Osteogenic potential of rat spleen stromal cells

Evidence is mounting that an increasing number of cell populations in the adult organism already committed and/or differentiated retain the ability to reprogram themselves and give rise to a different phenotype. Bone marrow stromal cells have long been recognized as early progenitor cells for osteoblasts, chondrocytes, hematopoietic-supportive fibroblasts and adipocytes. Recent reports though have demonstrated a potential of cell populations outside the bone marrow environment to sustain bone formation under specific circumstances. The formation of bone nodules in the spleen of IL-5 transgenic mice has been recently reported (Macias et al. (2001): J. Clin. Invest. 107, 949 - 959). We thus postulated that a cell population exists in the spleen that under particular microenvironmental conditions is able to reprogram itself and pursue a fate other than the tissue-specific one. Therefore we isolated and expanded in vitro spleen-derived stromal cells. After expansion, these cells were challenged with culture conditions designed to induce osteogenic differentiation. We hypothesized that the combination of a proliferating factor (fibroblast growth factor 2) and a differentiating hormone (dexamethasone) would allow us to induce spleen-derived stromal cells to proliferate and at the same time to express osteoblast-specific genes. Thus, spleen-derived stromal cells were isolated from rat spleen and expanded in the presence of fibroblast growth factor 2 and dexamethasone. Once primary cultures reached confluence they were either switched to an osteo-inductive medium or implanted in immunodeficient mice. Although no bone formation was observed in in vivo experiments, in vitro spleen-derived stromal cells were able to deposit a mineralized matrix. Gene expression, as revealed by RT-PCR analysis, evidenced that the deposition of a mineralized matrix was concomitant with the expression of CBFA1 and osteocalcin, along with alkaline phosphatase and bone sialoprotein. Our data suggest that rat spleen-derived stromal cells can undergo osteogenic differentiation in a permissive microenvironment.     

High intensity physical exercise induced effects on plasma levels of copper and zinc

Physical exercise leads to many metabolic, cardiovascular, and muscular changes in the body. The trace elements (TE) zinc and copper are directly involved, as enzymatic cofactors, in many of these processes, especially those related to nutrients metabolism, oxygen transport, and formation of usable energy. The effects of high-intensity physical exercise on plasma levels of CU²⁺ and Zn²⁺ in 19 subjects are investigated (9 males and 10 females). Plasma copper concentration decreases, and plasma zinc concentration increases, after exercise, in both sexes. After 30 min recovery, both TE concentration values shifts-toward rest values in both sexes. These results only partially agree with literature data, probably because we used the treadmill exercise, which makes many muscles work, whereas other authors made their subjects perform a cycloergometer exercise. Physical exercise causes a marked redistribution of TE (copper and zinc) between body stores, bloodstream, and tissues. The condition of high metabolism may lead to a deficiency of TE, requiring supplementation in order to maintain high level performance.     

Brain Cytochrome Oxidase in Alzheimer''s Disease

A recent demonstration of markedly reduced (-50%) activity of cytochrome oxidase (CO; complex 4), the terminal enzyme of the mitochondrial enzyme transport chain, in platelets of patients with Alzheimer's disease (AD) suggested the possibility of a systemic and etiologically fundamental CO defect in AD. To determine whether a CO deficiency occurs in AD brain, we measured the activity of CO in homogenates of autopsied brain regions of 19 patients with AD and 30 controls matched with respect to age, postmortem time, sex, and, as indices of agonal status, brain pH and lactic acid concentration. Mean CO activity in AD brain was reduced in frontal (-26%: p less than 0.01), temporal (-17%; p less than 0.05), and parietal (-16%; not significant, p = 0.055) cortices. In occipital cortex and putamen, mean CO levels were normal, whereas in hippocampus, CO activity, on average, was nonsignificantly elevated (20%). The reduction of CO activity, which is tightly coupled to neuronal metabolic activity, could be explained by hypofunction of neurons, neuronal or mitochondrial loss, or possibly by a more primary, but region-specific, defect in the enzyme itself. The absence of a CO activity reduction in all of the examined brain areas does not support the notion of a generalized brain CO abnormality. Although the functional significance of a 16-26% cerebral cortical CO deficit in human brain is not known, a deficiency of this key energy-metabolizing enzyme could reduce energy stores and thereby contribute to the brain dysfunction and neurodegenerative processes in AD.     

