Changes in the effect of isoflurane on N-methyl-D-aspartic acid-gated currents in cultured cerebral cortical neurons with time in culture: evidence for subunit specificity

BACKGROUND: Developmental changes in NR1 splice variants and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor have been associated with changes in the sensitivity of NMDA receptors to agonists, antagonists, and pharmacologic modulators. The authors have investigated changes in the effect of isoflurane on NMDA-gated currents from cultured cortical neurons with time in culture and related these changes to the subunit composition of the NMDA receptors. METHODS: N-methyl-D-aspartate-gated currents were measured using whole-cell voltage clamp recording in cortical neurons cultured for 1-4 weeks and HEK 293 cells transiently expressing NR1-1a + NR2A or NR1-1a + NR2B subunit-containing receptors. NMDA alone or NMDA with treatment agents (isoflurane or ifenprodil) was applied to cells using a U tube. RESULTS: The effect of isoflurane and the NR2B selective antagonist ifenprodil on NMDA-gated currents from cortical neurons decreased significantly with time in culture. NMDA-gated currents mediated by NR2A-containing receptors were less sensitive to isoflurane than those mediated by NR2B-containing receptors. Tachyphylaxis to repeated application of isoflurane was found in cortical neurons and HEK 293 cells with recombinant NMDA receptors. Hooked tail currents were induced by isoflurane in cultured cortical neurons and HEK 293 cells with expressed NMDA receptors. CONCLUSIONS: Isoflurane inhibits NMDA-gated currents at concentrations well below 1 minimum alveolar concentration (MAC). This effect of isoflurane was subunit dependent with the NR2B-containing receptors more sensitive to isoflurane than the NR2A-containing receptors. A potent tachyphylaxis occurred after brief exposure to isoflurane.     

Functional characterization of prethymic T cells committed to alloreactivity

The results of previous experiments on MHC fully allogenic bone marrow transplantation (BMT) in nonthymectomizd recipients indicated that anti-MHC alloreactivity starts to become irreversibly committed at the prethymic level. This is a matter of some controversy. Since it is possible that conflicting results depend on the methods chosen, we reexamined our previous results by applying two new approaches. Adult thymectomized (ATX) Balb/c mice received a syngeneic fetal thymus either 3 weeks before or 3 weeks after lethal irradiation and reconstitution with C57BL/6 BM incubated in antiserum. Since monoclonal antibodies such as anti-Thy 1 are of limited value for investigations of the above type (Thy 1 antigen crosses the prethymic/thymic border), we used two highly selective, excessively cytotoxic xenoantisera for incubation of the donor BM--either a specific anti-T cell serum (SAT) that eliminated only mature T cells, or a specific antilymphocyte serum (SAL) that reacted additionally with a subset of prethymic T cells (PTC). In both experimental approaches the results were similar: Recipients of SAT-BM developed antihost reactivity, in contrast to recipients of SAL-BM. SAT-BM recipients became immunodeficient, whereas SAL-BM chimeras were immunocompetent. Late mortality was observed only following SAT treatment. Preliminary morphological findings in the lymphoid tissue of BM recipients agree fully with the functional observations. We conclude that the data confirm our previous results in nonthymectomized BM recipients--i.e., PTCs initiate antihost reactivity in MHC fully allogeneic BMT--and PTC commitment is thymus/thymus factor independent. These conclusions are discussed with regard to the problems of MHC allogeneic clinical BMT.     

Functional characterization of the immunomodulatory properties of human urine-derived stem cells

BackgroundUrine-derived stem cells (USCs) have been widely researched as a novel cell source for stem cell therapy, but their immunomodulatory characteristics remain to be investigated. This study aimed to characterize the immunomodulatory properties of human USCs.MethodsHuman USCs were isolated from fresh voiding urine samples from healthy male donors and expanded. Their cell surface markers were characterized by flow cytometry analysis and the telomerase activities for several USCs clones were determined. The immunosuppressive potential of USCs was evaluated by the performing the mixed lymphocyte reaction (MLR) [co-culture with peripheral blood mononuclear cells (PBMNCs)] and natural killer cells (NK) cytotoxicity assay. USCs cytokines release profile was determined by using human cytokine proteome array.ResultsUSCs exhibited high cell surface expression of embryonic/mesenchymal stem cells (MSCs) markers CD29, CD44, CD54, CD73, CD90, CD146, and CD166, while lacked expression of hematopoietic stem cell markers CD11, CD14, CD19, CD31, CD34, CD45, B cell marker CD79, and co-stimulatory factors CD80 and CD86, thus, exhibiting the phenotype of MSCs. MLR indicated that USCs significantly inhibited the proliferation of PBMNCs, as compared to that of the human smooth muscle cells (SMCs). In cell cytotoxicity assays, NK cells displayed less cytotoxicity against USCs than against bone marrow mesenchymal stem cells (BMSCs) and SMCs. Furthermore, upon PBMNCs stimulation, USCs secreted higher levels of immunomodulatory cytokines, including IL-6, IL-8, MCP-1, RANTES, GROα, and GM-CSF, compared to those of BMSCs, especially when directly contact mix-culture with PBMNCs.ConclusionsUSCs secreted immunoregulatory cytokines and possessed immunomodulatory properties, comparable to those of BMSCs.     

