Search

The design, fabrication, and measurement of a phase-shift Bragg grating in silicon using the equivalent phase-shift method are presented. Fabrication constrains are largely relaxed with this method, compared to that with conventional phase-shift structure using electron-beam lithography, enabling the fabrication of high-precision phase-shift gratings with low-cost lithography. We fabricated an equivalent phase-shift Bragg grating in silicon with optical-contact-lithography and interference lithography. The transmission spectrum shows that an equivalent quarter-wave phase-shift is achieved. [ABSTRACT FROM PUBLISHER]

Abstract Background Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity. Results After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation. Conclusion Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

Targeting constitutively activated FMS-like tyrosine kinase 3 [(FLT3); FLT3-ITD] with tyrosine kinase inhibitor (TKI) in acute myeloid leukemia (AML) leads to clearance of blasts in the periphery but not in the bone marrow, suggesting a protective effect of the marrow niche on leukemic stem cells. In this study, we examined the effect of stromal niche cells on CD34(+) progenitors from patients with FLT3-ITD(+) or wild-type FLT3 (FLT3-WT) AML treated with the TKIs SU5614 or sorafenib. TKIs effectively and specifically inhibited FLT3 and increased the fraction of undivided progenitors in both FLT3-ITD(+) and FLT3-WT samples. Treatment with SU5614 and sorafenib also reduced the number of mature leukemic progenitors, whereas contact with stroma protected against this cell loss. In contrast, primitive long-term progenitors from both FLT3-ITD(+) and FLT3-WT AML were resistant to TKIs. Additional contact with niche cells significantly expanded long-term FLT3-ITD(+) but not FLT3-WT progenitors in the presence of SU5614 but not that of sorafenib. Thus, TKIs with first-generation inhibitors fail to eradicate early leukemic stem/progenitor cells in FLT3-ITD(+) AML. Further, we defined a specific interaction between FLT3-ITD(+) progenitors and niche cells that enables the maintenance of leukemic progenitors in the presence of TKI. Collectively, our findings suggest that molecular therapy may have unpredicted effects on leukemic progenitors, underscoring the necessity of developing strategies to selectively eliminate the malignant stem cell clone., (©2011 AACR.)

Mesenchymal stem cells (MSC) represent a mixture of different cell types, of which only a minority is therapeutically relevant. Surface markers specifically identifying non-differentiated MSC from their differentiated progeny have not been described in sufficient detail. We here compare the gene expression profile of the in vivo bone-forming bone marrow-derived MSC (BM-MSC) with non-bone-forming umbilical vein stromal cells (UVSC) and other non-MSC. Clustering analysis shows that UVSC are a lineage homogeneous cell population, clearly distinct from MSC, other mesenchymal lineages and hematopoietic cells. We find that 89 transcripts of membrane-associated proteins are represented more in cultured BM-MSC than in UVSC. These include previously identified molecules, but also novel markers like NOTCH3, JAG1, and ITGA11. We show that the latter three molecules are also expressed on fibroblast colony-forming units (CFU-F). Both NOTCH3 and ITGA11, but not JAG1, further enrich for CFU-F when combined with CD146, a known marker of cells with MSC activity in vivo. Differentiation studies show that NOTCH3+ and CD146+ NOTCH3+ cells sorted from cultured BM-MSC are capable of adipogenic and osteogenic progeny, while ITGA11-expressing cells mainly show an osteogenic differentiation profile with limited adipogenic differentiation. Our observations may facilitate the study of lineage relationships in MSC as well as facilitate the development of more homogeneous cell populations for mesenchymal cell therapy., (2010 Elsevier Inc. All rights reserved.)

Background: Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity., Results: After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1alpha expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation., Conclusion: Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

Multipotent mesenchymal stromal cells (MSC) have immunomodulatory effects. The aim of this study was to demonstrate safety and feasibility of MSC transfusion in pediatric patients who had undergone allogeneic stem cell transplantation from MMFD, MUD, MMUD and MSD. Patients with posttransplant complications based on deregulated immune effector cells who may benefit from an immunomodulatory effect of MSC had been selected. MSC were isolated from the hematopoietic stem cell donors in five cases and from a third party parental donor in two cases. We transfused ex vivo-expanded MSC in 11 doses into seven pediatric patients. Cell doses were escalated based on availability from 0.4x10(6) to 3.0x10(6) per kg bodyweight No adverse effects were detected with a maximum follow-up of 29 months. One out of three patients showed slight improvement of chronic GVHD. Two patients with severe acute GvHD did not progress to cGvHD. One patient received MSC to stabilize graft function after secondary haploidentical transplantation. One patient recovered from trilineage failure due to severe hemophagocytosis. This is the first case of a pediatric patient treated with MSC for trilineage failure after haploidentical stem cell transplantation from her father. We report the first series of 11 transfusions of expanded MSC in pediatric patients with immunological complications after allogeneic transplantation. Transfusion of MSC was safe and encouraging improvements in some patients were observed.

