Triterpenoid saponins from Acacia victoriae (Bentham) decrease tumor cell proliferation and induce apoptosis.

This report describes the isolation and partial purification of novel triterpenoid saponins [Fraction 35 (F035)] and two pure biologically active derivatives (termed avicins D and G) from Acacia victoriae, an Australian desert tree of the Leguminosae family. F035 and the avicins markedly inhibited the growth of several tumor cell lines with minimum growth inhibition in human foreskin fibroblasts, mouse fibroblasts, and immortalized breast epithelial cells at similar concentrations. F035 and the avicins induced cell cycle (G1) arrest of the human MDA-MB-453 breast cancer cell line and apoptosis of the Jurkat (T-cell leukemia) and the MDA-MB-435 breast cancer cell line. The triterpenoid saponins also partially inhibited phosphatidylinositol 3-kinase activity in Jurkat T cells in a time-dependent manner and phosphorylation in the downstream protein Akt, whereas no affect was seen on the Ras/mitogen-activated protein kinase cascade. These observations as well as other work from our laboratory demonstrating mitochondrial perturbation, chemoprevention, and inhibition of nuclear factor kappaB suggest that triterpenoid saponins from A. victoriae have potential as novel anticancer agents. Recent work linking Akt signaling with glucose metabolism, stress resistance, and longevity suggests other potential applications of these compounds.

Implantable defibrillator event rates in patients with idiopathic dilated cardiomyopathy, nonsustained ventricular tachycardia on Holter and a left ventricular ejection fraction below 30%.

OBJECTIVES: This study investigated the incidence of appropriate implantable cardioverter defibrillator (ICD) interventions for ventricular tachycardia (VT) or ventricular fibrillation (VF) in patients with idiopathic dilated cardiomyopathy (IDC) and nonsustained VT in the presence of a left ventricular ejection fraction below 30%, versus in patients with syncope and patients with a history of VT or VF. BACKGROUND: To date, only limited information is available about the prophylactic use of ICDs in patients with IDC. METHODS: From January 1993 to July 2000, 101 patients with IDC underwent implantation of ICDs with electrogram storage capability at our institution. Patients were placed into one of three groups according to their clinical presentation: asymptomatic or mildly symptomatic nonsustained VT in the presence of a left ventricular ejection fraction < or = 30% (49 patients, prophylactic group), unexplained syncope or near syncope (26 patients, syncope group) and a history of sustained VT or VF (26 patients, VT/VF group). RESULTS: During 36 +/- 22 months follow-up, 18 of 49 patients (37%) in the prophylactic group received appropriate shocks for VT or VF, compared with 8 of 26 patients (31%) in the syncope group and with 9 of 26 patients (35%) of the VT/VF group. Multivariate Cox analysis of baseline clinical variables identified left ventricular ejection fraction, atrial fibrillation and a history of sustained VT or VF as predictors for appropriate ICD interventions during follow-up. CONCLUSIONS: Patients with IDC and prophylactic ICD implantation for nonsustained VT in the presence of a left ventricular ejection fraction < or = 30% had an incidence of appropriate ICD interventions similar to that of patients with a history of syncope or sustained VT or VF. These findings indicate that ICDs may have a role in not only secondary but also primary prevention of sudden death in IDC.

Screening and confirmation of hereditary spherocytosis in children using a CELL-DYN Sapphire haematology analyser.

