodgkin's lymphoma, 53 of which had not received any prior chemotherapy or radiotherapy. DNA content was measured in ethanol fixed cells stained with chromomycin A3. According to the criteria of the International Working Formulation there were 43 low grade, 58 intermediate grade, and eight high grade lymphomas; six cases were in the miscellaneous group. Seventy seven (67%) had only diploid DNA content. Thirty eight (33%) showed DNA aneuploidy; 20 of these had been previously treated with chemotherapy or radiotherapy, or both. DNA aneuploidy was seen as hyperdiploidy in all cases except one, and it varied from slightly hyperdiploid to tetraploid. The incidence of aneuploidy increased significantly with increasing histological grade (p = 0.0002) and was not related to previous treatment. The low, intermediate, and high grade lymphomas had 14% (six of 43), 47% (27 of 58), and 62.5% (five of eight) cases, respectively, that showed DNA aneuploidy. The percentage of cells in S phase increased significantly with a higher histological grade (p less than 0.0001). The median S fraction in the low, intermediate, and high grade lymphomas was 1.0 (0.5 to 10)% 4 (0.4 to 35)%, and 27 (4.6-56)%, respectively. There is a significant correlation between histological grade and S fraction and the presence or absence of aneuploidy. There is heterogeneity, however, within both histological grade and a histological subtype.

The effects of very low fat diets enriched with fish or kangaroo meat on cold-induced vasoconstriction and platelet function.

Eighteen healthy volunteers consumed very low fat diets (less than 7% of daily energy) enriched with different sources of long chain (C20 and C22) polyunsaturated fatty acids (PUFA). Three diets provided 500 g/day of fish caught in the tropical waters of Australia (rich in arachidonic acid and docosahexaenoic acid), fish caught in the southern waters of Australia (rich in docosahexaenoic acid), or kangaroo meat (rich in linoleic and arachidonic acids). The fourth diet was vegetarian, similarly low in fat but containing no 20- and 22-carbon PUFA. An increase in the percentage of a particular C20 or C22 PUFA in the plasma phospholipid fraction in subjects consuming these low fat diets corresponded to the dietary PUFA composition. This study examined the effect of dietary modification of the level of arachidonic acid in plasma phospholipids on both traditional measures of platelet function and on cold-induced vasoconstriction. The cold pressor response, measured by venous occlusion plethysmography, was depressed in diets which elevated the levels of arachidonic acid in plasma lipids (kangaroo and tropical fish), enhanced after subjects consumed a diet which increased the levels of docosahexaenoic acid and eicosapentaenoic acid (southern fish diet), and was unchanged by the low fat vegetarian diet. There was no effect on bleeding time or platelet responsiveness.

Studies on an acquired inhibition of factor VIII induced by penicillin allergy.

An inhibitor of antihaemophilic globulin has been found in association with penicillin allergy. Inhibitor activity was detected after a severe reaction of penicillin. Neutralization studies showed the activity resided in an IgG globulin with kappa light chains. Experiments with insolubilized gammaglobulin demonstrated that the activity of the inhibitor was found in a specific penicillin antibody.

Neutropenia due to low-dose methotrexate therapy for psoriasis and rheumatoid arthritis may be fatal.

OBJECTIVE: To review experience with neutropenia related to low-dose methotrexate therapy in patients with psoriasis and rheumatoid arthritis. DESIGN: Retrospective review of medical records. SETTING: A 509-bed Melbourne teaching hospital. PATIENTS: Five patients admitted in 1987 and 1988, with neutrophil counts of less than 1 x 10(9)/L, given low doses of methotrexate for psoriasis or rheumatoid arthritis. MAIN OUTCOME MEASURES: Death, or length of hospital admission. FINDINGS: Four patients were women, and one a man; three had been treated for psoriasis, and two for rheumatoid arthritis. Ages ranged from 56 to 91 years. The eldest patients, aged 77, 81 and 91 years, died. The other two were discharged after 43 and 48 days. Prior to or shortly after admission, four patients were treated with penicillin antibiotics which may have interfered with methotrexate excretion. CONCLUSIONS: Methotrexate clearances (related to creatinine clearance rates and presumably low) were probably reduced sufficiently by concomitant therapy to result in neutropenia. Practitioners using methotrexate should be aware of drug interactions resulting in delayed methotrexate excretion. Blood counts should be monitored after changes in therapy, especially in patients with impaired renal function, such as the elderly.

A case of paroxysmal nocturnal haemoglobinuria terminating in a myeloproliferative syndrome.

This report discusses the case of a 60-year-old man who presented in 1969 with thrombocytopenia and mild marrow hypoplasia. A diagnosis of paroxysmal nocturnal haemoglobinuria (PNH), was established. The subsequent course included episodes of overt intravascular haemolysis. Thrombocytopenia reverted on several occasions during Oxymetholone therapy. The terminal phase of the illness was marked by the development of a leukocytosis and densely hypercellular bone marrow with splenomegaly. The features were those of a myeloproliferative disorder, although frank leukaemia did not develop.

Penicillin-induced haemolytic anaemia associated with microangiopathy.

The development of a penicillin-induced haemolytic anaemia was studied in a patient with microangiopathy following an average daily dose of five million units of penicillin G (total dose 46 million units). It has been proposed that the combination of the benzylpenicilloyl hapten with exposed or altered erythrocyte antigens, induces an autoimmune response. In this particular case, it is suggested that erythrocyte membrane damage has predisposed to immune drug-mediated red cell damage.