Histomorphometric study of trabecular channels in normal iliac bone

Summary Cancellous bone channels in the normal iliac crest have been studied histologically and by histomorphometry, and their biological role has been considered. Eighty percent of trabecular channels were typical osteons with the same structural and remodeling features as cortical osteons. The similarity of osteons in these two locations was corroborated by the comparability of morphometric features. The points of difference between osteons in the two types of bone were irregular configurations of trabecular osteons and marrow cells in the central canal in some. Since the number of trabecular osteons decreases with age, and since active trabecular resorption cavities were few in number, it is unlikely that additional osteons are formed in normal bone after the active modeling phase of bone growth. It is improbable that they make a significant contribution to bone remodeling since their number decreases with age, and since the available surface of trabecular channels for remodeling is extremely small. However, it is probable that they aid the intraosseous microcirculation and mineral exchange in thick trabecula and bifurcations, where they are mainly located. The demarcation curves at the 95% confidence interval, which suggest the normal range for numbers of channels, was calculated from the scatter diagram against bone area.     

In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow

Efforts toward routine islet cell transplantation as a means for reversing type 1 diabetes have been hampered by islet availability as well as allograft rejection. In vitro transdifferentiation of mouse bone marrow (BM)-derived stem (mBMDS) cells into insulin-producing cells could provide an abundant source of autologous cells for this procedure. For this study, we isolated and characterized single cell-derived stem cell lines obtained from mouse BM. In vitro differentiation of these mBMDS cells resulted in populations meeting a number of criteria set forth to define functional insulin-producing cells. Specifically, the mBMDS cells expressed multiple genes related to pancreatic beta-cell development and function (insulin I and II, Glut2, glucose kinase, islet amyloid polypeptide, nestin, pancreatic duodenal homeobox-1 [PDX-1], and Pax6). Insulin and C-peptide production was identified by immunocytochemistry and confirmed by electron microscopy. In vitro studies involving glucose stimulation identified glucose-stimulated insulin release. Finally, these mBMDS cells transplanted into streptozotocin-induced diabetic mice imparted reversal of hyperglycemia and improved metabolic profiles in response to intraperitoneal glucose tolerance testing. These results indicate that mouse BM harbors cells capable of in vitro transdifferentiating into functional insulin-producing cells and support efforts to derive such cells in humans as a means to alleviate limitations surrounding islet cell transplantation.     

Functional comparison of bone marrow-derived liver stem cells: Selection strategy for cell-based therapy

Several distinct subpopulations of bone marrow-derived liver progenitor cells were recently described. However, there is inadequate information comparing these subpopulations from a liver-function point of view. This study was undertaken to compare two subpopulations of liver progenitors: β₂-microglobulin (β₂m)-negative/Thy-1-positive cells, and liver progenitors obtained from the non-adherent cell fraction after a panning procedure. The cells were cultured under several conditions including high- and lowdose hepatocyte growth factor, various cellular densities, and different media. Growth characteristics, liver-specific metabolic capacity, and liver regeneration-associated gene expression were studied. Both isolation procedures yielded cells that produced albumin and metabolized ammonia into urea. The study demonstrated that the β₂m-negative/Thy-1-positive cell fraction metabolized ammonia into urea more efficiently and produced a superior amount of albumin compared with the panned cell fraction. The β₂m-negative/Thy-1-positive cell fraction could be optimal for the development of novel cell-based treatment strategies for congenital or acquired liver diseases. Presented at the Forty-Sixth Annual Meeting of The Society for Surgery of the Alimentary Tract, Chicago, IL, May 14–18, 2005 (poster presentation).     

