Down-regulation of bcl-2 in AML blasts by all-trans retinoic acid and its relationship to CD34 antigen expression

High levels of expression of the bcl-2 oncoprotein in acute myeloblastic leukaemia (AML) cells have been associated with low complete remission rates and poor survival. The sensitivity of AML blasts to drugs such as Ara-C can be increased by the down-regulation of bcl-2 expression by antisense oligonucleotides. All-trans retinoic acid (ATRA) has been reported to increase the sensitivity of AML cell lines to Ara-C and to induce differentiation in the HL60 promyelocytic cell line, with both effects being accompanied by a decrease in bcl-2 expression. Using flow cytometry and a monoclonal antibody to bcl-2, we have investigated the effects of ATRA (1 μM ) on bcl-2 expression in the blast cells of 25 AML patients and the K562 cell line after incubation for 72 or 24 h, respectively. Using Kolmogorov-Smirnov statistical analysis where a D value of > 0.12 was statistically significant, we found that in 8/25 AML samples and the K562 cells there was a significant decrease in bcl-2 protein expression after incubation with ATRA (D value range 0.14–0.44). The mean peak fluorescence (MPF) values for the bcl-2 levels of the ATRA responders (n = 8) was reduced to 35.5 ± 6.9 following incubation with ATRA compared to 47.6 ± 8.2 (mean ± SEM) for control samples incubated in the absence of ATRA (P = 0.014). There was no significant difference between the baseline bcl-2 molecules of equivalent soluble fluorochrome (MESF) levels in the ATRA responders (48.9 ± 5.7, n = 8) and the non-responders (41.3 ± 3.9, n = 17) (mean ± SEM) (P = 0.28). The down- regulation of bcl-2 expression by ATRA was particularly associated with CD34-negative AML and of the eight AML patients’ cells that responded to ATRA by down-regulating bcl-2, seven were CD34 negative (P<0.05). Our data suggest that the addition of ATRA to combination chemotherapy would increase the chemosensitivity of some patients with AML, particularly CD34-negative AML, due to down-regulation of bcl-2 expression.     

The putative role of arylsulfatase in interleukin-2- mediated cytotoxicity and interleukin-7-mediated bactericidal activity of natural killer cells

 We have examined the cytolytic and bactericidal activity of resting and cytokine-stimulated natural-killer (NK) cells against K562 and Daudi cell lines and Escherichia coli, respectively. Unstimulated NK cells showed considerable cytolytic activity against K562 (64±4%) and relatively low activity against the Daudi cell lines (22±9%). Pretreatment of NK cells with the arylsulfatase (AS) type-II-specific inhibitor NaH₂PO₄ reduced cytotoxicity towards K562 and Daudi 1.3- and 2.9-fold (p<0.05;n=12), respectively, indicating that AS participates in NK-mediated cytotoxicity. Interleukin-2 (IL-2) (200 units/ml) caused a 1.3- and 3.5-fold (p<0.5;n=12) enhancement of NK cytotoxic activity against K562 and Daudi, respectively. Pretreatment of these cells with the AS type-II-specific inhibitor NaH₂PO₄ reduced cytotoxicity 1.1-fold towards K562 (p>0.05;n=12) and 1.2-fold towards Daudi (p>0.05;n=12) indicating that AS does not participate in IL-2-mediated NK cytolytic activity against these cell lines. IL-7 (3 units/ml) did not cause any enhancement of NK cytolytic activity. Unstimulated NK cells showed considerable bactericidal activity against E. coli (23±4%). Incubation of resting NK cells with NaH₂PO₄ reduced the bactericidal effect only by 1.09-fold (p>0.05;n=12), indicating that AS does not mediate this effect. IL-2 (200 units/ml) and IL-7 (3 units/ml) enhanced the bactericidal activity 1.5- and 2.2-fold, respectively (p<0.05;n=12). This effect was not influenced by incubation of IL-2-stimulated cells with NaH₂PO₄, indicating that AS does not participate in the IL-2-mediated NK bactericidal effect. IL-2 seems to exert its stimulatory effect upon NK-mediated bactericidal activity by a different, non-AS-dependent mechanism. However, incubation of IL-7-stimulated NK cells with NaH₂PO₄ reduced the NK bactericidal effect by 1.2-fold (p<0.05;n=12), indicating that AS may have a role in this reaction. These data can be further confirmed by detection of AS through degranulation of NK cells, showing that IL-2 induced only mild degranulation of resting and f-MLP-stimulated NK cells (26±1% vs 22±2% and 31±2% vs 29±2, respectively) (p>0.05;n=8). In contrast, IL-7 showed significant enhancement of AS release in resting or f-MLP-induced NK cells (36±3% vs 22±2% and 49±3% vs 29±2%, respectively) (p<0.05;n=8). Received: 13 August 1996 / Accepted: 15 November 1996     