Multipotent mesenchymal stromal cells (MSCs) play an important role in stromal support for hematopoietic stem cells, immune modulation, and tissue regeneration. We investigated their potential as cellular therapeutic tools in neurometabolic diseases as a growing number of affected children undergo to bone marrow transplantation. MSCs were isolated from bone marrow aspirates and expanded ex vivo under various culture conditions. MSCs under optimal good medical practice (GMP)-conform culture conditions showed the typical morphology, immunophenotype, and plasticity. Biochemically, the activities of beta-hexosaminidase A, total beta-hexosaminidase, arylsulfatase A (ASA), and beta-galactosidase measured in MSCs were comparable to those in fibroblasts of healthy donors. These four enzymes were interesting for their expression in MSCs, as each of them is defective, respectively, in well-known neurometabolic diseases. We found that MSCs released significant amounts of ASA into the media. In coculture experiments, fibroblasts from patients with metachromatic leukodystrophy, who are deficient for ASA, took up a substantial amount of ASA that was released into the media from MSCs. Mannose-6-phosphate (M6P) inhibited this uptake, which was in accordance with the M6P receptor-mediated uptake of lysosomal enzymes. Taken together, we show that MSCs produce appreciable amounts of lysosomal enzyme activities, making these cells first-choice candidates for providing metabolic correction when given to enzyme-deficient patients. With the example of ASA, it was also shown that an enzyme secreted from MSCs is taken up by enzyme-deficient patient fibroblasts. Given the plasticity of MSCs, these cells represent an interesting add-on option for cellular therapy in children undergoing bone marrow transplantation for lysosomal storage diseases and other neurometabolic diseases.

Objectives: The present study investigated endogenous postprandial release of cholecystokinin (CCK) and pancreatic polypeptide (PP) in relation to gallbladder dynamics in healthy subjects and patients with gallstones., Methods: Gallbladder volume (by ultrasonography) and plasma concentrations of CCK and PP (by radioimmunoassay) were evaluated in 18 patients with gallstones and 14 healthy controls before and after administration of a semi-liquid test meal (250 ml, 1450 kJ). Gallbladder contractility was previously assessed on a separate day by intravenous infusion of ceruletide (2.5 ng/kg/min)., Results: Basal gallbladder volume was not different in patients (32 +/- 5.9 cm3) and controls (26 +/- 2.7 cm3). Postprandial gallbladder contractility was impaired in gallstone patients, who showed a reduced integrated response (-3718 +/- 349 vs. -5251 +/- 376 cm3/2 h, p < 0.01) and a delayed time to maximal gallbladder contraction (67 +/- 7.4 min vs. 37 +/- 2.4 min, p < 0.002). Maximal gallbladder contraction after ceruletide infusion was also reduced (44.1 +/- 5.0% vs. 72.5 +/- 3.2%, p < 0.001), but not delayed (15.8 +/- 2.4 vs. 15.7 +/- 1.4 min) in gallstone patients. Basal CCK and PP plasma levels were similar in both groups. Postprandial CCK release was impaired in gallstone patients, predominantly due to a decreased response over the first 30 min (3.8 +/- 1.8 vs. 20.0 +/- 4.9 pmol/L/30 min, p < 0.005). Postprandial PP release was not different between groups. A direct linear correlation between postprandial release of CCK and PP was found in healthy controls but not in patients with gallstones. Postprandial gallbladder volume at any moment was inversely correlated with CCK plasma levels in healthy subjects, but not in gallstone patients. No correlation between postprandial PP response and gallbladder dynamics was observed., Conclusions: Based on a multivariate logistic approach, a reduced and delayed postprandial gallbladder contractility and an impaired CCK release in the early postprandial phase are significantly associated with gallstone disease. Our data provide further evidence for the predominant role of endogenous postprandial CCK release in gallbladder contraction. A role for PP in modulating postprandial gallbladder dynamics is not supported.

Postprandial gastric emptying and gallbladder contraction were assessed in 14 healthy subjects by means of ultrasonography after oral administration of a semi-liquid test meal (250 ml, 1450 kJ). For this purpose, cross-sectional areas of the gastric antrum and gallbladder volume were calculated and recorded over a period of 120 minutes using an annular-array-transducer. The semi-liquid test meal allowed suitable sonographic measurement of cross-sectional areas of the antrum in all 14 subjects. Mean half-time of gastric emptying was 47 minutes (range 17-72 minutes). Mean peak gallbladder contraction was 36% (range 17-60%) of initial volume and mean time to peak contraction was 65 minutes (range 20-120 minutes). The method described was found to be a practical and reliable procedure for the investigation of postprandial gastric emptying and gallbladder contraction. It is therefore of potential interest for application to a variety of clinical questions. A minor drawback involves the relative length and variability of time required for testing, owing to the wide range in the time-course of gastric emptying and gallbladder contraction across subjects. Normal ranges for defined test meals must be established in large control groups.