High-dose mitoxantrone induces programmed cell death or apoptosis in human myeloid leukemia cells

Mitoxantrone has been shown in vitro to exhibit a steep dose-response relationship with respect to the clonogenic survival of acute myeloid leukemia cells. In this report, we show that 1-hour exposure of human myeloid leukemia HL-60 and KG-1 cells to mitoxantrone concentrations ranging between 0.1 and 10.0 mumol/L induced internucleosomal DNA fragmentation of approximately 200-bp integer multiples, characteristic of cells undergoing programmed cell death (PCD) or apoptosis. Mitoxantrone-mediated PCD was associated with a steep inhibition of the clonogenic survival of the leukemic cells. In addition, intracellularly, mitoxantrone-induced PCD was associated with a marked induction of c-jun and significant repression of c-myc and BCL-2 oncogenes. Pretreatment with the protein kinase C stimulator phorbol myristate acetate enhanced mitoxantrone-induced internucleosomal DNA fragmentation, whereas protein kinase C inhibitors staurosporine and H7 had no effect. These findings suggest that PCD is a potential mechanism underlying the steep dose-response relationship of mitoxantrone to the inhibition of clonogenic survival of acute myeloid leukemia cells.     

Identification of three candidate mutations causing type IIA von Willebrand disease using a rapid, nonradioactive, allele-specific hybridization method

Type IIA von Willebrand disease (vWD), the most common type II vWD variant, is characterized by decreased binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (Gplb) and by a decrease in large and intermediate vWF multimers. Mutations reported to cause vWD type IIA are clustered within the A2 domain of vWF, which is encoded by exon 28. Genomic DNA from affected members of 12 unrelated families with type IIA vWD were screened for these mutations by a rapid, nonradioactive, allele-specific oligonucleotide (ASO) hybridization method. Oligonucleotides containing each of eight mutations were cross-linked onto a nylon membrane by UV irradiation. A fragment of vWF exon 28 was amplified from peripheral blood leukocyte DNA using biotinylated primers and hybridized to the immobilized oligonucleotides. Positive signals were detected with an avidin-alkaline phosphatase conjugate and chemiluminescent substrate. Thus, in a single hybridization reaction, a patient sample could be analyzed for a large number of mutations simultaneously. Polymerase chain reaction (PCR) products from four patients did not contain any of the tested mutations and therefore were sequenced. Three additional candidate missense mutations, two of them novel, were identified: Arg(834)-->Gln in one patient, Gly(846)-->Arg in one patient, and Val(902)-->Glu in three ostensibly unrelated patients. By ASO hybridization, the mutations were confirmed in the affected patients and excluded in unaffected relatives and 50 normal controls. In one family, the Val(902)-->Glu mutation was shown to be a de novo mutation. This rapid screening method is applicable to other subtypes of vWD for which mutations have been identified.     

Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway

Fibrin(ogen) (FGN) is important for hemostasis and wound healing and is cleared from sites of injury primarily by the plasminogen activator system. However, there is emerging evidence in plasminogen activator- deficient transgenic mice that nonplasmin pathways may be important in fibrin(ogen)olysis, as well. Given the proximity of FGN and monocytes within the occlusive thrombus at sites of vascular injury, we considered the possibility that monocytes may play an ancillary role in the degradation and clearance of fibrin. We found that monocytes possess an alternative fibrinolytic pathway that uses the integrin Mac- 1, which directly binds and internalizes FGN, resulting in its lysosomal degradation. At 4 degrees C, FGN binds to U937 monocytoid cells in a specific and saturable manner with a kd of 1.8 mumol/L. Binding requires adenosine diphosphate stimulation and is calcium- dependent. At 37 degrees C, FGN and fibrin monomer (FM) are internalized and degraded at rates of 0.37 +/- 0.13 and 0.55 +/- 0.03 microgram/10(6) cells/h by U937 cells, 1.38 +/- 0.02 and 1.20 +/- 0.30 microgram/10(6) cells/h by THP-1 cells, and 2.10 +/- 0.20 and 2.52 +/- 0.18 micrograms/10(6) cells/h by human peripheral blood mononuclear cells, respectively. The serine protease inhibitors, PPACK and aprotinin, and the specific elastase inhibitor, AAPVCK, do not significantly inhibit degradation. However, degradation is inhibited by chloroquine, suggesting that a lysosomal pathway is involved. Factor X, a competitive ligand with FGN for the Mac-1 receptor, also blocks degradation, as does a monoclonal antibody to the alpha-subunit of Mac- 1. Autoradiography of radioiodinated, internalized FGN shows that FGN proteolysis by the pathway produces a unique degradation pattern distinct from that observed with plasmin. In a fibrin clot lysis assay, Mac-1-mediated fibrinolysis contributed significantly to total fibrinolysis. In summary, FGN is internalized and degraded by activated human monocytoid cells via Mac-1 in the absence of plasmin, thereby providing an alternative fibrinolytic pathway. Thus, in addition to the function of cell adhesion, integrins may also act as receptors that mediate the internalization and degradation of bound ligands.     