Type and position of promoter elements in retroviral vectors have substantial effects on the expression level of an enhanced green fluorescent protein reporter gene

Purpose: Although gene transfer with retroviral vectors has already been applied to patients as part of clinical protocols, low expression of transgenes in target cells still remains a problem. Therefore, we compared various retroviral vectors using different promoters and backbones for expression of the enhanced green fluorescent protein (EGFP) reporter gene in fibroblasts and CD34⁺ cells. Methods: The N2A retroviral vector was used to test expression from the herpes simplex virus thymidine kinase promoter (vector N2A-TK-EGFP), a human phosphoglycerate kinase promoter (vector N2A-PGK-EGFP), and the SV40 promoter (vector N2A-SV-EGFP). Additional constructs used the spleen focus-forming virus (SFFV) long terminal repeat (LTR) as promoter and expressed EGFP alone (vector SFβ1-EGFP) or EGFP and a downstream (vector SFβ1-EGFP-IRES) or upstream (vector SFβ1-IRES-EGFP) internal ribosomal entry site. Results: For NIH 3T3 cells the fluorescence-activated cell sorting analysis revealed that the most active internal promoter was the SV40 promoter in the vector N2A-SV-EGFP (mean fluorescence intensity, MFI, 66.7 ± 0.4), followed by N2A-PGK-EGFP (26.3 ± 1.8 MFI), and N2A-TK-EGFP (4.8 ± 0.1 MFI). Expression from the SFβ1-EGFP vector (82.6 ± 6.7 MFI) and the SFβ1-EGFP-IRES vector (102.8 ± 6.2 MFI) was higher than from SFβ1-IRES-EGFP vector (15.5 ± 1.8 MFI). In human CD34-positive cells, the EGFP expression from all vectors was considerably lower than in fibroblasts with the SFβ1-EGFP vector still being four- to fivefold more active than the internal promoters tested. Conclusion: The SFFV LTR seems to allow a high expression of transgenes, as long as the transgene is not expressed downstream of an internal ribosomal entry site. Internal promoters may be useful for targeted gene expression in specific cell types, but the reduced level of expression from some internal promoters has to be taken into consideration. Received: 7 January 2000 / Accepted: 1 February 2000     

Abnormal surface markers expression on bone marrow CD34<Superscript>+</Superscript> cells and correlation with disease activity in patients with systemic lupus erythematosus

Defects of hematopoietic stem cells (HSCs) have been suggested to contribute to the development of systemic lupus erythematosus (SLE). The aim of this study was to investigate the phenotypic characteristics of bone marrow (BM) CD34⁺ cells in patients with SLE and its relationship with SLE disease activity. Ten SLE patients and 10 healthy subjects were recruited and their BM CD34⁺ cells were analyzed by flow cytometric analysis with CD45/SSC gating for the expression of CD90, CD95, CD117, CD123, CD164, CD166, FAS-L, and HLA-DR. The percentage of BM CD34⁺ cells was significantly decreased in active SLE patients (1.48 ± 0.41%, n = 7) compared to the healthy controls (2.31 ± 0.75%, n = 10, p < 0.01), but no significant difference was found between the inactive patients (2.04 ± 0.44%, n = 3) and the controls. The expression of CD95, CD123, and CD166 on BM CD34⁺ cells were significantly increased in SLE patients (48.31 ± 10.59%, 44.9 ± 21.5%, 30.9 ± 19.54%, respectively, n = 10) when compared with the control subjects (24.33 ± 11.1%, 19.5 ± 4.4%, 10.7 ± 5.5%, respectively, n = 10, p < 0.05). The increased CD123 expression was negatively correlated with the number of peripheral white blood cells (r = −0.700, p < 0.05, n = 10). The percentage of CD166 expression was found significantly correlated with the index of SLE disease activity (r = 0.472, p < 0.05, n = 10) and 24 h proteinuria (r = 0.558, p < 0.05, n = 10), but negatively correlated with serum C3 level (r = −0.712, p < 0.01, n = 10). Our study found that the surface marker expression of CD95, CD123, and CD166 on BM CD34⁺ cells were significantly increased in patients. This supports the hypothesis that there are abnormalities of the HSC in SLE. Since CD166 and CD123 correlated with the overall lupus activity, their role as a biomarker of inflammatory disease activity also requires further study.     

