Detection of cytarabine resistance in patients with acute myelogenous leukemia using flow cytometry

Cytarabine (Ara-C) is currently used in the treatment of adult acute myeloid leukemia (AML). To predict the results of induction chemotherapy, it could be useful to detect leukemic cells that are resistant to Ara-C in patients with AML. Using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM), we have developed a cell resistance index to Ara-C (RI). The technique has been applied to 121 bone marrow (BM) samples from patients with de novo AML treated by a regimen containing Ara-C and daunorubicin (DNR). Ninety-seven patients achieved a complete remission (CR), and 24 patients did not and were considered drug-resistant (DR). The BM cells collected at diagnosis were cultured for 48 hours and underwent BrdUrd/DNA analysis. Among 25 patients with no or very low proliferative activity (<3% of cells in S-phase), the proportion of DR patients (nine of 25) was significantly higher than in a second group of 96 patients with detectable proliferative activity (15 of 96) (P < .025). Within this second group, there was a first group of nine patients with high RI values, which included only DR patients; a second group of 63 patients with low RI values, which included 62 CR patients; and a third group of 24 patients with intermediate RI values, which included 19 CR and five DR patients. In view of this series, our results show that it is possible to detect a majority of DR patients treated by Ara-C.     

Presence of Epstein-Barr virus in extranodal T-cell lymphomas: differences in relation to site

T-cell lymphomas with similar morphology but with different sites of origin have a different clinical behavior. The theoretical explanation for this finding originates from the hypothesis that non-Hodgkin's lymphomas (NHLs) are neoplastic equivalents of immunological reactions involving tissue-restricted lymphocytes. This hypothesis also implies that T-NHLs originating from different sites differ in their genesis, and thus may differ in oncogen expression, expression of adhesion molecules, or presence of certain DNA/RNA viral sequences. Therefore, we have investigated in T-cell lymphomas with similar morphology originating from different sites, ie, nose (n = 5; all pleomorphic small- or medium- and large-cell T-cell lymphomas [PTL]), skin (PTL, n = 6; anaplastic large-cell [ALCL], n = 11), gut (PTL, n = 8; ALCL, n = 4), and lung (PTL, n = 6), the presence of Epstein-Barr virus (EBV) at the DNA, RNA (EBER 1 and EBER 2), and protein level (LMP-1). A double- staining technique was used to detect EBER 1/2, LMP-1, and differentiation markers at the single-cell level. High numbers of EBER 1/2-positive tumor cells (> 100 per medium power field [mpf]) were found in five of five nasal T-cell lymphomas, none of 17 primary cutaneous T-cell lymphomas, one of 12 gastrointestinal T-cell lymphomas (ALCL), and two of six pulmonary T-cell lymphomas. These lymphomas are therefore called EBV-associated lymphomas. In contrast to our earlier findings in lymph nodes, no extranodal lymphomas were found, with only a few EBV-positive tumor cells. Five gastrointestinal cases positive for EBV by polymerase chain reaction (PCR) showed that EBER 1/2 was only found in sporadic nonneoplastic, ie, reactive lymphocytes. Angiocentricity was present in 18 PTL and one ALCL, but not associated with the presence of EBV. These results indicate that the presence of EBV in extranodal T-cell lymphomas is site-restricted and argues for a different pathogenesis of T-cell lymphomas with similar morphology but originating from different sites. The presence of EBV in most tumor cells in these EBV-associated lymphomas suggests that when present, EBV might be important in the pathogenesis of these lymphomas.     

Intracranial metastasis from prostatic adenocarcinoma simulating a meningioma

We report an unusual case of extra‐axial metastatic adenocarcinoma of the prostate that closely simulated a frontal, parasagittal, dural‐based meningioma. Such tumours, which satisfy several criteria for a diagnosis of meningioma, but which have proved instead to be metastatic adenocarcinoma of the prostate, form the focus of our report.     

Procaine in Cardioplegia: Does It Affect the Endothelial Function?

