Characterization of primitive subpopulations of normal and leukemic cells present in the blood of patients with newly diagnosed as well as established chronic myeloid leukemia

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors, including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC), have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study, which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis, we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC, often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly, both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+, as previously documented for LTC-IC in normal marrow. Thus, neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless, an association of these phenotypes with LTC- IC function did allow highly enriched (> 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph- , respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally, they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells.     

Platelet glycoproteins IIb and IIIa as a calcium channel in liposomes

Human platelet membrane glycoproteins IIb and IIIa (GPIIb and IIIa) were incorporated into phospholipid vesicles by the reverse-phase technique to assess the ability of GPIIb and IIIa to function as a Ca2+ channel. Movement of Ca2+ across the lipid bilayer was quantitated by injection of proteoliposomes with encapsulated Fura-2 into Ca2+ buffers and measurement of Fura-2 fluorescence as an indicator of Ca2+ influx. Reciprocally, to assess the function of proteins in an inside-out orientation, Ca2+-loaded vesicles were injected into Ca2+-free buffer and Ca2+ efflux monitored by a calcium electrode. Incorporation of the IIb-IIIa complex produced significant facilitation of Ca2+ movement across the lipid bilayer. No net transmembrane Ca2+ movement was seen with dissociated IIb and IIIa. Movement of Ca2+ was proportional to the transmembrane Ca2+ gradient. Ca2+ movement into the vesicles was inversely proportional to extravesicular NaCl from 25 to 150 mmol/L, analogous to several studies in the intact platelet. Adenosine triphosphate had no effect on Ca2+ movement into or out of the vesicles. Specific inhibition of a Ca2+ shift into the vesicles was seen with M148, a monoclonal antibody to IIb/IIIa, while no inhibition was observed with a panel of other anti-IIb/IIIa monoclonal antibodies. This suggests that a specific site on the complex or orientation of the complex is essential for calcium channel function. These data demonstrate that the GPIIb/IIIa complex can serve as a passive Ca2+ channel across a phospholipid bilayer and has the potential to play a role in Ca2+ flux across the platelet plasma membrane.     

Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid- dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506- FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.     

Lack of Evidence to Support Thromboprophylaxis in Hospitalized Medical Patients with Cancer

Background

The administration of anticoagulant thromboprophylaxis for all patients with cancer who are hospitalized for acute medical illness is considered standard practice and strongly recommended in clinical guidelines. These recommendations are extrapolated from randomized controlled prophylaxis trials not specifically conducted in cancer cohorts. Because hospitalized patients with cancer constitute a unique population with increased risk of venous thromboembolic events and major hemorrhage, validation of the efficacy and safety of primary thromboprophylaxis in this population is critical. We sought to summarize the rates of venous thromboembolic events and major bleeding episodes among hospitalized patients with cancer who were receiving anticoagulant therapy compared with placebo.

Methods

A systematic literature search strategy was conducted using MEDLINE, EMBASE, and the Cochrane Register of Controlled Trials. Two reviewers independently extracted data onto standardized forms. The primary end points were all venous thromboembolic events. Secondary end points included major bleeding episodes and symptomatic venous thromboembolic events. Pooled analysis with relative risk using a random effect model was used as the primary measurement.

Results

A total of 242 citations were identified by the literature search. Of these, 3 placebo-controlled randomized trials included venous thromboembolic events as a primary outcome and were analyzed according to cancer subgroups. The pooled relative risk of venous thromboembolic events was 0.91 (95% confidence interval, 0.21-4.0; I²: 68%) among hospitalized patients with cancer who were receiving thromboprophylaxis compared with placebo. None of the trials reported the rates of symptomatic venous thromboembolic events or major bleeding episodes according to cancer status.

Conclusions

The risks and benefits of primary thromboprophylaxis with anticoagulant therapy in hospitalized patients with cancer are not known. This is especially relevant because numerous Medicare-type pay-for-performance incentives mandate prophylaxis specifically in patients with cancer.     

Defining time in therapeutic range for busy clinicians: Frequency of dose changes is a good surrogate marker to identify patients with suboptimal anticoagulation with warfarin

Introduction

Patients on warfarin with sub-optimal time-in-therapeutic-range (TTR) are more likely to have adverse events. Target-specific oral anticoagulants (TSOACs) are approved and can be used as an alternative to warfarin for a number of indications. Further, the efficacy and safety profiles of the TSOACs compared to warfarin are more favourable when the TTR is ≤ 65% for certain indications.