Acute leukemias express a functional receptor for the human growth hormone

 The potential influence of the human growth hormone (hGH) on the behavior of acute leukemias is a matter of controversy. We investigated primary childhood and adult leukemias (n=44) and leukemic cell lines (n=13) for the expression of the hGH receptor (hGHR) by immunohistochemistry and flow cytometry. All leukemias expressed the hGHR in the cytoplasm; expression on the surface was undetectable in some of the leukemias. In leukemic cell lines, hGHR expression on the surface was demonstrated in a dose-dependent manner after incubation with rhGH. Physiologic concentrations of hGH were more efficient than higher doses in increasing hGHR surface expression. A proliferative response to hGH was accomplished in cell lines REH, Molt4, and K562. However, only one of 19 primary leukemias (ALL, n=12; AML, n=7) showed increased cell counts after the addition of 50–800 ng/ml recombinant hGH (rhGH). These cells were of an immature T-cell phenotype. We thus conclude that acute leukemias can be stimulated by hGH to up-regulate its receptor, but that most primary leukemias may require additional signals for the induction of proliferation. Received: 2 February 1997 / Accepted: 6 May 1997     

Functional expression of MDR-1 in acute myeloid leukemia: correlation with the clinical-biological, immunophenotypical, and prognostic disease characteristics

 Up till now, clinical data on the possible prognostic influence of multidrug resistance (MDR) in hematological malignancies have been inconsistent, probably due to technical pitfalls. Moreover, in most studies qualitative information on the presence/absence of MDR-1 expression has been used instead of quantitative results. In addition, results usually refer to the total BM population and not specifically to blast cells. In the present study we analyzed the expression of MDR-1 in a series of 50 newly diagnosed de novo AML using a double-staining technique: (a) monoclonal antibodies for the specific identification of blast cells and (b) the rhodamine-123 efflux assay, which allows a quantitative and calibrated measurement of MDR-1 function. Expression of MDR-1 was correlated with clinical, biological, and immunophenotypical disease characteristics. All patients were uniformly treated according to the AML 87/91 protocols of the Spanish Pethema Cooperative Group; the median age was 51±19 years and the FAB distribution was as follows: 2 M₀, 9 M₁, 9 M₂, 12 M₃, 11 M₄, 5 M₅, and 2 M₆ cases. Upon grouping the 50 AML patients analyzed according to the level of rh123 elimination, it was observed that those cases with ≥30% decrease in rh123 fluorescence displayed higher WBC counts (9±12 vs 37±73×10⁹/l, p=0.02) and platelet numbers (94±11 vs 35±25×10⁹/l, p=0.02), together with a higher incidence of extramedullary involvement (35% vs 24%, p=0.02). Half of the patients (47%) displaying a low rh123 elimination (<30%) showed M₃ morphology, while among the 33 patients with a higher rate of rh123 elimination (≥30%), only four (12%) corresponded to the M₃ morphological subtype (p=0.0006). From the immunophenotypic point of view, a low rate of rh123 elimination was associated with a lower expression of HLADR antigen (p=0.003) and a higher expression of CD117 (p=0.01). Regarding the possible prognostic influence of MDR1 expression, we found that a high rate of rh123 elimination (>30%) was associated with a tendency towards poor disease outcome, illustrated by both a lower complete remission rate with the first cycle of chemotherapy (36% vs 56%) and a lower median disease-free survival (22 months vs median DFS not reached), although differences did not reach statistical significance (p=0.1 in both comparisons). This data shows that although MDR-1 can be a relevant parameter in the evaluation of AML patients, larger series of patients using appropiate techniques for specifically analyzing the MDR of blast cells will be necessary in order to establish the final clinical value of this parameter. Received: 24 April 1997 / Accepted: 25 June 1997     

Variability in responsiveness to lovastatin of the primitive CD34+ AML subfraction compared to normal CD34+ cells

In the present study, we questioned whether the cholesterol synthesis inhibitor lovastatin potentiates the cytotoxicity of chemotherapeutic agents in the primitive CD34⁺ subpopulation of acute myeloid leukemia (AML) cells. AML mononuclear cells (n = 17) were sorted in CD34⁺ and CD34⁻ fractions and compared to normal CD34^+/− cells (n = 7). The percentage of surviving cells upon exposure to lovastatin (25–100 μM) and/or chemotherapeutics (cytarabin or daunorubicin) was determined with a luminescent cell viability assay. The results demonstrate that the primitive CD34⁺ subpopulation of normal and AML cells displayed a higher sensitivity to lovastatin than the more mature CD34⁻ subpopulation. The combination of lovastatin and chemotherapeutics resulted in a more pronounced inhibitory effect on both subpopulations. In contrast to the homogeneous results in normal CD34⁺ cells, a distinct heterogeneity in lovastatin sensitivity was found in AML samples. Therefore, a group of normal (n = 11) and abnormal (n = 6) responders were identified based on a reduced or increased cell survival compared to normal CD34⁺ cells. This distinction was not only observed in the CD34⁺ AML subfraction but also in CD34⁺CD38⁻AML cells. In the abnormal responder group, 50% of patients presented with unfavorable cytogenetics and significant higher peripheral blast cell counts, which coincided with poor treatment results. In summary, the findings indicate that the primitive subfraction of CD34⁺ AML cells is in the majority of cases affected by lovastatin treatment, which is potentiated when combined with chemotherapeutics. Heterogeneity of the response observed in AML patients allowed identification of a subgroup with poor prognosis.     