INTRODUCTION: Multidimensional optical scatter of sphered erythrocytes can identify and enumerate hyperchromic erythrocytes, which might be used for hereditary spherocytosis (HS) screening. The flow cytometric eosin-5'-maleimide test (EMA) is highly sensitive and specific for HS as a confirmatory method. The aims of this study were to assess the utility of hyperchromic erythrocytes in HS screening and to evaluate the EMA test performed on CELL-DYN Sapphire analyser compared with the reference method. METHODS: Blood from 740 paediatric patients presenting at our institution was analysed in reticulocyte mode of the CELL-DYN Sapphire haematology analyser (Abbott Diagnostics) to obtain hyperchromic erythrocyte counts. The EMA test was performed using a flow cytometer as a reference, as well as CELL-DYN Sapphire as an investigational method. RESULTS: Hyperchromic erythrocytes were the highest in patients with HS (median 11.5%; range 5.1-29.2%). Patients with autoimmune haemolytic disease had significantly less hyperchromic erythrocytes (median 4.9%; range 0.0-18.3%). Hyperchromic erythrocytes showed a high area under the ROC curve: 0.972. At 4.9% cut-off, hyperchromic erythrocytes detected HS with 96.4% sensitivity and 99.1% specificity. The EMA test on CELL-DYN Sapphire correlated strongly with the reference test and had identical diagnostic power. Stability studies with blood from HS patients showed a significant decrease in hyperchromic erythrocytes after 6 h storage. CONCLUSIONS: Measurement of hyperchromic erythrocytes is highly sensitive and specific for detecting HS and can be used for rapid and inexpensive screening. If required, the EMA test can be performed on CELL-DYN Sapphire or a standard flow cytometer for confirmation of HS.

Differential diagnosis of microcytic anemia: the role of microcytic and hypochromic erythrocytes.

INTRODUCTION: Various indices derived from red blood cell (RBC) parameters have been described for distinguishing thalassemia and iron deficiency. We studied the microcytic to hypochromic RBC ratio as a discriminant index in microcytic anemia and compared it to traditional indices in a learning set and confirmed our findings in a validation set. METHODS: The learning set comprised samples from 371 patients with microcytic anemia mean cell volume (MCV < 80 fL), which were measured on a CELL-DYN Sapphire analyzer and various discriminant functions calculated. Optimal cutoff values were established using ROC analysis. These values were used in the validation set of 338 patients. RESULTS: In the learning set, a microcytic to hypochromic RBC ratio >6.4 was strongly indicative of thalassemia (area under the curve 0.948). Green-King and England-Fraser indices showed comparable area under the ROC curve. However, the microcytic to hypochromic ratio had the highest sensitivity (0.964). In the validation set, 91.1% of microcytic patients were correctly classified using the M/H ratio. CONCLUSIONS: Overall, the microcytic to hypochromic ratio as measured in CELL-DYN Sapphire performed equally well as the Green-King index in identifying thalassemia carriers, but with higher sensitivity, making it a quick and inexpensive screening tool.

Plasma prekallikrein activity in patients with total hip arthroplasty.

The behaviour of the contact system was studied in 40 patients with total hip arthroplasty, by measuring plasma prekallikrein, spontaneous kallikrein activity and factor XII. In the literature it had been shown that patients with complications from this operation had decreased prekallikrein and increased kallikrein activity (M. Nakahara. Acta orthop scand 1982; 53:591-6). In the present study, comprising patients with and without pain and proven loosening of the hip prosthesis, these findings could only partially be confirmed. Patients with a loosened prosthesis had significantly lower prekallikrein (mean 0.78 +/- 0.28 U/ml; p less than 0.01) than patients without problems, but no detectable kallikrein activity in plasma. Patients with pain but no loosening had normal prekallikrein (1.04 +/- 0.26 U/ml) and also no demonstrable kallikrein activity. Factor XII was normal in all patient groups. It is concluded that decreased prekallikrein is limited to patients with a loosened hip prosthesis, with or without pain.

Comparison of reagents for determining the activated partial thromboplastin time.

Six commercially available reagents for the determination of the activated partial thromboplastin time have been evaluated and compared with respect to their sensitivity to the coagulation factors VIII, IX and XI and to their response to heparin. Some variation was observed among the reagents regarding their sensitivity to factor XI and even greater differences were obtained with factors VIII and IX. It was also clear that none of the reagents was sensitive to the same extent to the factors tested. The sensitivity to heparin shows considerable variation, in terms of time as well as mode of response to increasing heparin levels. In four reagents this response is linear, it is logarithmic in one and the remaining one is yet again different. It seems unlikely that any standardization of the APTT determination is at present possible with the reagents studied.