Objective: Cardioplegic solutions are widely used in cardiac surgery and hyperkelemia in cardioplegia has been demonstrated to impair the endothelium‐derived hyperpolarizing factor (EDHF)‐mediated endothelial function. The present study examined the effect of procaine in St. Thomas Hospital Cardioplegia on the EDHF‐mediated response in porcine coronary arteries. Methods: Isometric force study: Porcine coronary micro‐arteries were studied in a myograph. Two rings taken from the same artery (diameter 200–450 μm, n = 8 ) were incubated with Kreb's solution as control or Kreb's solution plus procaine (1 mM) at 37 °C for 1 h, respectively. The EDHF‐mediated relaxation was induced by bradykinin (BK, ?10 ～?6.5 log M) in the presence of indomethacin (Indo, 7 μM), NG‐nitro‐L‐arginine (L‐NNA, 300 μM), and hemoglobin (HbO, 20 μM) after U46619‐precontraction (?8 log M). Electrophysiological study: The membrane potential of a single smooth muscle cell in coronary arteries was measured by a microelectrode after superfusion with Kreb's solution or Kreb's containing procaine (1 mM) for 1  h. Results: Procaine had little effect on the resting force of porcine coronary micro‐arteries (0.94 ± 0.74 mN vs. 0.67 ± 0.23 mN in control, P > 0.05 ) and did not alter the U46619‐induced precontraction (10.7 ± 1.7 mN vs. 12.0 ± 1.7 mN, P > 0.05 ). The BK‐induced, EDHF‐mediated relaxation was increased by the treatment with procaine with the EC50 shifted leftward (97.3 ± 0.6% vs. 83.0 ± 5.1% at ?7 log M and 99.4 ± 0.6% vs. 96.7 ± 1.6% at ?6.5 log M, P < 0.05 ; EC50: ?8.57 ± 0.24 vs. ?7.92 ± 0.23 log M, P < 0.05 ). Procaine slightly depolarized the smooth muscle cell (?56.3 ± 1.0 vs. ?59.3 ± 0.7 mV, P > 0.05 ) and decreased the BK‐induced hyperpolarization from ?70.3 ± 0.4 mV to ?68.0 ± 0.8 mV (?7 log M, P < 0.05) and from ?72.3 ± 0.7 mV to ?68.8 ± 0.8 mV (?6.5 log M, P < 0.01) . Conclusions: In the coronary arteries, procaine has depolarizing effect but it enhances the EDHF‐mediated relaxation. Therefore, addition of procaine in cardioplegia may preserve the EDHF‐mediated endothelial function.     

The 47-kD fragment of talin is a substrate for protein kinase P

This laboratory has been characterizing protein serine/threonine kinase reactions of hematopoietic tissues, whose most distinguishing characteristics in vitro are stimulation with vesicular phosphatidyl glycerol, and the ability to function using Mn2+ as the sole divalent cation. The major protein substrates are a 73-kD protein and a protein migrating near ovalbumin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 47-kD protein was partially purified from cells harvested by leukapheresis from a patient with acute myelogenous leukemia, using ammonium sulfate precipitation and ion exchange chromatography. This partially purified ion-exchange fraction contained an endogenous kinase activity with characteristics similar to those we previously described of protein kinase P (protein kinase, phospholipid- stimulable: PK-P), but not typical of any form of protein kinase C (PK- C). With longer phosphorylation, the 47-kD band showed increasingly lower mobility demonstrable both by Coomassie blue staining and autoradiography, suggesting both that it was multiply phosphorylated, and that the excisable band was pure. The protein was thus eluted from preparative gel slices and digested with endoproteinase lys C. Sequence data from the fragments identified the protein as the 47-kD calpain fragment of talin, a protein found in focal adhesion plaques and some cell-cell contacts. PK-C phosphorylated the 47-kD protein, as has been reported previously, and phosphopeptide mapping disclosed a similar pattern of phosphorylation using either PK-C or the endogenous activity. The 47-kD protein labeled with the endogenous kinase contained predominantly phosphoserine, with some phosphothreonine and a trace of phosphotyrosine. Intact, purified talin was also phosphorylated by PK-P in a phospholipid-stimulable manner, but at 1/20 the rate of the 47-kD fragment.     

NON-HODGKINS LYMPHOMA PRESENTING WITH EVIDENCE OF RIGHT VENTRICULAR OUTFLOW TRACT OBSTRUCTION

SUMMARY Anterior mediastinal tumours have been reported that initially presented with signs suggestive of cardiac disease. The widespread availability of two-dimensional echocardiography has demonstrated that, in the majority of cases, right ventricular compression is the major cardiac complication of such masses. We report two cases of mediastinal lymphoma that presented with chest pain and signs of right ventricular outflow obstruction.     

