Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo

Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38- cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38- subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long- term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38- cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.     

Global Analysis of Methylation Profiles From High Resolution CpG Data

New high throughput technologies are now enabling simultaneous epigenetic profiling of DNA methylation at hundreds of thousands of CpGs across the genome. A problem of considerable practical interest is identification of large scale, global changes in methylation that are associated with environmental variables, clinical outcomes, or other experimental conditions. However, there has been little statistical research on methods for global methylation analysis using technologies with individual CpG resolution. To address this critical gap in the literature, we develop a new strategy for global analysis of methylation profiles using a functional regression approach wherein we approximate either the density or the cumulative distribution function (CDF) of the methylation values for each individual using B‐spline basis functions. The spline coefficients for each individual are allowed to summarize the individual's overall methylation profile. We then test for association between the overall distribution and a continuous or dichotomous outcome variable using a variance component score test that naturally accommodates the correlation between spline coefficients. Simulations indicate that our proposed approach has desirable power while protecting type I error. The method was applied to detect methylation differences, both genome wide and at LINE1 elements, between the blood samples from rheumatoid arthritis patients and healthy controls and to detect the epigenetic changes of human hepatocarcinogenesis in the context of alcohol abuse and hepatitis C virus infection. A free implementation of our methods in the R language is available in the Global Analysis of Methylation Profiles (GAMP) package at http://research.fhcrc.org/wu/en.html .     

Amiloride delays the ischemia-induced rise in cytosolic free calcium

An increase in cytosolic free calcium (Cai) has been shown to occur early during ischemia in perfused rat, ferret, and rabbit hearts. It has been proposed that this increase in Cai may occur as a result of exchange of Nai for Cao, which occurs as a result of an increase in Nai arising from exchange of Nao for H+i. The latter exchange is stimulated by the intracellular acidification that occurs during ischemia. To test this hypothesis, we examined Cai, Nai, ATP, and pHi during ischemia in rats in the presence and absence of 1 mM amiloride, a Na-H exchange inhibitor. Cai was measured using 19F nuclear magnetic resonance (NMR) of 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetra-acetic acid (5F-BAPTA)-loaded rat hearts. Nai was measured using 23Na NMR, and the shift reagent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetramethylenephosph onate (Tm[DOTP]-5) was used to separate Nai and Nao. ATP and pH were determined from 31P NMR measurements. During 20 minutes of ischemia, amiloride did not significantly alter the ATP decline but did significantly attenuate the rise in Nai and Cai. After 20 minutes of ischemia, time-averaged Cai was 1.0 +/- 0.2 microM (mean +/- SEM) in amiloride-treated hearts compared with 2.3 +/- 0.9 microM in nontreated hearts. After 20 minutes of ischemia, Nai in the untreated heart was threefold greater than control, whereas in the amiloride-treated heart, Nai was not significantly different from control. These data are consistent with the involvement of Na-Ca exchange in the rise in Cai during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of cholecystokinin in the gastrocolonic response to a fat meal

The fat component of the meal is the major stimulant of the gastrocolonic response. The aim of this study was to characterize the mechanism of the gastrocolonic response after eating fat in healthy human volunteers. A bipolar clip electrode recorded spike activity (SP) from the distal colon. An immediate increase in colonic spike activity occurred after eating a fat meal (18.0 +/- 3.0 SP/30 min) (p less than 0.02). Spike activity remained elevated for 90 min after eating the fat meal (18.6 +/- 2.7 SP/30 min) (p less than 0.01). The intravenous infusion of naloxone or atropine inhibited the fat-induced increase in colonic motility. Cholecystokinin is a candidate mediator of the fat-stimulated gastrocolonic response. The intravenous infusion of the octapeptide of cholecystokinin (20 ng/kg . h) stimulated an increase in distal colonic spike activity (16.0 +/- 3.7 SP/30 min) (p less than 0.025). Intravenous atropine did not affect stimulation of colonic motility by the octapeptide of cholecystokinin. Continuous infusion of naloxone, however, did inhibit stimulation of colonic spike activity by the octapeptide of cholecystokinin. There was no cross-reactivity between opiate or muscarinic receptors. These data suggest (a) fat stimulation of the gastro-colonic response required a muscarinic and opiate receptor, and (b) octapeptide of cholecystokinin can stimulate colonic motility, but it is not the major mediator of the gastrocolonic response.     