Oral supplements of vitamin E improve measures of oxidative stress in plasma and reduce oxidative damage to LDL and erythrocytes in β-thalassemia intermedia patients

Fifteen β-thalassemia intermedia patients, not requiring chronic transfusional therapy, were monitored in order to check their antioxidant status, and the lipid oxidation products in plasma, LDL, and erythrocytes before and during a 9-month oral treatment with 600 mg/day vitamin E. The low level of vitamin E, and high level of malondialdehyde in plasma clearly tended to normalize after three months (P<.001), and were quite similar to control after six months. The abnormally low level of vitamin E in LDL and the four times higher than control basal level of conjugated dienes (LDL-CD), were not modified after three months of treatment. Significant changes of LDL-VE (P<.05) and of the basal LDL-CD (P<.001) were evident after six months. LDL-VE was within the normal range after nine months, whereas LDL-CD still appeared twice as higher than control.

Plasma vitamin A, ascorbate, β-carotene, and lycopene increased markedly at the end of the trial (P<.005).

The level of vitamin E in red blood cells was normalized after six months of supplementation. A decrease of the baseline value of conjugated dienes was observed after nine months, although it remained 1.4-fold higher than control. The RBC count and hematocrit appeared higher at the end of the trial (P<.05 and P<.001, respectively). The hemoglobin value did not show variations. A shift to normal of the resistance of erythrocytes to osmotic lysis was observed.

Our findings provide evidence that an oral treatment with vitamin E improves the antioxidant/oxidant balance in plasma, LDL particles, and red blood cells, and counteracts lipid peroxidation processes in β-thalassemia intermedia patients.     

Embryo‐fetal erythroid megaloblasts in the human coelomic cavity

The coelomic cavity is part of the extraembryonic mesoderm, surrounding amniotic cavity, embryo, and yolk sac in the early gestation. It is now believed to represent an important transfer interface and a reservoir of nutrients for the embryo. Coelocentesis by ultrasound‐guided transvaginal puncture offers an easier access to the early human embryo, from 28 days post‐fertilization. However, despite some studies about its biochemical composition being reported, our knowledge about the presence of cellular elements and their quality in this compartment are still limited. Here we studied human coelomic fluids sampled from 6.6 (48 days) to 10 weeks of gestation, demonstrating the presence of functional embryonic erythroid precursors, that is, megaloblasts in the coelomic cavity. The ease of access of the coelomic cavity could allow the development of novel strategies for diagnostic or therapeutic purposes by ultrasound imaging and ultrasound‐guided puncture. J. Cell. Physiol. 225: 385–389, 2010. © 2010 Wiley‐Liss, Inc.     

Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2

Bone marrow stromal cells, obtained from postnatal bone marrow, contain progenitors able to differentiate into several mesenchymal lineages. Their use in gene and cell therapy requires their in vitro expansion and calls for the investigation of the culture conditions required to preserve these cells as a stem compartment with high differentiative potential during their life span. Here we report that fibroblast growth factor 2 (FGF-2)-supplemented bone marrow stromal cell primary cultures display an early increase in telomere size followed by a gradual decrease, whereas in control cultures telomere length steadily decreases with increasing population doublings. Together with clonogenic culture conditions, FGF-2 supplementation prolongs the life span of bone marrow stromal cells to more than 70 doublings and maintains their differentiation potential until 50 doublings. These results suggest that FGF-2 in vitro selects for the survival of a particular subset of cells enriched in pluripotent mesenchymal precursors and is useful in obtaining a large number of cells with preserved differentiation potential for mesenchymal tissue repair.     