A prospective study evaluating the lowering of hemoglobin standards for blood donors

Strict regulations exist for the selection of blood donors. These include minimum hemoglobin (Hb) standards of 13.5 and 12.5 g/dL for males and females respectively. In Canada 2% of all blood donors, or approximately 25,000 individuals annually attempt to make a blood donation but are deferred because their Hb level does not meet these minimum accepted standards. In a previous study we provided a biostatistical approach to ascertain objectively the optimal minimum Hb standards for blood donors to best discriminate between iron deficient and non iron deficient individuals. The derived values were lower than the existing minimum Hb standards and because of concern that blood donors accepted using these lower Hb standards might have asymptomatic disease, the proposed new levels were not adopted. This present prospective study was undertaken to assess the impact on the blood donors of the new Hb standards. Over 26 months we evaluated 1,558 donors (695 males and 863 females). On entry into the study they were screened for a variety of medical conditions which could result in asymptomatic anemia and 6 months later they answered a health questionnaire. Blood donors were entered into 1 of 3 groups. Group I were donors accepted by the existing criteria. Group II were donors who did not meet the existing Hb criteria but satisfied the newly derived standards. Group III were donors deferred even by the new Hb standards.(ABSTRACT TRUNCATED AT 250 WORDS)     

Obesity,Diabetes and Atrial Fibrillation; Epidemiology,Mechanisms and Interventions

The last few decades have witnessed a global rise in adult obesity of epidemic proportions. The potential impact of this is emphasized when one considers that body mass index (BMI) is a powerful predictor of death, type 2 diabetes (T2DM) and cardiovascular (CV) morbidity and mortality [1, 2]. Similarly we have witnessed a parallel rise in the incidence of atrial fibrillation (AF), the commonest sustained cardiac arrhythmia, which is also a significant cause of cardiovascular morbidity and mortality. Part of this increase is attributable to advances in the treatment of coronary heart disease (CHD) and heart failure (HF) improving life expectancy and consequently the prevalence of AF. However, epidemiological studies have demonstrated an independent association between obesity and AF, possibly reflecting common pathophysiology and risk factors for both conditions. Indeed, weight gain and obesity are associated with structural and functional changes of the cardiovascular system including left atrial and ventricular remodeling, haemodynamic alterations, autonomic dysfunction, and diastolic dysfunction. Moreover, diabetic cardiomyopathy is characterized by an adverse structural and functional cardiac phenotype which may predispose to the development of AF [3]. In this review, we discuss the pathophysiological and mechanistic relationships between obesity, diabetes and AF, and the challenges posed in the management of this high-risk group of individuals.     

Current prospects for interferon‐free treatment of hepatitis C in 2012

Present interferon‐based therapy for chronic hepatitis C is limited by both efficacy and tolerability. Telaprevir and boceprevir are the first two direct‐acting antiviral drugs (DAAs) that inhibit hepatitis C virus replication to be licensed for use in conjunction with pegylated interferon and ribavirin. Numerous other DAAs are in clinical development, and phases 2 and 3 trials are evaluating interferon‐free combination DAA therapy. Interferon‐free sustained virologic responses have now been achieved with combinations of asunaprevir and daclatasvir; sofosbuvir and ribavirin; sofosbuvir and daclatasvir; faldaprevir and BI207127; ABT‐450, ritonovir and ABT‐333; ABT‐450, ritonovir and ABT‐072; miracitabine, danoprevir and ritonavir; and alisporivir and ribavirin. Some drugs are genotype‐specific in their activity, whereas others are pan‐genotypic, and differential responses for the genotype 1 subtypes 1a and 1b have emerged with many DAA combinations. Viral breakthrough and resistance are important considerations for future trial design. The prospect of interferon‐free combination DAA therapy for hepatitis C virus is now finally becoming a reality.     