Total effective compliance of the vascular bed in essential hypertension

Total effective vascular compliance, hemodynamic parameters, cardiopulmonary (CPBV) and total blood volumes (TBV) were determined in 31 men, including nine normotensive controls and 22 permanent essential hypertensive patients. The effective compliance was calculated from the changes in central venous pressure recorded simultaneously with the changes in blood volume obtained after a rapid dextran infusion. In hypertensives, compliance was significantly reduced (1.55 +/- 0.6 vs 2.25 +/- 0.11 ml./mm. Hg/Kg. in controls) (P less than 0.001) and negatively correlated with blood pressure (P less than 0.01), cardiac index (P less than 0.01), and the CPBV/TBV ratio (P less than 0.01). These results suggest that venous compliance contributes to the control of cardiac output in essential hypertension.     

Therapeutic studies and arterial stiffness in hypertension: recommendations of the European Society of Hypertension. The Clinical Committee of Arterial Structure and Function. Working Group on Vascular Structure and Function of the European Society of Hypertension

BACKGROUND: Increased pulse pressure and arterial stiffness are identified as predictors of cardiovascular risk in older hypertensive populations, particularly that of myocardial infarction. Because increased pulse pressure involves an increase in systolic (SBP) and a decrease in diastolic blood pressure (DBP), and because the former promotes cardiac hypertrophy and the latter alters coronary perfusion, a drug regimen reducing pulse pressure and decreasing arterial stiffness might further reduce cardiovascular risk. Under conventional treatment, normalization of DBP (< or = 90 mmHg) is not consistently associated with normalization of SBP (< or = 140 mmHg). THERAPEUTIC DESIGNS: In individuals older than 50 years, the goal of antihypertensive treatment should be, not only to decrease mean blood pressure (to less than 100 mmHg), but also to decrease pulse pressure (to less than 50 mmHg). Using appropriate pharmacological tools, trials should test whether an active decrease in arterial stiffness might produce an attenuation of the age-related increase in SBP and decrease in DBP, thus delaying the age-related increase in pulse pressure and decreasing further cardiovascular risk. This procedure requires concomitant non-invasive evaluations of aortic stiffness. CONCLUSION: The studies that are required in hypertension should use two different approaches: novel titrations of conventional drugs to achieve a decrease in either SBP or pulse pressure, and development of new drugs acting selectively on the large artery wall, to facilitate the conduct of subsequent controlled trials.     

Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA and oxidized glutathione: indirect mode of action on polypeptide chain initiation.

In the presence of added double-stranded RNA or oxidized glutathione, protein synthesis in heminsupplemented reticulocyte lysates declines abruptly after 8-12 min of incubation at 30 degrees. The kinetics of amino-acid incorporation are very similar to those seen when lysates incorporation are very similar to those seen when lysates are incubated in the absence of added hemin. The inhibitory effects of double-stranded RNA (dsRNA) and oxidized glutathione (GSSG) are partially overcome by a homogeneous initiation factor, IF-MP, which also stimulates protein synthesis in hemin-deficient lysates. This factor is involved in the binding of Met-tRNAfmet to 40S ribosomal subunits during protein chain initiation. However, neither dsRNA alone nor GSSG alone significantly inhibits formation of [40S subunit-Met-tRNAf] complexes induced in reticulocyte lysates by dsRNA or GSSG involves one or more components present in the lysates but absent from the fractionated in vitro system. Such components may be related to the translational inhibitor that is active in hemin-deficient lysates.