Hereditary increase of plasma histidine-rich glycoprotein associated with abnormal heparin binding (HRG Eindhoven).

Plasma histidine-rich glycoprotein (HRG) was found to be persistently increased in a patient with a history of recurrent arterial thromboembolic events. The mean concentration was 270% of normal pooled plasma. Increased HRG was found in eight of the 17 relatives studied, but none of them has experienced thrombo-embolism yet. Apparently, increased HRG was hereditary with autosomal dominant inheritance. A significant correlation was found between the increased plasma concentration of the protein and the age of the subjects (P < 0.02), whereas no such relation is present in a normal population. The plasma HRG of the proposita and 9 of her family members displayed abnormal binding to heparin, as assessed in a crossed affinity immuno-electrophoresis system: the usual increase in mobility after binding to heparin was absent. The binding of this variant HRG to plasminogen was normal. This case represents the first abnormal HRG variant reported and it is proposed to designate it: HRG Eindhoven.

Identification and genetic analysis of a common molecular variant of histidine-rich glycoprotein with a difference of 2kD in apparent molecular weight.

Two forms of histidine-rich glycoprotein (HRG) were detected on SDS-PAGE by silver staining and immunoblotting after isolation of the protein from pooled plasma using immuno-affinity chromatography followed by chromatography with heparin-Sepharose. Both forms were single-chain molecules and the apparent molecular weights of form 1 and form 2 were 77 kD and 75 kD respectively. Mendelian inheritance of both HRG forms was observed in four families with 24 informative meioses, strongly suggesting that the two forms are encoded by different alleles. The frequency of form 1 and form 2 in a group of 36 individuals was 0.35 and 0.65 respectively. The difference between the two molecular variants was studied by direct sequence analysis of amplified exons of the HRG gene from 6 individuals who were homozygous either for form 1 or form 2. Five amino acid polymorphisms in three different exons were observed: Ile/Thr in exon4; Pro/Ser in exon 5; His/Arg, Arg/Cys and Asn/Ile in exon 7. Analysis of these polymorphisms in 20 volunteers showed that only the Pro/Ser polymorphism at position 186 in exon 5 was coupled to the form of the HRG protein. Ser was found in form 1 and Pro in form 2. The presence of Ser at position 186 introduces a consensus sequence for a N-glycosylation site (Asn-X-Ser/Thr). By removing N-linked sugars with N-glycanase, it could be demonstrated that the difference between the two forms of HRG is caused by an extra carbohydrate group at Asn 184 in form 1.

Blood viscosity during thrombolytic therapy with anistreplase in acute myocardial infarction.

It has been postulated that a reduction in blood viscosity due to degradation of plasma fibrinogen may be of benefit to patients with acute myocardial infarction (AMI), who are treated with thrombolytic agents. The aims of this study were to investigate the time course of rheologic parameters, to identify the principal factors determining blood viscosity, and to find possible correlations between viscosity and cardiac function during thrombolytic therapy with anistreplase. Therefore, the viscosity of whole blood and plasma and the hematocrit were measured before and at 10 time points after thrombolysis in 10 patients with AMI. In addition, plasma fibrinogen and fibrin(ogen) degradation products were determined. Immediately after the start of thrombolysis, the viscosity of blood (both at high and low shear rate) and plasma decreased significantly and continued to do so for 24 hours. The mean hematocrit also decreased markedly, and even after correction for these hematocrit changes, the reduction in blood viscosity remained significant: it decreased to 72% of pretreatment (measured at low shear rate), whereas the high-shear viscosity decreased to 95% of baseline. The viscosity of plasma significantly decreased from 1.39 +/- 0.13 mPa.s (mean +/- SD) before thrombolysis to 1.22 +/- 0.08 mPa.s after 2 hours. There was a rapid, nearly complete depletion in fibrinogen, followed by a striking rebound after the second day. The decrease in blood viscosity lasted for 2 days after anistreplase and was mainly accounted for by the reduction in hematocrit. The contribution of fibrinogen to blood viscosity appeared less prominent. Despite these rheologic changes, no improvement in cardiac output was noticed in the patients.