Functional ultrastructure of cerebrospinal fluid drainage channels in human arachnoid villi

The functional ultrastructure of the human arachnoid villi was studied to clarify drainage channels of cerebrospinal fluid (CSF). The apical portion of each villus was usually covered by the arachnoid cell layer alone with no endothelial investment, whereas most of the stromal central core was further encompassed by a fibrous capsule with an endothelial investment. Accordingly, the CSF-blood interface was assumed to be in both the endothelial cells and the arachnoid cell layer. The former were characterized by abundant micropinocytotic vesicles and occasional intracytoplasmic vacuoles, whereas the latter was characterized by numerous extracellular cisterns measuring 10 micron in maximal diameter. There were no free communications such as endothelial open junctions or endothelium-lined tubules. In the villi affected by subarachnoid hemorrhage, endothelial cells were intact and continuous despite the erythrocyte-packed subendothelial space, which appeared to be on the verge of rupturing. Intracytoplasmic vacuoles, measuring less than 1 micron diameter, sometimes contained serum protein-like substance. Furthermore, the extracellular cisterns were distended by intact or disintegrating erythrocytes that served as a natural tracer, suggesting CSF drainage channels. It is conceivable that, in human arachnoid villi, the extracellular cisterns of the arachnoid cell layer contribute to the passive transport of CSF, whereas micropinocytosis and vacuolization mechanisms of the endothelial cells are available for active transport.     

Functional anatomy of the pisiform bone

The pisiform is the only moving structure of the canalis carpi. In the power grip, it undergoes a posterior and medial movement, limited by the pisohamate ligament. This motion is indispensable for a good prehension. The strength of the pisohamate ligament effectively balances the dislocation tendency the flexors carpi ulnaris exert on the pisiform.     

The isolation and partial characterization of a calcium-rich particulate fraction from bone cells

Ninety-five percent of the total calcium and phosphate in homogenates of cells isolated mechanically from bone was found to sediment with subcellular particles in a centrifugal field of 15000×G. A method was devised for the isolation and partial purification of this calcium-rich fraction by enzymatic digestion with deoxyribonuclease and ultracentrifugation through 70% sucrose. The resultant pellet was 10% mineral, present as some form of calcium-phosphate complex, and was rich in protein and lipid. Significant amounts of phospholipid and RNA were present also, but only small amounts of hexosamine and sialic acid. Enzyme assays and electron microscopy indicated that the fraction contained several different types of cytoplasmic particles but those which contained calcium could not be determined from available data.     

Isolation and characterization of turkey bone marrow‐derived mesenchymal stem cells

Flexor tendon injury is often associated with suboptimal outcomes and results in substantial digit dysfunction. Stem cells have been isolated from several experimental animals for the growing interest and needs of utilizing cell‐based therapies. Recently, turkey has been developed as a new large animal model for flexor tendon research. In the present study, we reported the isolation and characterization of bone marrow‐derived mesenchymal stem cells (BMSCs) from 8‐ to 12‐month‐old heritage‐breed turkeys. The isolated cells demonstrated fibroblast‐like morphology, clonogenic capacity, and high proliferation rate. These cells were positive for surface antigens CD90, CD105, and CD44, but were negative for CD45. The multipotency of turkey BMSCs was determined by differentiating cells into osteogenic, adipogenic, chondrogenic, and tenogenic lineages. There was upregulated gene expression of tenogenic markers, including mohawk, tenomodulin, and EGR1 as well as increased collagen synthesis in BMP12 induced cells. The successful isolation and verification of bone marrow‐derived MSCs from turkey would provide opportunities of studying cell‐based therapies and developing new treatments for tendon injuries using this novel preclinical large animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1419–1428, 2019.     

Functional neointima characterization of vascular prostheses in human

BACKGROUND: The purpose of this study was to evaluate neointimal functionality of synthetic vascular grafts repopulated by host cells after implantation. METHODS: We obtained reseeded inflow and outflow cannulas of 2 patients undergoing orthotopic heart transplantation after left ventricular assist device implantation 9 and 10 months before. After cell isolation we examined cellular function of reseeded cells and their capability to form a functional endothelial layer applying immunohistologic markers and quantitative Western blot for endothelial nitric oxide synthase activity. RESULTS: Neointima formation in inflow and outflow cannulas differs macroscopically and by histologic appearance. The neointima formation on the surface of the polyethylene terephthalate fiber (Dacron) grafts differs substantially from native aortic vessel wall with respect to cellular and extracellular matrix composition and cellular function. CONCLUSIONS: The neointima of Dacron prostheses is composed of cells with rudimentary physiologic endothelial function. We conclude that synthetic matrices are not suitable scaffolds for generating functional cardiovascular implants.     