Heme oxygenase-1 plays a crucial role in chemoresistance in acute myeloid leukemia

Abstract

Objectives

The heme oxygenase-1 (HO-1) gene may contribute to the development of acquired chemoresistance in solid tumor cells, but its function in acute myeloid leukemia (AML) remains unclear. Therefore, we investigated whether the expressions of HO-1 mRNA and protein were associated with AML chemoresistance.

Methods

Bone marrow or peripheral blood was obtained from newly diagnosed (n = 26), relapsed (n = 10), and completely remitted (n = 18) patients with AML (M3 exclusion) and healthy donors (n = 10). Small interfering RNA was used to stably silence HO-1 gene expression in AML cell lines. The expressions of HO-1, hypoxia inducible factor-1ɑ (HIF-1ɑ), glucose transporter-1 (GLUT1) mRNA and proteins were measured by quantitative real-time PCR and Western blot. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction was analyzed by flow cytometry.

Results

The drug-resistant AML cell line HL-60R was significantly less sensitive to cytarabine and daunorubicin than HL-60 cells. HO-1 mRNA and proteins were highly expressed in HL-60R cells. However, down-regulating HO-1 significantly enhanced the sensitivity of HL-60R to chemotherapy, and the expressions of HIF-1ɑ and GLUT1 mRNA and proteins decreased. Meanwhile, the expressions of caspase-3 and caspase-8 proteins increased, while that of bcl-2 decreased. Overexpressions of HO-1, HIF-1ɑ, and GLUT1 were associated with poor response of AML to chemotherapy.

Conclusions

Overexpressions of HO-1, HIF-1ɑ, and GLUT1 might be involved in the chemoresistance of AML. HO-1 is a potential target to overcome the drug resistance of AML.     

Effect of type 2 diabetes mellitus on exercise intolerance and the physiological responses to exercise in peripheral arterial disease

Aims/hypothesis There are conflicting data about the effect of type 2 diabetes mellitus on exercise tolerance in peripheral arterial disease. To elucidate this problem, we compared the tolerance and physiological responses to treadmill and cycle exercise in 31 patients with peripheral arterial disease and intermittent claudication. Materials and methods One group of these patients had type 2 diabetes (n = 12) and its members were matched for sex and age with a group of patients who did not have diabetes (n = 12). Since BMI and body weight were greater in the diabetic group (28.4 ± 3.7 vs 25.2 ± 2.4 kg/m²; 84.0 ± 14.6 vs 73.8 ± 8.0 kg), we also studied a third, ‘heavy’ group of non-diabetic patients with claudication of similar age (n = 7; BMI = 30.9 ± 5.3 kg/m²; body weight = 85.2 ± 8.2 kg). Results Compared with the ‘light’ non-diabetic group, maximum treadmill times were shorter for the diabetic and heavy non-diabetic groups (1,448 vs 845 and 915 s; ANOVA p = 0.01); maximum cycle time also tended to be shorter (ANOVA, p = 0.08) in the diabetic and heavy non-diabetic groups (median = 1,231 vs 730 and 797 s). The majority of physiological responses assessed were not different between the groups, although the time constant of oxygen uptake during submaximal treadmill and cycle exercise was significantly larger (ANOVA p < 0.05) for the diabetic group. Conclusions/interpretation These data demonstrate that exercise tolerance is lower in diabetic than non-diabetic patients with claudication, but that this difference is due to obesity rather than diabetes itself.     

Does acute ingestion of large amounts of alcohol cause pancreatic injury? A prospective study

Summary The contribution of ethanol to the pathogenesis of acute pancreatitis has been questioned for a long time. The authors asked whether acute ingestion of large amounts of alcohol may lead to pancreatic injury, as assessed by serum amylase levels, clinical picture, and abdominal ultrasound. Therefore, all patients (N=112) admitted to our medical emergency ward with the diagnosis of alcohol intoxication were evaluated prospectively during a 12-mo period. Of these, 78 (56 M, 22 F; mean age 36±15) could be evaluated. The other 44 were excluded because of incomplete data (n=18), mixed intoxications (n=8), repeated admission (n=9), incorrect diagnosis on admission (n=7), and chronic pancreatitis (n=2). Serum ethanol, amylase, and GOT were measured. Serum ethanol was 246±122 mg/dL (3–500 mg/dL), amylase 83±44 U/L (27–361 U/L), and GOT 25±37 U/L (5–271 U/L) without significant differences among the genders. No correlation between serum ethanol and serum amylase levels could be detected.     