Systemic effects of anisoylated plasminogen streptokinase activator complex and streptokinase therapy in acute myocardial infarction. Coagulation aspects of the Dutch Invasive Reperfusion Study.

The systemic effects of intravenous anisoylated plasminogen streptokinase activator complex (APSAC; 30U) and intracoronary streptokinase (250,000U) were compared in 54 patients with acute myocardial infarction. In 3 patients, no signs of a systemic lytic state were observed. In all other patients, significant reductions of coagulation and fibrinolytic factors occurred: fibrinogen levels decreased by 86% in the APSAC group and 81% in the streptokinase group; for plasminogen the decreases were 68 and 66%, and for alpha 2-antiplasmin activity greater than 95 and 94%, respectively. Fibrin(ogen) degradation products were increased 68- and 38-fold, respectively. Although there was a trend for the lytic state to be more profound in the APSAC-treated patients, there was no difference between treatment groups with regard to bleeding complications or therapeutic efficacy, the latter being 79 and 73%, respectively, for APSAC and streptokinase. Total fibrinolytic activity, measured as euglobulin clot lysis time, was sustained for longer in the APSAC group, which may explain the low reocclusion rate in this group in comparison with the streptokinase group.

VO(2) kinetics of mild exercise are altered by RER.

We propose that variations in fat and carbohydrate (CHO) oxidation by working muscle alter O(2) uptake (VO(2)) kinetics. This hypothesis provides two predictions: 1) the kinetics should comprise two exponential components, one fast and the other slow, and 2) their contribution should change with variations in fat and CHO oxidation, as predicted by steady-state respiratory exchange ratio (RER). The purpose of this study was to test these predictions by evaluating the VO(2) kinetic model: VO(2)(t) = alpha(R) + alpha(F)(1 - exp[(t - TD)/-tau(F)]) + alpha(C)(1 - exp[(t - TD)/-tau(C)]) for short-term, mild leg cycling in 38 women and 44 men, where VO(2)(t) describes the time course, alpha(R) is resting VO(2), t is time after onset of exercise, TD is time delay, alpha(F) and tau(F) are asymptote and time constant, respectively, for the fast (fat) oxidative term, and alpha(C) and tau(C) are the corresponding parameters for the slow (CHO) oxidative term. We found that 1) this biexponential model accurately described the VO(2) kinetics over a wide range of RERs, 2) the contribution of the fast (alpha(F), fat) component was inversely related to RER, whereas the slow (alpha(C), CHO) component was positively related to RER, and 3) this assignment of the fast and slow terms accurately predicted steady-state respiratory quotient and CO(2) output. Therefore, the kinetic model can quantify the dynamics of fat and CHO oxidation over the first 5-10 min of mild exercise in young adult men and women.

Systemic blood activation with open and closed venous reservoirs.