Antipeptide Antibodies Against a Torpedo Cysteine-String Protein

Abstract: An antipeptide antiserum was raised against the C-terminal undecapeptide of a Torpedo cysteine-string protein (csp), a putative subunit or modulator of presynaptic calcium channels. This antiserum was shown to identify selectively the 27-kDa in vitro translation product of the csp cRNA both by immunoprecipitation and on immunoblots. When affinity-purified anti-csp antibodies were used to probe immunoblots of membrane proteins from Torpedo electric organ or liver, specific immunoreactivity was detected only in electric organ. This immunoreactivity was associated principally with a single protein species of about 34 kDa. These results indicate that csp immunoreactivity is detectably expressed in electroplax, a heavily innervated tissue, but not in liver, which should have an appreciably lower abundance of presynaptic calcium channel proteins. Moreover, the increased relative molecular mass of csp in electric organ (compared with in vitro translated material) implies that csp is posttranslationally modified. Finally, immunoblot analysis of either intact, alkali-treated, or solubilized membrane fractions of electric organ reveals that csp is predominantly a membrane protein.     

Pattern of expression of cyclin D1/CDK4 complex in human placenta during gestation

Progression through the cell cycle in eukaryotic cells is controlled by a family of protein kinases, termed cyclin-dependent kinases (CDKs), and their specific partners, the cyclins. In particular, the control of mammalian cell proliferation occurs largely during the G1 phase of the cell cycle. Five mammalian G1 cyclins have been enumerated to date: cyclins D1, D2, and D3 (D-type cyclins), and cyclins E and E2. By the use of immunohistochemistry and immunoelectron microscopy, we observed that in the first trimester of gestation of human placenta, cyclin D1 was distributed in the nuclei of the cytotrophoblast compartment together with a weak positivity of endothelial cells surrounding blood vessels. The endothelial positivity of cyclin D1 strongly increased in the third trimester of gestation. Moreover, we observed the subcellular localization of cyclin D1 that was present both in the stroma of placental villi and in the nuclei of syncytiotrophoblast cells. Therefore, we observed that CDK4 was localized in the nuclei of the cytotrophoblast compartment during the first and third trimesters and it also had a nuclear positivity in the endothelial cells of blood vessels at the end of the third trimester of gestation. In conclusion we may hypothesize that cyclin D1/CDK4 complex functions to regulate the cell cycle progression in the proliferative compartment of human placenta, the cytotrophoblast, during the first trimester through interaction with p107 and p130. Therefore, cyclin D1 and CDK4 seem to be involved in the control of placental angiogenesis during the third trimester of gestation.This work was supported by the University of Naples Federico II (M.D.F., V.F. and V.L.), by the Second University of Naples (L.C. and A.D.L.) and I.S.S.C.O. (President H.E. Kaiser)     

Kinetics of in vivo bone deposition by bone marrow stromal cells within a resorbable porous calcium phosphate scaffold: an X-ray computed microtomography study

Resorbable ceramic scaffolds based on Silicon stabilized tricalcium phosphate (Si-TCP) were seeded with bone marrow stromal cells (BMSC) and ectopically implanted for 2, 4, and 6 months in immunodeficient mice. Qualitative and quantitative evaluation of the scaffold material was performed by X-ray synchrotron radiation computed microtomography (microCT) with a spatial resolution lower than 5 microm. Unique to these experiments was that microCT data were first collected on the scaffolds before implantation and then on the same scaffolds after they were seeded with BMSC, implanted in the mice and rescued after different times. Volume fraction, mean thickness and thickness distribution were evaluated for both new bone and scaffold phases as a function of the implantation time. New bone thickness increased from week 8 to week 16. Data for the implanted scaffolds were compared with those derived from the analysis of the same scaffolds prior to implantation and with data derived from 100% hydroxyapatite (HA) scaffold treated and analyzed in the same way. At variance with findings with the 100% HA scaffolds a significant variation in the density of the different Si-TCP scaffold regions in the pre- and post-implantation samples was observed. In particular a post-implantation decrease in the density of the scaffolds, together with major changes in the scaffold phase composition, was noticeable in areas adjacent to newly formed bone. Histology confirmed a better integration between new bone and scaffold in the Si-TCP composites in comparison to 100% HA composites where new bone and scaffold phases remained well distinct.