In vitro proliferation of hematopoietic stem cells in the absence of an adherent monolayer

Experiments on long-term murine bone marrow cultures indicate that the production and maintenance of the hematopoietic stem cell is dependent on the establishment of an adherent monolayer and a secondary repopulation of the culture with fresh marrow. In contrast, we have found that bone marrow cultures derived from the Syrian hamster do not require a repopulation step and produce stem cells that proliferate and differentiate for more than 12 wk in the absence of an adherent layer. Stem cells were grown in Fisher's medium (pH 7.0-7.2) containing 20% horse serum in a fully humidified atmosphere of 5% CO2 in air at 37 degrees C. Cultures were fed twice weekly by removal of half of the medium and supernatant cells and replacement with an equal volume of fresh medium. No hormones or exogenous growth factors were required for the maintenance of myeloid cells, monocytes, and megakaryocytes in either the adherent or suspension cells cultures.     

Presence of mixed colony-forming cells in long-term hamster bone marrow suspension cultures: response to pokeweed spleen conditioned medium

In contrast to the murine system, long-term hamster bone marrow suspension cultures maintain proliferation of both pluripotent and committed stem cells in the absence of an adherent layer and without addition of exogenous factors, such as hydrocortisone. Addition of pokeweed-mitogen-stimulated hamster spleen conditioned medium (SCM) to these long-term suspension cultures produces an increase in the number of mixed colonies assayed in soft-agar, These mixed colonies, which contained four cell lineages--granulocytic, erythroid, megakaryocytic, and macrophage--could be generated from cells grown in suspension for over 6 mo. Addition of SCM also induces an initial rapid expansion of the myeloid compartment, and this expansion results in 70% of the cells being terminally differentiated granulocytes. In contrast, addition of SCM to hamster bone marrow cultures containing both adherent cells and hematopoietic stem cells produced no change in the number of mixed colonies generated in the culture. This system allows the in vitro study of the process of stem cell proliferation and differentiation and also provides a means to examine the relationship of adherent and supernatant bone marrow populations.     

The effect of oral magnesium, alone or as an adjuvant to sotalol, after cardioversion in patients with persistent atrial fibrillation.

AIMS: To determine whether magnesium given orally decreases the recurrence rate of atrial fibrillation after elective direct current cardioversion of persistent atrial fibrillation. METHODS AND RESULTS: Consecutive outpatients were randomized to treatment with oral magnesium (10.3 mmol) or placebo twice daily in a double-blind fashion. Two groups were studied; magnesium study: 170 patients with atrial fibrillation persistent for >1 month, scheduled for their first direct current cardioversion. No concomitant antiarrhythmic drugs of class I or III were allowed. Sotalol and magnesium study: 131 patients with recurrence of persistent atrial fibrillation after previous direct current cardioversion, or a history of paroxysmal atrial fibrillation, treated with sotalol. Patients were followed until recurrence of atrial fibrillation or for at least 6 months. Magnesium study: at cardioversion 67 of 85 (79%) in the placebo group and 64 of 85 (75%) in the magnesium group had converted to sinus rhythm. At the end of the study, with a follow-up of 6 to 42 months, 15% of patients in the placebo group and 19% of patients in the magnesium group remained in sinus rhythm (Log rank test: P=0.37). Sotalol and magnesium study: pharmacological conversion to sinus rhythm, after oral treatment, was achieved in 34 of 131 (26%) patients. Sinus rhythm, with or without cardioversion, was restored in 89% and 85% of the patients in the placebo and magnesium groups, respectively. At the end of the study, with a follow-up of 6 to 42 months, 37% of patients in the placebo group and 30% of patients in the magnesium group remained in sinus rhythm (Log rank test: P=0.64). CONCLUSION: In patients with persistent atrial fibrillation, oral treatment with magnesium alone or as an adjuvant to sotalol, does not influence the recurrence rate of atrial fibrillation after elective cardioversion.     

Rationale, design, methods and baseline demography of participants of the Anglo-Scandinavian Cardiac Outcomes Trial. ASCOT investigators