In 20 patients undergoing coronary artery bypass grafting, we studied prospectively systemic blood activation, blood loss, and the need for donor blood when using an extracorporeal circuit equipped at random with one of two different venous reservoirs. In 10 patients we used an open venous reservoir system (ORS) consisting of a hard shell venous reservoir with an integral cardiotomy filter, and in 10 patients we used a closed reservoir system consisting of a collapsible venous reservoir and separate cardiotomy reservoir. Concentrations of complement 3a, elastase, thromboxane B2, and fibrin degradation products showed a biphasic course, especially in ORS patients. During bypass, we observed a first peak of levels of complement 3a, thromboxane B2, fibrin degradation products, and elastase, which was higher in ORS patients than in patients with the closed system, because their blood continuously contacted the foreign materials of the filter and air in the open reservoir, which was avoided in the closed reservoir. Intensive blood-foreign material contact also caused the highest (p < 0.05) hemolysis in ORS patients. The larger amount of hemolytic products in ORS patients theoretically resulted in a temporary decrease in capacity of their Kupffer cells to clear endotoxin released after aortic declamping. This theory might explain the significantly (p < 0.01) higher second peak of activated products after declamping that was observed in ORS patients. Due to increased blood activation, the largest (p < 0.001) amount of shed blood loss, greatest (p < 0.05) need for colloid-crystalloid infusion, and largest (not significant) need for donor blood were found in ORS patients (0.8 +/- 0.4 versus 0.2 +/- 0.2 units of packed cells).(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric analysis of consecutive lymph node samples from patients with Hodgkin's disease: reproducible within one biopsy?

In Hodgkin's disease DNA aneuploidy is not a prognostic factor. However, the prognostic significance of DNA content in Hodgkin's disease may be missed by either intratumor DNA heterogeneity or DNA analysis of limited samples. For flow cytometry usually one section of 40-60 microns is used for the analysis. In breast cancer this proved to be insufficient. In Hodgkin's disease no data are available. Therefore, we examined if analysis of more sections does increase the yield of aneuploidy. Archival, formalin-fixed, parafin embedded tissues were used. From 13 patients four sections of 50 microns could be analysed for DNA content. In 12 of 13 patients the results were consistent in all four sections of one patient case; seven diploid, four aneuploid and one multiploid. In one case ploidy status changed: two sections were diploid and two were aneuploid. The DNA-index of the aneuploid samples ranged from 0.75 to 1.38 and varied from 0.02 to 0.14 within one case. The S-phase fraction remained constant within all evaluable cases (sd: 0.5-1.5%), except for one (sd: 4.7%). In conclusion, in Hodgkin's disease the ploidy status of the first section can be regarded to represent the whole tissue sample. Therefore, the absence of prognostic value of ploidy status is not explained by sampling errors in tissues analysed.

DNA aneuploidy in Hodgkin's disease: a multiparameter flow cytometric analysis.

Paraffin-embedded lymph nodes from patients with Hodgkin's disease were examined for flow cytometric DNA content. In order to increase the sensitivity of the assay we tried to enrich for the neoplastic cells by bivariate analysis using a polyclonal anti-nucleolar antibody (AN-AB) and the forward scatter (FSC). DNA aneuploidy was found to be present in 67 of all 137 cases (49%), in 24 cases only demonstrable by dual-parameter analysis. The DNA index varied from 0.69 to 1.89 with a total of 22 hypo-diploid cases. The number of aneuploid nuclei exceeded the expected frequency of Reed-Sternberg (RS) and Hodgkin (H) cells in most of the analysed specimens. In conclusion, flow cytometry in Hodgkin's disease appears to give useful information regarding the ploidy status and evidence has been provided that the malignant cell population in Hodgkin's disease is not limited to the classical RS/H cells.

Interactions between thrombolytic agents and platelets: effects of plasmin on platelet glycoproteins Ib and IIb/IIIa.

The mechanisms by which thrombolytic agents affect platelet function are not yet elucidated. The aim of the present study was to investigate the effects of plasmin, generated by thrombolytic agents in plasma, on platelet glycoproteins (GP) Ib and IIb/IIIa. Platelet-rich plasma was incubated with pharmacological amounts of streptokinase, anistreplase and tissue-type plasminogen activator and the platelet surface GP's were investigated with a panel of monoclonal antibodies using flow cytometry. As assessed from the mean fluorescence intensity of incubated and control platelets, no significant changes in the binding of antibodies to GP Ib and GP IIb/IIIa were found. The functional integrity of these glycoproteins was severely impaired by treatment with the thrombolytic agents, as shown by significant inhibition of ADP- and ristocetin-induced platelet aggregation. Experiments with purified plasmin and washed platelets indicated significant degradation of GP IIb/IIIa and upregulation of GP Ib, which is in agreement with previous findings. In addition, platelet activation by plasmin was shown using two monoclonal antibodies to activation-specific antigens. We conclude that degradation of platelet GP's by plasmin offers no likely explanation for the defect in platelet function, which is induced by thrombolytic agents in platelet-rich plasma.