Functional outcome of bone marrow stem cells: mononuclear versus mesenchymal stem cells after cellular therapy in myocardial scar in Wistar rats

Experimental studies have suggested that a bone marrow stem cell transplant into the heart produces a favorable impact on tissue perfusion, yielding a new perspective on myocardial regeneration. Studies in human beings have demonstrated an improved clinical and functional cardiac state, which has been explained mainly by the angiogenic potential of the stem cells. Our objective was to compare the functional outcome of mononuclear stem (MoSC) and mesenchymal stem (MeSC) cell therapy after myocardium infarction in rats. Forty-two rats with myocardial infarctions underwent autologous transplantation of MoSC and MeSC in animals with ejection fractions lower than 40%. The functional analysis was performed using echocardiography at baseline and at 1 month after direct injection into the ventricular wall using: 5 x 10(6) MoSC (n = 08) or 2.5 x 10(6) MeSC (n = 13) or medium controls (n = 21). Statistical significance was accepted when P < .05. Intragroup comparisons of baseline versus 1-month follow-up were performed with paired t tests. Kruskal-Wallis was used as appropriate. There was a difference in baseline left ventricular ejection fraction (LVEF) and left ventricular-end dyastolic volume between all groups. After 1 month, LVEF decreased in the control group but remained unchanged in MoSC and MeSC groups. In all groups we observed myocardial remodeling. In conclusion, we have not demonstrated functional effectiveness with either MoSC or MeSC cell type, but potentially improved myocardial perfusion needs to be analyzed.     

Functional characterization of late outgrowth endothelial progenitor cells in patients with end‐stage renal failure

Renal transplantation is potentially curative in renal failure, but long‐term efficacy is limited by untreatable chronic rejection. Endothelial damage contributes to chronic rejection and is potentially repairable by circulating endothelial progenitor cells (EPC). The frequency and function of EPC are variably influenced by end‐stage renal failure (ESRF). Here, we isolated and functionally characterized the late outgrowth EPC (LO‐EPC) from ESRF patients to investigate their potential for endothelial repair. Patients with ESRF generated more LO‐EPC colonies than healthy controls and had higher plasma levels of IL‐1rα, IL‐16, IL‐6, MIF, VEGF, Prolactin, and PLGF. Patients' LO‐EPC displayed normal endothelial cell morphology, increased secretion of PLGF, MCP‐1, and IL‐1β, and normal network formation in vitro and in vivo. They demonstrated decreased adhesion to extracellular matrix. Integrin gene profiles and protein expression were comparable in patients and healthy volunteers. In some patients, mesenchymal stem cells (MSC) were co‐isolated and could be differentiated into adipocytes and osteocytes in vitro. This is the first study to characterize LO‐EPC from patients with ESRF. Their behavior in vitro reflects the presence of elevated trophic factors; their ability to proliferate in vitro and angiogenic function makes them candidates for prevention of chronic rejection. Their impaired adhesion and the presence of MSC are areas for potential therapeutic intervention.     

Skeletal site-specific characterization of orofacial and iliac crest human bone marrow stromal cells in same individuals

Autologous grafts from axial and appendicular bones commonly used to repair orofacial bone defects often result in unfavorable outcome. This clinical observation, along with the fact that many bone abnormalities are limited to craniofacial bones, suggests that there are significant differences in bone metabolism in orofacial, axial and appendicular bones. It is plausible that these differences are dictated by site-specificity of embryological progenitor cells and osteogenic properties of resident multipotent human bone marrow stromal cells (hBMSCs). This study investigated skeletal site-specific phenotypic and functional differences between orofacial (maxilla and mandible) and axial (iliac crest) hBMSCs in vitro and in vivo. Primary cultures of maxilla, mandible and iliac crest hBMSCs were established with and without osteogenic inducers. Site-specific characterization included colony forming efficiency, cell proliferation, life span before senescence, relative presence of surface markers, adipogenesis, osteogenesis and transplantation in immunocompromised mice to compare bone regenerative capacity. Compared with iliac crest cells, orofacial hBMSCs (OF-MSCs) proliferated more rapidly with delayed senescence, expressed higher levels of alkaline phosphatase and demonstrated more calcium accumulation in vitro. Cells isolated from the three skeletal sites were variably positive for STRO 1, a marker of hBMSCs. OF-MSCs formed more bone in vivo, while iliac crest hBMSCs formed more compacted bone that included hematopoietic tissue and were more responsive in vitro and in vivo to osteogenic and adipogenic inductions. These data demonstrate that hBMSCs from the same individuals differ in vitro and in vivo in a skeletal site-specific fashion and identified orofacial marrow stromal cells as unique cell populations. Further understanding of site-specific properties of hBMSCs and their impact on site-specific bone diseases and regeneration are needed.