Glycogen synthase kinase-3 (GSK-3) regulates TGF-β1-induced differentiation of pulmonary fibroblasts

Background

Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-β is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-β₁-induced myofibroblast differentiation is currently largely unknown.

Purpose

To determine the contribution of GSK-3 signalling in TGF-β₁-induced myofibroblast differentiation.

Experimental Approach

We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively.

Results

Stimulation of MRC5 and primary human lung fibroblasts with TGF-β₁ resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β₁-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β₁-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-β/smad signalling.

Conclusion and Implication

We demonstrate that GSK-3 signalling regulates TGF-β₁-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.     

Effect of oxygenated sterol compounds on human bone marrow granulocytic progenitor cells

Oxygenated sterol compounds are potent inhibitors of sterol and DNA synthesis in mammalian cells. We studied the effects of oxygenated sterols on human marrow granulocytic progenitor cells in vitro (CFU-C). 25-Hydroxycholesterol was found to be a potent inhibitor of sterol synthesis in marrow mononuclear cells, with 50% inhibition occurring at approximately 10(-7) M. This compound, as well as 6-ketocholestanol, 7- ketocholesterol, and 20 alpha-hydroxycholesterol, also demonstrated marked inhibition of CFU-C proliferation. The latter effect, which was not a result of direct cytoxicity of the compounds, was reversible by cholesterol, but not by mevalonic acid. We conclude that inhibition of sterol synthesis by oxygenated sterol compounds may be insufficient to explain their suppression of CFU-C proliferation.     

Monitoring polio supplementary immunization activities using an automated short text messaging system in Karachi,Pakistan

Problem

Polio remains endemic in many areas of Pakistan, including large urban centres such as Karachi.

Approach

During each of seven supplementary immunization activities against polio in Karachi, mobile phone numbers of the caregivers of a random sample of eligible children were obtained. A computer-based system was developed to send two questions – as short message service (SMS) texts – automatically to each number after the immunization activity: “Did the vaccinator visit your house?” and “Did the enrolled child in your household receive oral polio vaccine?” Persistent non-responders were phoned directly by an investigator.

Local setting

A cluster sampling technique was used to select representative samples of the caregivers of young children in Karachi in general and of such caregivers in three of the six “high-risk” districts of the city where polio cases were detected in 2011.

Relevant changes

In most of the supplementary immunization activities investigated, vaccine coverages estimated using the SMS system were very similar to those estimated by interviewing by phone those caregivers who never responded to the SMS messages. In the high-risk districts investigated, coverages estimated using the SMS system were also similar to those recorded – using lot quality assurance sampling – by the World Health Organization.

Lessons learnt

For the monitoring of coverage in supplementary immunization activities, automated SMS-based systems appear to be an attractive and relatively inexpensive option. Further research is needed to determine if coverage data collected by SMS-based systems provide estimates that are sufficiently accurate. Such systems may be useful in other large-scale immunization campaigns.     

Experience as a doctor in the developing world: does it benefit the clinical and organisational performance in general practice?

Background

Lipid-lowering medication remains underused, even in high-risk populations. The objective of this study was to determine factors underlying general practitioners' decisions not to prescribe such drugs to patients with type 2 diabetes.

Methods

A qualitative study with semi-structured interviews using real cases was conducted to explore reasons for not prescribing lipid-lowering medication after a guideline was distributed that recommended the use of statins in most patients with type 2 diabetes. Seven interviews were conducted with general practitioners (GPs) in The Netherlands, and analysed using an analytic inductive approach.

Results

Reasons for not-prescribing could be divided into patient and physician-attributed factors. According to the GPs, some patients do not follow-up on agreed medication and others object to taking lipid-lowering medication, partly for legitimate reasons such as expected or perceived side effects. Furthermore, the GPs themselves perceived reservations for prescribing lipid-lowering medication in patients with short life expectancy, expected compliance problems or near goal lipid levels. GPs sometimes postponed the start of treatment because of other priorities. Finally, barriers were seen in the GPs' practice organisation, and at the primary-secondary care interface.

Conclusion

Some of the barriers mentioned by GPs seem to be valid reasons, showing that guideline non-adherence can be quite rational. On the other hand, treatment quality could improve by addressing issues, such as lack of knowledge or motivation of both the patient and the GP. More structured management in general practice may also lead to better treatment.