Systemic effects of BRL 26921 during thrombolytic treatment of acute myocardial infarction.

The effects of BRL 26921, an active-site acylated streptokinase-plasminogen complex, on the fibrinolytic and coagulation systems were studied in 13 patients treated for acute myocardial infarction with a short intravenous infusion of 30 mg of the new drug. In 12/13 patients (92%) significant changes were observed in the concentrations of fibrinogen, fibrin(ogen) degradation products, plasminogen, alpha 2-antiplasmin and F VIII:C; all changes indicating a substantial degree of systemic fibrinolysis. In only one patient were no signs of systemic activation of the fibrinolytic system observed. The therapeutic efficacy of the drug was 85% in this study.

Antibacterial and antifungal flavanones from Eysenhardtia texana.

An activity-guided fractionation of a methanol-dichloromethane extract obtained from the aerial parts of Eysenhardtia texana led to the isolation of two novel antibacterial and antifungal flavanones together with a known flavanone. Their structures were established as 4',5,7-trihydroxy-8-methyl-6-(3-methyl-[2-butenyl])-(2S)-flavanone, 4',5,7-trihydroxy-6-methyl-8-(3-methyl-[2-butenyl])-(2S)-flavanone and 4',5-dihydroxy-7-methoxy-6-(3-methyl-[2-butenyl])-(2S)-flavanone on the basis of their UV, 1D and 2D-NMR spectra.

Application of isoelectric focusing in alpha1-antitrypsin phenotyping.

alpha1-Antitrypsin, the major protease inhibitor in human serum, occurs in a considerable number of variant forms, some of which are associated with lung and liver diseases. The identification of these genetic variants, generally called Pi-types, by means of isoelectric focusing is described, as well as investigations concerning the practical application of isoelectric focusing as a routine procedure for typing the variants of alpha1-antitrypsin. Finally, isoelectric focusing is compared with the most widely used Pi-phenotyping technique, namely acid starch-gel electrophoresis followed by immunoelectrophoresis in antibody-containing agarose gel.

Automated determination of the antitrypsin activity of serum.

An automated determination of the serum antitrypsin activity is described in which all reaction parameters have been optimized. The determination is carried out by measuring the difference between the activity of a standard amount of trypsin with and without addition of serum. Trypsin activity is measured using N-alpha-benzoyl-L-arginine-p-nitroanilide as a substrate. The assay is performed by means of an automatic enzyme reaction rate analyser the Vitatron AKES. The antitrypsin activity is expressed in Inhibitor Units making standardization of the trypsin preparation superfluous. Normal values of the antitrypsin activity for some of the most frequently occurring genetic variants of alpha1-antitrypsin are given.

Radioenzymatic assay of plasma adrenaline and noradrenaline: evidence for a catechol-O-methyltransferase (COMT) inhibiting factor associated with essential hypertension.

During the evaluation of a modified radioenzymatic determination of plasma adrenaline and noradrenaline, it has been found that there exists a highly significant (p less than 0.001) negative correlation between the amount of radioactivity incorporated in the internal standard added to plasma and the period during which the plasma had been stored at -25 degrees C, but only in plasma from patients with essential hypertension. Plasma from normotensive persons exhibits a complete lack of correlation between these factors. The consequences of the hypertension-associated COMT-inhibiting factor for the assays' specifications are discussed and data are presented for comparison with a recently described uremia-associated COMT-inhibitor (Demassieux et al, Clin Chim Acta 115, 377--391; 1981).