OBJECTIVE: To test the primary hypothesis that a newer antihypertensive treatment regimen (calcium channel blocker +/- an angiotensin converting enzyme inhibitor) is more effective than an older regimen (beta-blocker +/- a diuretic) in the primary prevention of coronary heart disease (CHD). To test a second primary hypothesis that a statin compared with placebo will further protect against CHD endpoints in hypertensive subjects with a total cholesterol < or = 6.5 mmol/l. DESIGN: Prospective, randomized, open, blinded endpoint trial with a double-blinded 2 x 2 factorial component. SETTING: Patients were recruited mainly from general practices. PATIENTS: Men and women aged 40-79 were eligible if their blood pressure was > or = 160 mmHg systolic or > or = 100 mmHg diastolic (untreated) or > or = 140 mmHg systolic or > or = 90 mmHg diastolic (treated) at randomization. INTERVENTIONS: Patients received either amlodipine (5/ 10 mg) +/- perindopril (4/8 mg) or atenolol (50/ 100 mg) +/- bendroflumethiazide (1.25/2.5 mg) +K+ with further therapy as required to reach a blood pressure of < or = 140 mmHg systolic and 90 mmHg diastolic. Patients with a total cholesterol of < or = 6.5 mmol/l were further randomized to receive either atorvastatin 10 mg or placebo daily. MAIN OUTCOME MEASURE: Non-fatal myocardial infarction (MI) and fatal coronary heart disease (CHD). RESULTS: 19 342 men and women were initially randomized, of these 10297 were also randomized into the lipid-lowering limb. All patients had three or more additional cardiovascular risk factors. CONCLUSIONS: The study has 80% power (at the 5% level) to detect a relative difference of 20% in CHD endpoints between the calcium channel blocker-based regimen and the beta-blocker-based regimen. The lipid-lowering limb of the study has 90% power at the 1% level to detect a relative difference of 30% in CHD endpoints between groups.     

The cost-effectiveness of ramipril in the treatment of patients at high risk of cardiovascular events: a Swedish sub-study to the HOPE study

OBJECTIVE: To evaluate if long-term treatment with ramipril is cost-effective in patients at high risk of cardiovascular events. DESIGN: Randomized double-blind and placebo controlled. Information was gathered prospectively for a number of direct medical, direct nonmedical and indirect costs. SETTING AND SUBJECT: This is a sub-study to the Heart Outcomes Prevention Evaluation (HOPE) study performed in Swedish patients. All Swedish centres (19; n= 554) were invited to take part and 18 centres agreed to do so (n=537). The patients were managed in a specialist setting with a mean follow-up period of 4.5 years. Main outcome measures. The number of life-years saved was derived from the global HOPE study (n=9297) and subsequently the estimated life expectancy of those who completed the clinical study alive was added to the calculation. Direct medical costs related to cardiovascular disease only were considered in the primary analysis, whilst all kinds of costs and costs for all kinds of diseases were included in subsequent analyses. The cost of added years of life, according to the future cost method, was included in sensitivity analyses. RESULTS: The cost per life-year gained was SEK 16 600 (Euro 1940) when direct medical costs for cardiovascular reasons only were considered and SEK 45 400 (Euro 5300) when direct medical costs for all diseases were considered. The corresponding costs when direct nonmedical and indirect cost were added to the estimate were SEK 16 100 (Euro 1880) and SEK 54 600 (Euro 6380), respectively. When the future cost method was applied, the cost per life-year gained was SEK 208 300 (Euro 24 300). CONCLUSION: Ramipril is highly cost-effective in the treatment of patients at high risk of cardiovascular events.     

Inducibility of Atrial Fibrillation After GP Ablations and “Autonomic Blockade”: Evidence for the Pathophysiological Role of the Nonadrenergic and Noncholinergic Neurotransmitters

Autonomic Blockade and Atrial Fibrillation . Background: Recent clinical reports that used cholinergic and adrenergic blockade (CAB) as an alternative to ganglionated plexi (GP) ablation to terminate atrial fibrillation (AF) showed mixed results. We investigated the role of other neurotransmitters in AF inducibility. Methods: In 23 pentobarbital anesthetized dogs, a left and right thoracotomy allowed the attachment of electrode catheters to the left and right pulmonary veins and atrial appendages (AA). Programmed stimulation was used to determine the effective refractory periods (ERP) and AF inducibility, measured by the window of vulnerability (WOV). AF duration in response to acetylcholine (Ach; 100 mM) applied to the AA was measured before and after GP ablation + CAB and with vagus nerve stimulation (VNS). After GP ablation + CAB, Ach induced AF duration was determined in response to vasoactive intestinal peptide (VIP) and its specific antagonist ([Ac‐Tyr1,D‐phe2]‐VIP). Results: GP ablation + CAB significantly prolonged ERP, eliminated WOV, and suppressed the duration of Ach induced AF (P ≤ 0.01 for all). Also slowing of the heart rate by VNS was essentially blocked; however, with Ach 100 mM applied to the AA, VNS, and VIP applied to the AA markedly prolonged AF duration. This effect was blocked by the VIP antagonist. Conclusions: Neither GP ablation nor CAB can fully suppress AF inducibility arising from the atrial neural network. Our findings suggest that other neurotransmitters, such as VIP released during VNS, can promote sustained AF despite GP ablation and “autonomic blockade,” which may further define the substrate for AF outside the pulmonary vein‐atrial junctions. (J Cardiovasc Electrophysiol, Vol. 24, pp. 188‐195, February 2013)