Early Increase in Myocardial Perfusion After Stem Cell Therapy in Patients Undergoing Incomplete Coronary Artery Bypass Surgery

Incomplete revascularization is associated with worse long-term outcomes. Autologous bone marrow cells (BMC) have recently been tested in patients with severe coronary artery disease. We tested the hypothesis that intramyocardial injection of autologous BMC increases myocardial perfusion in patients undergoing incomplete coronary artery bypass grafting (CABG). Twenty-one patients (19 men), 59 ± 7 years old, with limiting angina and multivessel coronary artery disease (CAD), not amenable to complete CABG were enrolled. BMC were obtained prior to surgery, and the lymphomonocytic fraction separated by density gradient centrifugation. During surgery, 5 mL containing 2.1 ± 1.3 × 10⁸ BMC (CD34+ = 0.8 ± 0.3%) were injected in the ischemic non-revascularized myocardium. Myocardial perfusion was assessed by magnetic resonance imaging (MRI) at baseline and 1 month after surgery. The increase in myocardial perfusion was compared between patients with <50% (group A, n = 11) with that of patients with >50% (group B, n = 10) of target vessels (stenosis ≥ 70%) successfully bypassed. Injected myocardial segments included the inferior (n = 12), anterior (n = 7), and lateral (n = 2) walls. The number of treated vessels (2.3 ± 0.8) was significantly smaller than the number of target vessels (4.2 ± 1.0; P < 0.0001). One month after surgery, cardiac MRI showed a similar reduction (%) in the ischemic score of patients in group A (72.5 ± 3.2), compared to patients in group B (78.1 ± 3.2; P = .80). Intramyocardial injection of autologous BMC may help increase myocardial perfusion in patients undergoing incomplete CABG, even in those with fewer target vessels successfully treated. This strategy may be an adjunctive therapy for patients suffering from a more advanced (diffuse) CAD not amenable for complete direct revascularization.     

Diabetic polyneuropathy is associated with respiratory muscle impairment in type 2 diabetes

Aims/hypothesis Diabetes has a major negative effect on intensive care unit outcome. This has been partly attributed to impaired respiratory neuromuscular function. However, data on respiratory neuromuscular involvement in diabetes are lacking. This study therefore aimed to assess respiratory neuromuscular function related to diabetic polyneuropathy in patients with type 2 diabetes. Methods Respiratory neuromuscular function was assessed by the use of volitional tests and twitch mouth (TwPmo) and twitch transdiaphragmatic (TwPdi) pressures during non-volitional bilateral anterior magnetic phrenic nerve stimulation in 21 male type 2 diabetic patients without pulmonary disease and in 23 healthy, well-matched controls (forced expiratory volume in 1 s 103 ± 11 vs 103 ± 12% predicted; p = 0.9). Results Both volitionally assessed maximal inspiratory and expiratory mouth pressures, and sniff nasal and transdiaphragmatic pressures were comparable between diabetic patients and controls (p > 0.1 for all). TwPmo was reduced in diabetic patients compared with controls (1.3 ± 0.5 vs 1.0 ± 0.4 kPa; p = 0.04), while TwPdi was comparable (1.7 ± 0.5 vs 1.6 ± 0.7 kPa; p = 0.6). Following subgroup analysis, patients with no or mild polyneuropathy (n = 10) as assessed by neurological disability scoring had normal respiratory neuromuscular function, whereas patients with moderate or severe polyneuropathy (n = 11) presented with markedly impaired respiratory neuromuscular function as indicated by TwPmo (1.3 ± 0.4 vs 0.8 ± 0.3 kPa; p = 0.01) and TwPdi (1.9 ± 0.6 vs 1.1 ± 0.4 kPa; p < 0.01). Conclusions/interpretation With regard to volitional tests, diabetes does not affect respiratory neuromuscular function. In contrast, the application of non-volitional phrenic nerve stimulation provides strong evidence that diabetic polyneuropathy, as simply assessed by neurological disability scoring, is associated with substantially impaired respiratory neuromuscular function in type 2 diabetic patients.     

