Effects of HOXB4 downstream targets on the haemopoietic differentiation of pluripotent stem cells

Attempts for the in vitro differentiation of reconstituting human HSCs from ESCs have been unsuccessful as key factors of HSC specification remain unclear. Enforced HOXB4 expression can enhance haemopoietic differentiation of mouse and human ESCs and generate reconstituting HSCs from mouse ESCs. We have previously shown that HOXB4 can enhance haemopoietic differentiation of mouse ESCs in a paracrine manner. Microarray analysis identified a number of secreted factors upregulated by HOXB4 potentially mediating this paracrine effect. The aim of this study was to assess whether these factors alone and/or in combination can enhance the in vitro haemopoietic differentiation of mouse and human ESCs. We first developed a defined, serum and feeder-free protocol to test the effects of these secreted factors on haemopoietic differentiation. This defined protocol allowed us to compare the haemopoietic potential of mouse ESCs with the recently derived epiblast stem cells (EpiSCs), thought to be comparable to hESCs. Haemopoietic colony forming assay and flow cytometry analysis showed that serum-free conditions generated 8- 10 fold more CD144+CD41+ and c-Kit+CD41+ haemopoietic progenitor cells (HPCs) compared to serum conditions from both ESCs and EpiSCs, with ESCs giving the most significant increase. We then validated the panel of HOXB4 target genes by QRT-PCR and selected those increased in expression by at least 2.5-fold when HOXB4 was activated. We used this defined, serum-free protocol to assess the effects of our panel of secreted factors, FGF17, RSPO3 and APLN, on the haemopoietic differentiation of mouse ESCs. We demonstrated that FGF17 can mediate HOXB4 haemopoietic activity by enhancing the generation of c-Kit+ HPCs. On the other hand increasing concentrations of RSPO3 inhibited haemopoietic development by reducing the numbers of CD41+ and CD41+CD45+ HPCs, while, APLN did not have any effects on the haemopoietic activity of the cells. We finally used the secreted factors in human ESC and iPSC differentiation cultures. We observed differences in the activity of the tested factors not only between species but also between human cell lines. These results suggest that HOXB4 haemopoietic activity is partly mediated by paracrine signalling but more complex cell interactions are probably required for it to fully exert its effects. More importantly, HOXB4 regulatory pathways differ between mouse and human cells stressing the need for careful translation of data between the two species and more detailed analysis of key human haemopoietic factors for the successful generation of reconstituting HSCs

Pharmacological and Non-Pharmacological Agents versus Bovine Colostrum Supplementation for the Management of Bone Health Using an Osteoporosis-Induced Rat Model

Osteoporosis is defined by loss of bone mass and deteriorated bone microarchitecture. The present study compared the effects of available pharmacological and non-pharmacological agents for osteoporosis [alendronate (ALE) and concomitant supplementation of vitamin D (VD) and calcium (Ca)] with the effects of bovine colostrum (BC) supplementation in ovariectomized (OVX) and orchidectomized (ORX) rats. Seven-month-old rats were randomly allocated to: (1) placebo-control, (2) ALE group (7.5 μg/kg of body weight/day/5 times per week), (3) VD/Ca group (VD: 35 μg/kg of body weight/day/5 times per week; Ca: 13 mg/kg of body weight/day/3 times per week), and (4) BC supplementation (OVX: 1.5 g/day/5 times per week; ORX: 2 g/day/5 times per week). Following four months of supplementation, bone microarchitecture, strength and bone markers were evaluated. ALE group demonstrated significantly higher Ct.OV, Ct.BMC, Tb.Th, Tb.OV and Tb.BMC and significantly lower Ct.Pr, Tb.Pr, Tb.Sp, Ct.BMD and Tb.BMD, compared to placebo (p < 0.05). BC presented significantly higher Ct.Pr, Ct.BMD, Tb.Pr, Tb.Sp, and Tb.BMD and significantly lower Ct.OV, Ct.BMC, Tb.Th, Tb.OV and Tb.BMC compared to ALE in OVX rats (p < 0.05). OVX rats receiving BC experienced a significant increase in serum ALP and OC levels post-supplementation (p < 0.05). BC supplementation may induce positive effects on bone metabolism by stimulating bone formation, but appear not to be as effective as ALE

Decisional responsibility for mechanical ventilation and weaning: an international survey

