The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTR-HSC and can reconstitute lethally irradiated recipients with fewer than 20 cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6 (90% to 100% HPP-CFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or IL-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or IL-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the early time points of culture was significantly augmented by Tpo. Low Ho/low Rh cells, when assayed in agar directly after sorting, did not form megakaryocyte colonies in any growth condition tested. Several days of culture in the presence of multiple cytokines were required to obtain colony-forming units-megakaryocyte (CFU-Mk). In contrast, more differentiated, low Ho/high Rh cells, previously shown to contain short- term repopulating hematopoietic stem cells (STR-HSC), were able to form megakaryocyte colonies in agar when cultured in Tpo alone directly after sorting. These data establish that Tpo acts directly on primitive hematopoietic stem cells selected using the Ho/Rh method, but this effect is dependent on the presence of pluripotent cytokines. These cells subsequently differentiate into CFU-Mk, which are capable of responding to Tpo alone. Together with the results of previous reports of its effects on erythroid progenitors, these results suggest that the effects of Tpo on hematopoiesis are greater than initially anticipated.     

Hypertension in dialysis and kidney transplant patients

For the first time, the Canadian Hypertension Education Program has studied the evidence supporting blood pressure control in people requiring renal replacement therapy for end-stage kidney disease, including those on dialysis and with renal transplants. According to the Canadian Organ Replacement Registry’s 2008 annual report, there were an estimated 33,832 people with end-stage renal disease in Canada at the end of 2006, an increase of 69.7% since 1997. Of these, 20,465 were on dialysis and 13,367 were living with a functioning kidney transplant. Thus, it is becoming more likely that primary care practitioners will be helping to care for these complex patients. With the lack of large controlled clinical trials, the consensus recommendation based on interpretation of the existing literature is that blood pressure should be lowered to below 140/90 mmHg in hypertensive patients on renal replacement therapy and to below 130/80 mmHg for renal transplant patients with diabetes or chronic kidney disease.     

Adolescents with type 1 diabetes and risky behaviour

Aim: The aim of the student is to assess whether adolescents with type 1 diabetes mellitus (T1DM) in Italy differ from their healthy peers in regard to risky behaviour. Methods: Data were collected from 215 patients, aged 14 ± 2 years with a mean disease duration of 7 ± 5 years. The control group was comprised of 464 healthy adolescents recruited among high school students. Each patient completed an anonymous confidential questionnaire to determine the prevalence of sexual behaviour, alcohol and tobacco consumption, illicit drug use, and, among patients with diabetes and frequency of mismanagement related to diabetes care. Results: Compared with controls, subjects with diabetes showed a similar rate of sexual intercourse among males and lower rates among females (34.8% vs 35.5%, p NS and 29.4% vs 41.4%, p < 0.05, respectively). Males in the diabetes group reported a higher rate of tobacco use, whereas females showed similar or higher rates of use for every illicit drug studied. Among patients with diabetes, those who are engaged in risky behaviour showed a higher rate of treatment mismanagement (76% vs 34%, p < 0.01). Conclusion: Adolescents with T1DM are as likely as their healthy peers to engage in risky behaviour, indicating the potential benefit of anticipatory guidance concerning glycaemic control and increased risk of acute and chronic complications.     

Oligosaccharides in human milk during different phases of lactation

Twenty-one oligosaccharides of human milk were quantified by high-performance anion-exchange chromatography. Milk samples were collected from 18 mothers during the first 3 mo of lactation. The data show that the highest amount of all oligosaccharides is present at day 4 postpartum (20 g l⁻¹) and then decreases by about 20% at day 30 of lactation. The protective role played by these substances against different infectious agents, in different organs and systems of the breastfed baby, is emphasized.     

