M.R. Boisseau

Levels of plasma thrombomodulin are increased in atheromatous arterial disease

The plasma thrombomodulin (TM) level depends on the integrity of the endothelium and the clearance of the molecule. In several different pathological conditions, plasma TM levels increase with damage to the endothelium. We studied plasma TM levels in patients with various localizations of atheromatous arterial disease who had normal serum creatinine levels. Two groups of patients had a single symptomatic localization, which was either peripheral occlusive arterial disease (POAD) or ischemic heart disease (IHD) and a third group of patients had multiple symptomatic localizations (polyvascular). We compared the plasma TM levels with the plasma levels of other specific markers of endothelial cell activation such as: prostacyclin (PGI2), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1). Plasma TM levels were significantly increased in all three individual groups and when all patients were considered (total patients), as compared with normal controls. When all patients were considered, there was a significant positive correlation between plasma TM levels and t-PA and between plasma TM levels and PGI2. A significant positive correlation was also found between the plasma TM levels and PAI-1 for patients with POAD. Thus, our findings suggest that an increased influx of TM into the plasma may be caused by endothelial cell damage in patients with atheromatous arterial disease. However in our study, the plasma TM levels obtained were similar for all three types of atheromatous arterial disease. Though plasma thrombomodulin is a marker of endothelial cell injury, it cannot be of a clinical interest until its levels are related to the extend of the atheromatous lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Three months supplementation of hyperhomocysteinaemic patients with folic acid and vitamin B6 improves biological markers of endothelial dysfunction.

Hyperhomocysteinaemia is a risk factor for premature atherosclerosis and venous thromboembolic disease. Supplementation with folic acid and vitamin B6 has been shown to decrease plasma homocysteine but data fail to assess an effect on the progression of vascular disease. We measured plasma homocysteine and two markers of endothelial injury (plasma soluble thrombomodulin and von Willebrand factor) at baseline and after 3 months of treatment with folic acid and vitamin B6. After this treatment there was a significant decrease in fasting soluble thrombomodulin (−15 ng/ml, 95%CI 5–22.2). Von Willebrand factor was significantly raised after methionine load at baseline but did not significantly rise after supplementation.

Endothelial dysfunction during acute methionine load in hyperhomocysteinaemic patients

Hyperhomocysteinaemia has been associated with arterial and venous thrombosis possibly by causing damage to the endothelium. We hypothesised that an oral load in methionine, that increases plasma homocysteine, would also result in an increase in biological markers of endothelial or platelet dysfunction. Then we investigated two groups of patients with arterial or venous occlusive disease: 17 with hyperhomocysteinemia and 12 without hyperhomocysteinemia. We measured in both groups plasma soluble thrombomodulin, von Willebrand factor, P-selectin and tissue factor plasma inhibitor before and 6 hours after a load with 100 mg/kg oral methionine. Methionine load resulted in a significant increase in von Willebrand factor in both groups (P<0.02), suggesting that endothelial dysfunction occurs during the load.

CYTOMETRIC STUDY OF INTRACELLULAR P-GP EXPRESSION AND REVERSAL OF DRUG RESISTANCE

Expression of the multidrug resistance (MDR) phenotype is responsible for chemotherapy failure in numerous cancers. This phenotype is generally due to the expression of the mdr1 gene-encoded P-gp. Modulation of P-gp activity by chemotherapy has limited possibilities because of toxicity and poor specificity. In contrast, specific transcription blockage of the mdr1 gene can be obtained by oligonucleotides forming a triple helix structure at the DNA level. We used here immunofluorescence and both flow cytometry and image analysis to evaluate surface and total P-gp content in K562 MDR cells. The mdr1 mRNA content was measured by RT-PCR. We confirm the capacity of a 27-mer oligodeoxynucleotide, targeted to an mdr1 DNA fragment, to cause a 10-fold decrease in mdr1 mRNA level. However, this specific genetic inhibition was functionally limited because cellular growth was not modified in a cytotoxic environment. We found that total P-gp content was reduced in resistant cells treated with the mdr1-targeted oligonucleotide, while it remained in high levels on the cell surface, suggesting the existence of a large cytoplasmic pool of P-gp (approximately 50% of the total cellular P-gp). Moreover, when cycloheximide was used for 72 h to suppress protein synthesis, surface P-gp expression showed no decrease, whereas total P-gp was considerably lowered. A rapid 35% decrease in surface P-gp level was reached when resistant cells were treated for 24 h with brefeldin A, an inhibitor of intracellular protein trafficking. Simultaneously, the total P-gp level remained stable, thus indicating a probable accumulation of cytoplasmic P-gp, in agreement with the interruption of protein migration. We propose that the cytoplasmic P-gp pool could be a storage pool consumed for maintaining a steady-state level of surface P-gp. Cytometry could be a useful tool to study such a mechanism of P-gp trafficking and cellular distribution, which could explain the difficulties encountered in achieving stable and rapid effects of MDR reversal with oligonucleotides.

