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.     

Mixed-Micellar Proliposomal Systems for Enhanced Oral Delivery of Progesterone

The objective of our study was to develop a mixed-micellar proliposomal formulation of poorly water-soluble drug progesterone and evaluate the dissolution profile and membrane transport. Several formulations of proliposomes were prepared by mixing different concentrations of lipid, progesterone, polysorbate 80, and microcrystalline cellulose. The mixed-micellar formulation of drug:dimyristoyl-phosphatidycholine:polysorbate 80 (1:20:3.3) exhibited the maximum dissolution (75.27%), while pure progesterone resulted in low dissolution. The above formulation showed a 4-fold increase in transport in Caco-2 cells and a 6-fold increase in transport across the everted rat intestinal sac experiments compared with control. Proliposomal formulations enhance the extent of dissolution and membrane transport of progesterone and serve as ideal carriers for oral delivery of drugs with low water solubility.     

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.     

Comparative assessment of in vitro release kinetics of calcitonin polypeptide from biodegradable microspheres

The objective of our study was to compare the in vitro release kinetics of a sustained-release injectable microsphere formulation of the polypeptide drug, calcitonin (CT), to optimize the characteristics of drug release from poly-(lactide-co-glycolide) (PLGA) copolymer biodegradable microspheres. A modified solvent evaporation and double emulsion technique was used to prepare the microspheres. Release kinetic studies were carried out in silanized tubes and dialysis bags, whereby microspheres were suspended and incubated in phosphate buffered saline, sampled at fixed intervals, and analyzed for drug content using a modified Lowry protein assay procedure. An initial burst was observed whereby about 50% of the total dose of the drug was released from the microspheres within 24 hr and 75% within 3 days. This was followed by a period of slow release over a period of 3 weeks in which another 10-15% of drug was released. Drug release from the dialysis bags was more gradual, and 50% CT was released only after 4 days and 75% after 12 days of release. Scanning electron micrographs revealed spherical particles with channel-like structures and a porous surface after being suspended in an aqueous solution for 5 days. Differential scanning calorimetric studies revealed that CT was present as a mix of amorphous and crystalline forms within the microspheres. Overall, these studies demonstrated that sustained release of CT from PLGA microspheres over a 3-week period is feasible and that release of drug from dialysis bags was more predictable than from tubes.     

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.     

Transdermal lontophoretic delivery of colchicine encapsulated in liposomes

Iontophoresis is useful for the transdermal delivery of charged drugs. However, nonionized drugs either have a low flux (due to electro-osmosis) or cannot be delivered using this technique. Because ionized or nonionized drugs can be encapsulated in charged liposomes, it was hypothesized that charged liposomes can deliver neutral or nonionized drug efficiently by iontophoresis. Colchicine, a neutral drug, was encapsulated in large unilamellar vesicles (LUVs), prepared with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) along with cholesterol (1:0.5 mole ratio). Multilamellar vesicles (MLVs) were prepared by the thin-film hydration method and LUVs were obtained by extruding MLVs through polycarbonate filters of 200 nm pore size. Positive charge was induced in the liposomes by adding stearylamine and negative charge by adding dicetyl phosphate. Nonencapsulated drug was separated from LUVs by the Ficoll density gradient method. Positively charged LUVs were delivered under the anode, negatively charged LUVs under the cathode, and neutral LUVs without current using Franz cells and human cadaver skin. Plain colchicine as well as colchicine encapsulated in positively charged LUVs was delivered better under the anode compared with the cathode and passive conditions. Delivery of colchicine encapsulated in positively charged DSPC liposomes was four to five times greater than that of plain colchicine and two to three times greater than that of colchicine encapsulated in DMPC or DPPC liposomes. Because LUVs prepared with DMPC and DPPC were fluid at 37°C, the encapsulated drug leaked during iontophoresis and therefore the delivery was less. Delivery of colchicine was lower under the cathode due to the change in pH during iontophoresis, which, as observed in high-performance liquid chromatographic analysis, caused degradation of the drug. Thus, it can be concluded that iontophoresis of colchicine encapsulated in positively charged DSPC liposomes can improve its delivery across human cadaver skin     

Iontophoretic in Vivo Transdermal Delivery of β-Blockers in Hairless Rats and Reduced Skin Irritation by Liposomal Formulation

Purpose. To demonstrate the in vivo transdermal delivery and establish the comparative pharmacokinetics of five -blockers in hairless rat. Methods. Intravenous dosing was initially done via jugular cannula. For iontophoretic delivery, current (0.1 mA/cm²) was applied for 2 h through a drug reservoir patch containing the -blocker (10 mg/ml). Blood samples were collected and analyzed by stereoselective HPLC assays. Any irritation resulting from patch application was quantified by a chromameter. Multilamellar liposomal formulation was prepared by the thin-film hydration method and converted to unilamellar liposomes by extrusion. Results. With transdermal iontophoresis, therapeutically relevant amounts of propranolol (83.78 ± 7.4 ng/ml) were delivered within an hour and lasted for up to 4 h. C_max (185.1 ± 56.8 ng/ml) was reached at hour 3. A significantly higher amount (p < 0.05) of sotalol HCl was delivered compared to other -blockers. There was no significant difference in the S/R ratio of AUC_0-t for enantiomers after both intravenous and transdermal delivery. Skin irritation was significantly reduced (p < 0.05) when a liposomal formulation of the propranolol base was used rather than the base itself. Conclusions. The comparative pharmacokinetics of intravenous and transdermal iontophoretic delivery of five -blockers in hairless rats was established. It was shown that there is no stereoselective permeation.     

Formulation, characterization, and in vitro release of glyburide from proliposomal beads

The objective of our study was to formulate and evaluate proliposomes in the form of enteric-coated beads using glyburide as a model drug. The beads were enteric coated with Eudragit L-100 by a fluidized bed coating process using triethyl citrate as plasticizer. Content uniformity of glyburide was estimated using HPLC analysis of beads dissolved in methanol. These proliposomal beads formed liposomes on disintegration in phosphate buffered saline (pH 7.4), which was confirmed by transmission electron microscopy. The dissolution study of enteric-coated beads exhibited enhanced dissolution compared with pure drug and a marketed product. Liposomes can be successfully prepared for oral administration in the form of enteric-coated beads that may offer a stable system to produce liposomes for oral administration.