The coeliac iceberg in Italy. A multicentre antigliadin antibodies screening for coeliac disease in school-age subjects

Background : Recent studies suggest that coeliac disease (CD) is one of the commonest, life-long disorders in Italy. The aims of this multicentre work were: (a) to establish the prevalence of CD on a nationwide basis; and (b) to characterize the CD clinical spectrum in Italy. Patients and methods : Fifteen centres screened 17201 students aged 6–15 years (68.6% of the eligible population) by the combined determination of serum IgG- and IgA-antigliadin antibody (AGA) test; 1289 (7.5%) were IgG and/or IgA-AGA positive and were recalled for the second-level investigation; 111 of them met the criteria for the intestinal biopsy: IgA-AGA positivity and/or AEA positivity or IgG-AGA positivity plus serum IgA deficiency. Results : Intestinal biopsy was performed on 98 of the 111 subjects. CD was diagnosed in 82 subjects (75 biopsy proven, 7 not biopsied but with associated AGA and AEA positivity). Most of the screening-detected coeliac patients showed low-grade intensity illness often associated with decreased psychophysical well-being. There were two AEA negative cases with associated CD and IgA deficiency. The prevalence of undiagnosed CD was 4.77 × 1000 (95% CI 3.79–5.91), 1 in 210 subjects. The overall prevalence of CD, including known CD cases, was 5.44 × 1000 (95% CI 4.57–6.44), 1 in 184 subjects. The ratio of known to undiagnosed CD cases was 1 in 7. Conclusions : These findings confirm that, in Italy, CD is one of the most common chronic disorders showing a wide and heterogeneous clinical spectrum. Most CD cases remain undiagnosed unless actively searched.     

Evolutionary constraints in conserved nongenic sequences of mammals

Mammalian genomes contain many highly conserved nongenic sequences (CNGs) whose functional significance is poorly understood. Sets of CNGs have previously been identified by selecting the most conserved elements from a chromosome or genome, but in these highly selected samples, conservation may be unrelated to purifying selection. Furthermore, conservation of CNGs may be caused by mutation rate variation rather than selective constraints. To account for the effect of selective sampling, we have examined conservation of CNGs in taxa whose evolution is largely independent of the taxa from which the CNGs were initially identified, and we have controlled for mutation rate variation in the genome. We show that selective constraints in CNGs and their flanks are about one-half as strong in hominids as in murids, implying that hominids have accumulated many slightly deleterious mutations in functionally important nongenic regions. This is likely to be a consequence of the low effective population size of hominids leading to a reduced effectiveness of selection. We estimate that there are one and two times as many conserved nucleotides in CNGs as in known protein-coding genes of hominids and murids, respectively. Polymorphism frequencies in CNGs indicate that purifying selection operates in these sequences. During hominid evolution, we estimate that a total of about three deleterious mutations in CNGs and protein-coding genes have been selectively eliminated per diploid genome each generation, implying that deleterious mutations are eliminated from populations non-independently and that sex is necessary for long-term population persistence.     

Selenium metabolism in zebrafish: multiplicity of selenoprotein genes and expression of a protein containing 17 selenocysteine residues

BACKGROUND: Fish are an important source of selenium in human nutrition and the zebrafish is a potentially useful model organism for the study of selenium metabolism and its role in biology and medicine. Selenium is present in vertebrate proteins in the form of selenocysteine (Sec), the 21st natural amino acid in protein which is encoded by UGA. RESULTS: We report here the detection of 18 zebrafish genes for Sec-containing proteins. We found two zebrafish orthologs of human SelT, glutathione peroxidase 1 and glutathione peroxidase 4, and single orthologs of several other selenoproteins. In addition, new zebrafish selenoproteins were identified that were distant homologues of SelP, SelT and SelW, but their direct orthologs in other species are not known. This multiplicity of selenoprotein genes appeared to result from gene and genome duplications, followed by the retention of new selenoprotein genes. We found a zebrafish selenoprotein P gene (designated zSelPa) that contained two Sec insertion sequence (SECIS) elements and encoded a protein containing 17 Sec residues, the largest number of Sec residues found in any known protein. In contrast, a second SelP gene (designated zSelPb) was also identified that contained one SECIS element and encoded a protein with a single Sec. We found that zSelPa could be expressed and secreted by mammalian cells. CONCLUSIONS: The occurrence of zSelPa and zSelPb suggested that the function of the N-terminal domain of mammalian SelP proteins may be separated from that of the C-terminal Sec-rich sequence: the N-terminal domain containing the UxxC motif is likely involved in oxidoreduction, whereas the C-terminal portion of the protein may function in selenium transport or storage. Our data also suggest that the utilization of Sec is more common in zebrafish than in previously characterized species, including mammals.