Large numbers of primitive stem cells are active simultaneously in aggregated embryo chimeric mice

The possibility has been repeatedly raised that erythropoiesis results from clonal succession--the differentiation of one or a very small number of the most primitive stem cells that are sequentially activated to proliferate forming clones of differentiated cells and then eventually decline, to be replaced by new stem cell clones. We studied this possibility in chimeric mice made by combining embryos from two different strains so that they would have two distinct stem cell populations, each of which produces a different hemoglobin type (d and s). These were compared with F1 hybrids in which every stem cell produces both types. We measured the percentage of type d in seven to ten serial samples of circulating reticulocytes taken at three- to seven-day intervals and found that the variability in percent of this hemoglobin was only slightly higher in the chimeric mice than in F1 controls; SD ranged from 2.7% to 5.5% in the chimeric mice and from 3.4% to 3.9% in the controls. Using the binomial formula, the numbers of new clones formed during the reticulocyte life span, approximately three days, ranged from 33 to 118 in the individual chimeric mice. However, these numbers are underestimates because estimated numbers of clones depend inversely on variabilities, and the calculations did not exclude the contribution of experimental error to the overall variability. Total percentages of type d hemoglobin were also measured in seven to nine successive serial samples at 60- to 136-day intervals. These gave mean values similar to measures of newly synthesized hemoglobin in the same mice, but SD were larger, ranging from 5.3% to 8.4%. This reflects experimental error, both because of excess day-to- day variability found in this type of measurement and because there could not be fewer primitive stem cells activated to form clones of erythrocytes during the 45-day erythrocyte life span than during the three-day life span of reticulocytes. Since most and maybe all of the variation between successive samples in the same chimeric mouse appear to result from experimental error, many or even all of the primitive stem cells may simultaneously contribute to erythropoiesis.     

Artificial microvascular graft thrombosis: the consequences of platelet membrane glycoprotein Ib inhibition and thrombin inhibition

Early thrombosis of artificial microvascular grafts (AMG, grafts < or = 2 mm internal diameter) prevents their reliable clinical use. The present studies were undertaken to examine the effect of hirudin, a thrombin-specific inhibitor, and of the F(ab')2 fragment of PG-1, a monoclonal antibody (MoAb) directed against guinea pig platelet membrane glycoprotein Ib (GPIb), on AMG patency in an animal model. One- centimeter long segments of expanded polytetrafluoroethylene (ePTFE), 0.88 mm internal diameter, were serially implanted as interposition grafts in the guinea pig femoral arterial systems bilaterally. A control group was treated with 0.5 mL saline intravenously (IV) 30 minutes before limb 1 and limb 2 graft implantation. Three experimental groups were treated with 0.5 mL saline IV before limb 1 graft implantation as an animal control and with either 0.5 mL saline containing 1.25 mg/kg IV PG-1 F(ab')2, (which inhibits ristocetin- induced platelet agglutination and von Willebrand factor binding), hirudin 1 mg/kg IV, or a combination of both agents before limb 2 graft implantation. GPIb inhibition, thrombin inhibition, and the combination resulted in a significant prolongation of AMG patency (P < .005). Whereas thrombin inhibition with hirudin prolonged AMG patency similar to that observed with GPIb inhibition, the combination of GPIb and thrombin inhibition provided the overall longest prolongation of AMG patency. These results indicate that both platelet membrane GPIb and thrombin play a role in AMG thrombosis.     

Do centimetres matter? Self‐reported versus estimated height measurements in parents

Aim: An impressive discrepancy between reported and measured parental height is often observed. The aims of this study were: (a) to assess whether there is a significant difference between the reported and measured parental height; (b) to focus on the reported and, thereafter, measured height of the partner; (c) to analyse its impact on the calculated target height range. Methods/Results: A total of 1542 individual parents were enrolled. The parents were subdivided into three groups: normal height (3–97th Centile), short (<3%) and tall (>97%) stature. Overall, compared with men, women were far better in estimating their own height (p < 0.001). Where both partners were of normal, short or tall stature, the estimated heights of their partner were quite accurate. Women of normal stature underestimated the short partner and overestimated the tall partner, whereas male partners of normal stature overestimated both their short as well as tall partners. Women of tall stature estimated the heights of their short partners correctly, whereas heights of normal statured men were underestimated. On the other hand, tall men overestimated the heights of their female partners who are of normal and short stature. Furthermore, women of short stature estimated the partners of normal stature adequately, and the heights of their tall partners were overestimated. Interestingly, the short men significantly underestimated the normal, but overestimated tall female partners. Conclusion: Only measured heights should be used to perform accurate evaluations of height, particularly when diagnostic tests or treatment interventions are contemplated. For clinical trails, we suggest that only quality measured parental heights are acceptable, as the errors incurred in estimates may enhance/conceal true treatment effects.