Automated estimation of factor Xa using the chromogenic substrate S-2222.

A rapid automated method for the estimation of factor Xa was developed using the "Centrifichem' rotary fast analyser and the substrate S-2222. The problem of interference from fibrin was overcome by performing the activation of factor X during centrifugation, so that defibrination and activation were achieved in a single step. A range of factor Xa concentrations was determined on 50 normal plasmas. This assay was correlated with a clotting assay of factor Xa using serial dilutions of normal plasma and plasma from 30 patients receiving warfarin therapy.

Use of double gradient denaturing gradient gel electrophoresis to detect (AT)n polymorphisms in the UDP-glucuronosyltransferase 1 gene promoter associated with Gilbert's syndrome.

Gilbert's syndrome, due to reduced hepatic bilirubin glucuronidation is associated with the presence of two extra nucleotides (TA) in the promoter region of the UDP-glucuronosyltransferase 1 (UGT1A1) gene. A rapid method was developed to detect this genetic polymorphism, using double gradient denaturing gradient gel electrophoresis (DG-DGGE). The promoter region of the UGT1A1 gene was amplified with a 40-mer GC-clamp attached to the 5'-end of the reverse primer. The polymerase chain reaction (PCR) product was then separated by DG-DGGE using denaturant concentrations of 15-25% and polyacrylamide concentrations of 6-12%. The (TA)6/(TA)6 homozygotes were clearly distinguished from both (TA)7/(TA)7 homozygotes and (TA)6/(TA)7 heterozygotes. The (TA)7 allele frequency was consistent with that previously reported and elevated bilirubin levels correlated with the presence of the (TA)7 allele. The DG-DGGE method described will make detection for this polymorphism fast, simple, nonradioactive and suitable for a clinical routine diagnostic laboratory, helping to establish the role of this polymorphism in individuals with jaundice due to multiple causes.

A comparison of five devices for the bedside monitoring of heparin therapy.

Five instruments were tested for their capacity to monitor heparin therapy on whole blood at the bedside. The instruments were 512 Coagulation Monitor (Ciba-Corning), Thrombotrack (Nycomed), Automated Coagulation Timer (Hemotec), Hemochron-ACT and Hemochron-APTT (International Technidyne Corporation). Fifty subjects with various levels of heparinisation were tested on each instrument and were also assayed for antithrombin III, fibrinogen, haematocrit, platelet count and plasma heparin level. The results were compared with a reference APTT performed on the Automated Coagulation Laboratory 300R (Instrumentation Laboratories). The Hemochron-ACT correlated least well. The Hemotec and Thrombotrack were unsuitable in a clinical setting because of pipetting requirements, although the Thrombotrack did correlate well with the reference parameters. The 512 Coagulation Monitor was the simplest to use, but its maximum response corresponded to the midpoint of the reference APTT therapeutic range. The Hemochron-APTT was simple to use, had an adequate response range and correlated well with reference parameters.

Autoantibodies in systemic vasculitis.

We have studied 495 sera that were referred to us from patients suspected on clinical and/or histological grounds to have a small vessel vasculitis. These sera were tested for antibodies against neutrophil cytoplasm antigens (anti-neutrophil cytoplasm antibodies, ANCA) using assays based on neutrophil acid extract, myeloperoxidase and elastase. Such antibodies are commonly found in Wegener's granulomatosis (WG) and microscopic polyarteritis (MPA), and sometimes in other small vessel vasculitides. One hundred and twenty-six of these sera (25%) were positive in the acid extract ELISA, 68 (14%) in the assay for anti-myeloperoxidase antibodies and 35 (16%) in the assay for anti-elastase antibodies. A total of 166 sera (34%) were positive for antibodies against neutrophil cytoplasm constituents. No ANCA, anti-myeloperoxidase or anti-elastase antibodies were detected in 26 convalescent sera from patients either with WG or MPA, or who had previously been positive. The mean time between positive and negative sera was eight weeks (range three weeks to six months) and three out of three who relapsed again developed ANCA of the same specificity as the original sera. Of the 228 sera also tested for anti-GBM antibodies, 13 (5.7%) were positive. All these contained antibodies against neutrophil cytoplasm constituents (three against the acid extract, eight against myeloperoxidase and two against elastase). Forty-nine of the 74 sera (66%) tested for ANA were positive. Twenty-nine (39%) had a speckled and 20 (27%) had a homogeneous pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

P-glycoprotein expression in classical multi-drug resistant leukaemia cells does not correlate with enhanced chloride channel activity.

1. P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump responsible for classical multi-drug resistance (MDR). 2. Pgp is part of a supergene family of membrane transport proteins that includes the cystic fibrosis gene product. 3. Transfection of cells with the MDR1 gene has been previously shown to generate volume-regulated chloride channel activity in association with Pgp expression. 4. We have used whole-cell patch clamping to examine the drug-sensitive T lymphoblastic cell line CEM-CCRF and its classical MDR derivative CEM/VLB100. The results suggest that expression of Pgp is not associated with increased chloride channel activity in this multi-drug resistant cell line. 5. We were unable to confirm previously reported results in MDR1 transfected cell lines that suggested that Pgp was associated with the presence of volume-regulated chloride channels.