Cilostazol reduces the progression of carotid intima-media thickness without increasing the risk of bleeding in patients with acute coronary syndrome during a 2-year follow-up

Cilostazol, a phosphodiesterase III inhibitor, is known to have anti-proliferative activity. We investigated the effects of cilostazol 200 mg, in addition to aspirin 100 mg and clopidogrel 75 mg, on carotid intima-media thickness (IMT) progression during a 2-year follow-up period in patients with acute coronary syndrome (ACS) requiring stent implantation. Patients with ACS (n = 130) were randomly assigned to the cilostazol group (n = 64) or the control group (n = 66). Longitudinal images of left and right carotid IMT were measured at baseline, at 6, 12, and 24 months using a 10-MHz linear vascular probe. The primary endpoint was to compare the changes in maximum carotid IMT at 2 years. Other parameters such as inflammatory markers [interleukin (IL)-6, tumor necrosis factor (TNF)-α, C-reactive protein (CRP), and adiponectin] and bleeding risk were also compared. The carotid IMT showed no significant progression from baseline in the cilostazol group compared to significant progression in the control group at 12 months (0.78 ± 0.38 and 0.85 ± 0.41 mm, p = 0.034, respectively) and 24 months (0.82 ± 0.41 and 0.96 ± 0.39 mm, p = 0.022, respectively). Major bleeding (p = 1.00), minor bleeding (p = 0.68), and total bleeding rates (p = 0.74) were similar between the two groups during the 2-year follow-up. Decreases from baseline in IL-6 (−2.79 ± 2.83 and −2.14 ± 3.36 pg/ml, p = 0.010, respectively) and TNF-α (−2.81 ± 1.97 and −2.21 ± 2.68 pg/ml, p = 0.029, respectively) were significantly greater in the cilostazol group than the control group during the follow-up. Cilostazol treatment, with greater anti-inflammatory effect, inhibited the progression of carotid IMT without increasing the risk of bleeding in patients with ACS during the 2-year follow-up.     

Platelet-monocyte cross talk and tissue factor expression in stable angina vs. unstable angina/non ST-elevation myocardial infarction

Tissue factor (TF), the major procoagulant in vivo, is usually absent from blood cells. However, since both monocyte TF (MoTF) expression and platelet activation are present in acute coronary syndrome we hypothesized that MoTF expression may in part depend on monocyte platelet aggregate (MPA) formation in coronary artery disease (CAD). Patients with unstable angina/non-ST-elevation myocardial infarction (UA/NSTEMI, n?=?20) had significantly higher levels of MoTF (17.4?±?3.1MFI) and MPAs (CD42b:273?±?183MFI; CD62P:256.3?±?48.5MFI) than patients with stable angina (SA, n?=?40; MoTF:13.2?±?2.2MFI, p?=?0.001; CD42b:160?±?113MFI, p?=?0.025; CD62P:118.7?±?24.5MFI, p?=?0.018) as measured by whole blood flow cytometry on CD14⁺-cells. TF-activity of isolated mononuclear cells (MNC) was elevated in UA/NSTEMI (75?±?27?pg/mL) in comparison to SA (47?±?17?pg/mL, p?=?0.001) as determined by chromogenic assay, and TF mRNA expression in isolated MNC was more frequent in UA/NSTEMI than in SA (50% vs. 18.2%; p?=?0.017). MoTF expression significantly correlated with the constitutive platelet marker CD42b (r?=?0.69, p?<?0.001) and the platelet activation marker CD62P (r?=?0.47, p?=?0.001) on CD14⁺-cells suggesting its association with MPAs in UA/NSTEMI. In addition, MoTF expression correlated with MoTF activity of isolated MNC (r?=?0.41, p?=?0.01) and plasma levels of the F1.2 prothrombin fragment (r?=?0.35, p?=?0.02). In conclusion, MoTF and MPAs are elevated in UA/NSTEMI compared with SA. MoTF expression correlates with platelet mass and activity attached to monocytes.     

Sex differences in serum 7α-hydroxycholesterol levels in the rat reflect hepatic activity of 3β-hydroxy-Δ5-C27-steroid dehydrogenase and cholesterol 7α-hydroxylase