Objective

We aimed to determine simple, sensitive and specific diagnostic tools to identify TTR ≤ 65% during the initial three months of warfarin therapy.

Methods

A cross-sectional study including patients newly initiated on warfarin without any interruption for three months was conducted. TTR was calculated using the Rosendaal method. Patients were stratified by TTR (≤ 65% or > 65%). Number of INR measurements, dose changes and INR measurements of ≤ 1.7 or ≥ 4.0 were evaluated as potential diagnostic tools to identify TTR ≤ 65%.

Results

670 patients were included. The most common indication for anticoagulation was venous thromboembolism. The mean TTR in the first three months was 68 ± 21% (Range: 10 to 100%). Three or more dose changes identified TTR ≤ 65% and demonstrated a sensitivity and specificity of 90% (95%CI 86 to 93%) and 56% (95%CI 51 to 61%), respectively. Three or more INR measurements of ≤ 1.7 during the initial three months of anticoagulation showed a sensitivity and specificity of 37% (95%CI 32 to 43%) and 98% (95%CI 96 to 99%), respectively.

Conclusion

Three or more dose changes and three or more INR measurements of ≤ 1.7 could identify patients with a TTR ≤ 65% in the first three months of warfarin therapy.     

Validity of standard gamble estimated quality of life in acute venous thrombosis

Introduction

The standard gamble is considered the ‘gold standard’ technique for measuring quality of life. We recently used the standard gamble to estimate quality of life in acute venous thrombosis, and found unexpected variability in the responses. The current study aimed to explore the reasons for variability by comparing the standard gamble technique in patients with acute venous thrombosis to other quality of life measurement tools.

Materials and Methods

Thrombosis clinic patients treated for venous thrombosis were eligible to participate. Patients evaluated their current health state by performing a standard gamble interview, reporting on a visual analogue scale, completing the SF-36 and disease specific questionnaires (PEmb-Qol and VEINES-QOL/Sym). Validity was assessed by correlating the standard gamble utilities with the other methods. Test-retest reliability, responsiveness and acceptability were also assessed.

Results

Forty-four patients were interviewed, with 16 attending for a repeat interview. The median standard gamble utility was 0.97 (0.84-1.0), SF-6D 0.64 (0.59 - 0.80) and visual analogue score 70 (60 - 80). Participants with pulmonary embolism had lower standard gamble estimates than those with deep vein thrombosis. There was good discriminant validity in that the standard gamble estimates were not associated with risk taking behavior, negative outlook, sex or education. Test-retest reliability with the standard gamble was moderate and there was evidence of a ceiling effect.

Conclusions

Standard gamble utilities are higher than other methods of measuring quality of life in venous thrombosis. The choice of utility values adopted in studies will impact on future economic studies.     

Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca(2+) sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+) sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca(2+) transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.     

Exotic and indigenous problem plants species used,by the Bapedi,to treat sexually transmitted infections in Limpopo Province,South Africa

Background

The ethnic usage of exotics and indigenous problem plants is a highly debated topic, as legislative requirements over-shadow their potential medicinal value, particularly to treat sexually transmitted infections (STIs). Limited information exists regarding their medicinal value among the Bapedi.

Objectives

To ascertain the importance of exotics and indigenous problem plants in the treatment of STIs, a major global primary health care challenge.

Methods

A field observation and semi-structured questionnaire focussing on species diversity, types of STIs treated and medicinal preparation as well as application was used to collect data from 34 traditional healers.

Results

Seven exotics and three indigenous problem species were identified. These species were used to treat four STIs; with Catharanthus roseus illustrating its dominance in the treatment of gonorrhoea. Some medicinal species used by Bapedi traditional healers have been validated through scientific research or through their extensive use by various cultures in South Africa and other parts of Africa. To the best of our knowledge Alternanthera pungens, Caesalpinia decapetala, Cinnamomum verum, and Citrullus lanatus are reported for the first time in the treatment of the investigated STIs.

Conclusions

Exotic and indigenous problem species constitute an important component of the STIs treatment protocol. Their utilization by Bapedi cautions against the narrow-minded approach of indiscriminate eradication, as these species can play a significant role in the primary health care needs of socio-economic vulnerable people.