IntroductionOptimal management of mechanical ventilation and weaning requires dynamic and collaborative decision making to minimize complications and avoid delays in the transition to extubation. In the absence of collaboration, ventilation decision making may be fragmented, inconsistent, and delayed. Our objective was to describe the professional group with responsibility for key ventilation and weaning decisions and to examine organizational characteristics associated with nurse involvement.MethodsA multi-center, cross-sectional, self-administered survey was sent to nurse managers of adult intensive care units (ICUs) in Denmark, Germany, Greece, Italy, Norway, Switzerland, Netherlands and United Kingdom (UK). We summarized data as proportions (95% confidence intervals (CIs)) and calculated odds ratios (OR) to examine ICU organizational variables associated with collaborative decision making.ResultsResponse rates ranged from 39% (UK) to 92% (Switzerland), providing surveys from 586 ICUs. Interprofessional collaboration (nurses and physicians) was the most common approach to initial selection of ventilator settings (63% (95% CI 59 to 66)), determination of extubation readiness (71% (67 to 75)), weaning method (73% (69 to 76)), recognition of weaning failure (84% (81 to 87)) and weaning readiness (85% (82 to 87)), and titration of ventilator settings (88% (86 to 91)). A nurse-to-patient ratio other than 1:1 was associated with decreased interprofessional collaboration during titration of ventilator settings (OR 0.2, 95% CI 0.1 to 0.6), weaning method (0.4 (0.2 to 0.9)), determination of extubation readiness (0.5 (0.2 to 0.9)) and weaning failure (0.4 (0.1 to 1.0)). Use of a weaning protocol was associated with increased collaborative decision making for determining weaning (1.8 (1.0 to 3.3)) and extubation readiness (1.9 (1.2 to 3.0)), and weaning method (1.8 (1.1 to 3.0). Country of ICU location influenced the profile of responsibility for all decisions. Automated weaning modes were used in 55% of ICUs.ConclusionsCollaborative decision making for ventilation and weaning was employed in most ICUs in all countries although this was influenced by nurse-to-patient ratio, presence of a protocol, and varied across countries. Potential clinical implications of a lack of collaboration include delayed adaptation of ventilation to changing physiological parameters, and delayed recognition of weaning and extubation readiness resulting in unnecessary prolongation of ventilation

Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells

We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ERT2 expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43+CD34+ progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a+/CD71+ erythroid cells. However, this did not result in a significant increase in more mature CD235a+/CD71− cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (ε) and fetal (γ) but not adult (β) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs

Cost and reimbursement analysis of end-of-life cancer inpatients. The case of the Greek public healthcare sector

BackgroundWhile hospital-based Palliative Care services are usually covered through the main funding healthcare framework, traditional reimbursement methods have been criticized for their appropriateness. The present study investigates for the first time the case of treating end-of-life cancer patients in a Greek public hospital in terms of cost and reimbursement.MethodsThis retrospective observational study used health administrative data of 135 deceased cancer patients who were hospitalized in the end of their lives. Following the cost estimation procedure, which indentified both the individual patient and overhead costs, we compared the relevant billing data and reimbursement requests to the estimated costs.ResultsThe average total cost per patient per day was calculated to be 97 EUR, with equal participation of individual patient’s and overhead costs. Length of stay was identified as the main cost driver. Reimbursement was performed either by per-diem fees or by Diagnosis Related Groups’ (DRGs), which were correspondingly associated with under or over reimbursement risks. In the case of the combined use of the two available reimbursement alternatives a cross-subsidization phenomenon was described.ConclusionAlthough the cost of end-of-life care proved to be quite low, the national per-diem rate fails to cover it. DRGs designed for acute care needs are rather unsuitable for such sub acute hospitalizations.Policy summaryThere is a concrete need for reconsidering the current reimbursement schemes for this group of patients as part of any national plan concerning the integration and reformation of Palliative Care services. Otherwise, there is a serious danger for public institutions’ reluctance to admit them with a serious impact on access and equity of end-of-life cancer care

Nutraceutical Supplementation Based on Colostrum as Osteoporosis Treatment: A Pilot Study

Introduction: Naturally based treatments for osteoporosis are currently limited. The purpose of this investigation was to ascertain whether bovine colostrum supplementation can improve bone health in humans. Methods: In total 63 individuals volunteered in a 4-month supplementation project. They were stratified into three groups: 1) healthy post-menopausal women (n = 24); 2) individuals with osteopenia (n = 25); 3) people with osteoporosis (n = 14). Participants of each group were randomly assigned into two experimental sub-groups: a) the bovine colostrum (BC) supplementation (200 mL/day; 5 days/week); b) the placebo sub-group. Before and after the 4-month supplementation, blood samples were obtained and bone mineral density (BMD) was measured. Dual-Energy X-ray Absorptiometry (DXA) was performed on three different anatomical sites: lumbar spine (LS), left femur neck (FN), and left forearm (Arm). Bone health markers (bone alkaline phosphatase (BAP), osteocalcin, C-terminal telopeptide (CTX-I), deoxypyridinoline (DPD)) as well as immunological markers (interleukin 6 (IL6) and immunoglobulin E (IgE)), were assessed in blood serum with enzyme immunoassays, at baseline and 4-months after BC supplementation. Results: No significant changes were found in bone densitometry factors (p > 0.05), for all studied blood parameters and their calculated effect sizes. Conclusions: It is concluded that, as studied herein, BC does not seem to affect human bone health. This pilot study though warrant the need for further research into the efficacy of BC in patients with osteoporosis