Factors that affect serum levels of 7α-hydroxycholesterol were studied in the rat. Serum levels of 7α-hydroxycholesterol differed in male and female rats fed regular chow (male; 0.2±0.1 nmol/ml (mean ±SD)n=8; female; 0.4±0.1 nmol/ml;n=8). When rats were fed with chow to which 3% cholestyramine had been added, the level increased significantly, particularly in female rats (male: 0.6±0.3 nmol/ml;n=8; female; 2.4±1.5 nmol/ml;n=8). The liver activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme for degradation of cholesterol, did not show any sex differences, irrespective of whether the animals were fed with regular chow (male; 51±15 pmol/min per mg protein;n=8; female; 58±21 pmol/min per mg protein;n=8), or the cholestyramine-supplemented chow (male; 162±33pmol/min per mg protein;n=8; female; 172±33 pmol/min per mg protein;n=8). In contrast, the activity of 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase, which acts after cholesterol 7α-hydroxylase in the catabolism of cholesterol, showed a marked difference between the sexes. In both sexes this enzyme activity was higher in cholestyramine-treated rats (male; 963±78 pmol/min per mg protein;n=8; female; 708±106 pmol/min per mg protein,n=8) compared to that in that rats received regular chow (male; 622±83pmol/min per mg protein;n=8). If the serum level of 7α-hydroxycholesterol depended solely on the enzyme activity of cholesterol 7α-hydroxylase, it would be difficult to explain these sex differences, since there were no sex differences in levels of cholesterol, 7α-hydroxylase. These results clearly indicate that, in the rat, the serum level of 7α-hydroxycholesterol depends not only on cholesterol 7α-hydroxylase activity but also on 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase activity. Presented, in part, at the 32nd Annual Meeting of the Japanese Society of Gastroenterology, November 1990, Nara, Japan     

Comparative quantitative expression of bcl-2 by normal and leukaemic myeloid cells

Summary. Expression of the bcl-2 oncoprotein by AML blasts has previously been demonstrated to be heterogenous with high levels of bcl-2 expression being associated with a low complete remission rate and poor survival. We have quantified bcl-2 expression in AML blasts in relation to expression of the CD34 antigen and in comparison to CD34-positive cells from normal bone marrow. When expressed as molecules of equivalent soluble fluorochrome (MESF) per cell, AML blast cell bcl-2 expression varied from 11.1 to 99.9 × lO³ (median 39.4 × 10³, n=56) with 28.5% of patients expressing high MESF values (>50 × 10³) and 16% of patients expressing low MESF values (<20 × 10³), the remainder expressing intermediate values. There was no significant difference between intensity of bcl-2 expression and FAB classification in the de novo AML cases, and there was no significant differences between de novo and secondary AML cases. Blasts from CD34⁺ AML patients expressed significantly higher levels of bcl-2 (mean MESF 43.6 × 10³, n =36) than CD34^? AML patients (mean MESF 31.7 × 10³, n=19). In five cases of CD34⁺ AML, bcl-2 expression was determined on purified CD34⁺ and CD34^? blast cell populations. In all cases CD34⁺ blasts were found to express significantly higher bcl-2 MESF values compared to the CD34^? fraction. Purified CD34⁺ cells from normal bone marrow consistently expressed high levels of bcl-2 (MESF >75 × 10³ n = 4), which was comparable to that found on CD34⁺ AML cells. Our results suggest that the poor prognosis previously associated with AML blasts expressing the CD34 antigen may in part be related to high expression of bcl-2. Also the ability to measure bcl-2 in AML blasts quantitatively by flow cytometry and to categorize patients into discrete groups may be of value as a prognostic indicator in AML.     

Effects of carbamylcholine chloride on human antral gastrin mRNA levels

The effects of the muscarinic receptor agonist, carbamylcholine chloride (carbachol), on gastrin release and gastrin mRNA levels in human antral mucosa (n=15) were determined. During a-2-h incubation period, carbachol (10⁻⁶−10⁻⁴M) decreased gastrin mRNA levels to 71±8% (10⁻⁶M), 40±8% (10⁻⁵M), and 33±5% (10⁻⁴M) of control levels. Carbachol (10⁻⁵M) decreased intracellular gastrin (from 1634±103 to 1272±126 pg/mg tissue protein), while it increased gastrin release into the medium (from 609±48 to 918±68 pg/ml per mg tissue protein). After 6-and 9-h culture, carbachol gradually increased gastrin mRNA levels, by 96±12% and 126±23%, respectively. Atropine sulfate (10⁻⁵ M) completely inhibited the carbachol-induced changes. Cycloheximide markedly decreased tissue gastrin concentration, but increased gastrin mRNA levels, whereas it had no effects on gastrin release. These findings suggested that carbachol may have a time-related biphasic action on human antral gastrin biosynthesis.     