Interactions between aspirin and COX‐2 inhibitors or NSAIDs in a rat thrombosis model

Recent in vitro studies, clinical trials and epidemiological studies have suggested possible interactions between aspirin and other cyclo‐oxygenase (COX) inhibitors, such as ibuprofen of the COX‐2 inhibitors celecoxib and rofecoxib. The objective of this study was to test the effects of aspirin (1, 2.5 and 5 mg/kg), and ibuprofen (4 and 15 mg/kg), diclofenac (2.5 mg/kg), flurbiprofen (2 mg/kg), celecoxib (7.5 mg/kg), and rofecoxib (1 mg/kg), alone or combined on a rat model of arterial thrombosis. Drugs were given orally daily for 7 days, before insertion of an arterio‐venous shunt thrombosis system, left in place for 15 min. Main parameter was thrombus weight. Five to 12 rats were used per experiment, and 35 controls overall. Aspirin inhibited thrombus formation in a dose‐dependent manner. All NSAIDS given alone also inhibited thrombus formation to approximately the same level as aspirin 1 mg/kg/day. Ibuprofen, celecoxib and rofecoxib inhibited the effects of aspirin, but not diclofenac or flurbiprofen. The interactions with aspirin do not seem to affect all NSAIDs to equal levels. The clinical impact of this needs to be confirmed in adequately powered clinical trials or pharmaco‐epidemiological studies.

Fibrinogen synthesis by megakaryocyte rich human marrow cell concentrates

A rapid method is described for the production of a human marrow cell suspension highly enriched in megakaryocytes. These concentrates were incubated with radiolabelled amino-acids, and cell lysates were then analysed for fibrinogen synthesis. Neosynthesized proteins were detected by immunoprecipitation, immuno-affinity chromatography and electrophoresis. Fluorography of the electrophoresis gels showed three radioactive bands corresponding to the three chains of cold fibrinogen. Immunoblotting and autoradiography of bidimensional, nonreduced-reduced electrophoresis gels showed that these three proteins were joined by disulfide bonds in the cell. These results suggest that megakaryocytes synthesize fibrinogen, and imply that platelet fibrinogen is of megakaryocytic origin.

Prognostic value of age and bone marrow karyotype in 78 adults with acute myelogenous leukemia

Bone marrow karyotypes of 78 adult patients with acute myelogenous leukemia (AML) were studied at the time of diagnosis, with special reference to the Sakurai and Sandberg classification (NN, karyotype completely normal; AN, mixture of normal and abnormal metaphases in the karyotype; AA, totally abnormal karyotype). The results showed no difference in complete remission rate (CR) or in survival time between the NN and the AN groups, but highly significant differences between the AA group and the NN and AN groups (whether taken together or separately). When studying the relationship between age and survival time, we found that 59 years of age was a frontier between two homogeneous groups having quite different prognoses. The NN/AN/AA classification had a good prognostic value in patients under 59 years of age. In older patients, no correlation was found between the classification and complete remission rate or survival time.

Acute myeloid leukemia with marrow hypereosinophilia and chromosome 16 abnormality

This article reports six cases of acute nonlymphocytic leukemia (ANLL) and an abnormal chromosome #16. All had the same hematologic pattern at diagnosis, i.e., peripheral blood hyperleukocytosis with a high percentage of monocytes and blast cells. The bone marrow showed three different cell populations: (a) myeloblasts, (b) monocytes and promonocytes, and (c) abnormal eosinophils. In three cases, an ultrastructural study confirmed the cytologic data. In all six cases, the diagnosis was acute myelomonocytic leukemia with bone marrow eosinophilia (M4-Eo). All cases showed an abnormal chromosome #16 in the bone marrow cells; in four cases, well-banded chromosomes were obtained, showing a pericentric inversion inv(16)(p13;q22). One patient had a 4-year remission, and another is still in remission 14 months after diagnosis. Three patients relapsed 7, 9, and 20 months after diagnosis. The last patient died soon after diagnosis. Thus, we do not support the hypothesis that patients with M4-Eo ANLL and chromosome #16 abnormality have a favorable prognosis.

Megakaryoblastic Transformation of Primary Thrombocythemia

The progression of primary thrombocythemia (PT) into acute leukemia was diagnosed in a 64-year-old man who was previously treated by hydroxyurea without alkylating agents or radioactive phosphorus. Such an event has only been reported in very rare cases. The blast cells had a lymphocytic morphology but were identified as promegakaryoblasts by the ultrastructural demonstration of platelet peroxidase. These data suggest that a megakaryoblastic transformation could occur in PT as had previously been reported in chronic granulocytic leukemia.

Ultrastructural cytochemical prospective study of adult acute lymphoblastic leukemia: Detection of peroxidase activity in patients failing to respond to treatment

Ultrastructural cytochemical studies revealed peroxidase activity in five of 25 adult patients with apparent null lymphoblastic leukemia (ALL) in whom the peroxidase reaction studied with light microcopy was negative. None of these 5 patients responded to a chemotherapy regimen used for adult ALL. The importance of ultrastructural cytochemistry which allows the recognition of myeloblastic differentiation in undifferentiated blast cells is also demonstrated. The correct classification of such cases may be important for prognosis because they appear to be resistant to the chemotherapy used in treating ALL.