An orientation session improves objective sleep quality and mask acceptance during positive airway pressure titration

The aim of this study was to determine whether an orientation session led by a polysomnography (PSG) technician during the night of positive airway pressure (PAP) titration can improve objective sleep quality and acceptance of nasal mask in patients referred to a sleep laboratory. Consecutive patients (n = 1,481), referred for PAP titration during PSG, were retrospectively evaluated. Patients were distributed in two groups: the control group, patients referred for PAP titration (n = 699) who did not undertake an orientation session led by a PSG technician, and the oriented group, patients referred to PAP titration (n = 782) who followed the orientation session. Demographic data were similar (p > 0.05) between groups (control vs oriented) for: male/female proportion (76:24 vs 75:25%), age (mean ± SD; 53 ± 12 vs 52 ± 12 years), Epworth Sleepiness Scale score (12 ± 6 vs 12 ± 6), and body mass index (31 ± 6 vs 31 ± 6 kg/m²). PSG data were different (p < 0.05) between the groups for: total sleep time (312 ± 81 vs 326 ± 85 min), sleep efficiency (74 ± 17 vs 77 ± 14%), sleep latency (22 ± 24 vs 18 ± 29 min), S1 (8 ± 8 vs 6 ± 5%), S3  4 (19 ± 11 vs 21 ± 13%), rapid eye movement sleep (17 ± 9 vs 18 ± 9%), and wake after sleep onset (106 ± 68 vs 93 ± 58 min). After the orientation session, the number of patients who did not accept nasal mask during PSG recording was higher in the control group than the oriented group (80 vs 44; p = 0.001). An orientation session led by a PSG technician can improve objective sleep quality and nasal mask acceptance during the night of PAP titration. Such an addition to PAP titration could be an efficient intervention to improve PAP compliance.     

Native LDL-Cholesterol Mediated Monocyte Adhesion Molecule Overexpression is Blocked by Simvastatin

Aim of the study  This study sought to evaluate the effect of nLDL concentrations on monocyte adhesion molecule expression in hypercholesterolemic patients with stable coronary artery disease (CAD) and to determine whether lipid-lowering therapy with simvastatin would change this effect. Methods  Blood samples from patients with hypercholesterolemia (mean LDL 152 mg/dL) and CAD (HC, n = 23) were collected before and after a 12-week treatment with 40 mg of simvastatin. Healthy individuals (mean LDL 111 mg/dL) were used as controls (CT, n = 15). Isolated nLDL, at a fixed concentration of 100 mg/dL, was added to monocyte suspensions obtained before and after the simvastatin treatment. Monocyte activation was determined by changes in cellular adhesion molecule expression. Results  In response to nLDL, CD11b and CD14 adhesion molecule expression was higher in HC patients than in CT patients before treatment (174.2 ± 8.4 vs 102.2 ± 6.3, P < 0.03 and 140.4 ± 5.0 vs 90.4 ± 6.7, P < 0.04). After simvastatin treatment, CD11b expression decreased to 116.9 ± 12.5 (P < 0.03) and CD14 expression to 107.5 ± 6.2 (P < 0.04). Alternatively, L-selectin expression was lower in HC patients than in CT patients before therapy (46.0 ± 3.5 vs 62.1 ± 5.5, P < 0.04), and it increased markedly after lipid reduction to 58.7 ± 5.0 (P < 0.04 vs baseline). After simvastatin treatment, LDL was reduced to mean 101.5 mg/dL. Conclusions  These data demonstrate that monocytes from HC patients are more prone to marked nLDL-mediated changes of adhesion molecule expression than monocytes from controls. Simvastatin is capable of inhibiting such nLDL effects. This proinflammatory response to nLDL may have a role in the early onset of atherosclerosis.     

Serum Concentrations of Insulin-Like Growth Factor-I (IGF-I) as a Marker of Liver Fibrosis in Patients With Chronic Hepatitis C

Liver biopsy was until recently the only way of evaluating liver fibrosis. Noninvasive tests for hepatic fibrosis, without potential risks, are desired by clinicians as well as patients. Insulin-like growth factor-I (IGF-I) synthesis is disturbed in liver fibrosis and reflects the severity of the clinical stage. We assessed serum IGF-I levels in patients with chronic hepatitis C (CHC) to correlate with liver fibrosis and antiviral therapy. Forty patients with CHC and persistently abnormal alanine aminotransferase values were enrolled and treated with peginterferon α-2a 180 μg per week plus ribavirin for 24 (n=20) or 48 (n=20) weeks. All patients underwent liver biopsy before treatment (METAVIR fibrosis stage F0, n=13; F1–F2, n=14; F3, n=7; F4, n=6). Serum IGF-I was measured at baseline, at the end of treatment period, and 24 weeks after finishing treatment. Mean IGF-I values were significantly lower in patients with advanced fibrosis (F4, 65.9±17.9 ng/mL) than in the others (F0, 145.2±47.1; F1–F2, 150.3±89.6; and F3, 121.4±35.2 ng/mL; P < .05). Serum IGF-I levels increased during combined therapy, being this increment markedly higher in patients with sustained virologic response. In conclusion, IGF-I synthesis is disturbed in CHC and reflects the severity of the liver fibrosis. Combined therapy improves serum IGF-I levels. IGF-I could represent a good, noninvasive marker of liver fibrosis.