Mesangiogenic Progenitor Cells and musculoskeletal tissue regeneration: differences between adipose-derived and bone marrow-derived cells?

Mesangiogenic Progenitor cells (MPCs) have been isolated from human bone marrow mononuclear cells (hBM-MNCs) and attracted particular attention for their ability to efficiently differentiate into exponentially growing mesenchymal stromal cells (MSCs) and toward endothelial lineage, suggesting the term "mesangiogenic". Coupling mesengenesis and angiogenis, MPCs has been hypothesized retaining a great tissue regenerative potential in musculoskeletal tissues regeneration. Bone marrow and adipose tissue (AT) represent most promising adult multipotent cell sources attempting to repair bone and cartilage, with controversial results regarding advantages applying BM- or AT-derived cells. As different culture determinants as well as tissue of origins, could strongly affect regenerative potential of cell preparations, we hypothesize that MPCs counterpart could have a role in defining efficacy of applying a cell-based medicinal product in musculoskeletal tissue repair. Here we present convincing data demonstrating that the ex vivo progenitors of MPCs are tissue specific and can be detected exclusively in hBM-MNCs.

Effects of ultrasound and selenium on human neurons in vitro.

As the effects of ultrasound on human brain functions might bear therapeutic potential, in this study, we examined the effects of diagnostic, i.e. non-thermal, ultrasound, on morphology, networking, and metabolic activity of SH- SY5Y human neurons in culture, as well as on the expression of GAP-43, Hsp90 and VEGF proteins, with and without selenium in the culture medium. The rationale for studying selenium lays in the observation that selenium improves functional neurologic outcome in traumatic brain injury and, therefore, analysis of the interactions between ultrasound and selenium may be of clinical interest. In the presence of selenium, ultrasound increased the overall number and length of elongations arising from the neuron bodies, thus reflecting an increase in the complexity of neuronal networks and circuits. The expression of GAP-43, Hsp90 and VEGF and metabolic activity of SH-SY5Y neurons, studied as markers of cell damage, were not affected by ultrasound or selenium. This study suggests that ultrasound may modulate neuronal networking in vitro without inducing cellular or molecular damage and highlights the potential role of selenium in the ultrasound-elicited cellular responses.

Morphological analysis of neurons: Automatic identification of elongations.

Our study is focused on the development of a new method for the automatic analysis of cell images. We focused on neurons (cells line SH-SY5Y) treated/untreated with ultrasound and stained with Haematoxylin-Eosin. The aim of the algorithm is the automatic detection of the cell body as well as the determination of the number and the length of neuron elongations. Starting point of the algorithm was the convolution of an image with a bank of rotating Gaussian kernels and the construction of a module map. Then several strategies were implemented to detect cell bodies and to detect and extract data about cell elongations. We have also realized a graphical user interface allowing the loading, saving and processing of images. Results show that this method is able to properly and efficiently detect cell contours and elongations. The automated evaluation is in strong agreement with manual evaluation performed by an expert operator, with an average error of 11% with most parameter combinations. This tool constitutes an important support in biological research activities, where operators need to analyze a large number of images to investigate about cell morphology before and after a treatment.

WITHDRAWN: Aids denialism at the ministry of health.

This Article-in-Press has been permanently withdrawn. The editorial policy of Medical Hypotheses makes it clear that the journal considers "radical, speculative, and non-mainstream scientific ideas", and articles will only be acceptable if they are "coherent and clearly expressed." However, we received serious expressions of concern about the quality of this article. Given these important signals of concern, we commissioned an external expert panel to investigate the circumstances in which this article came to be published online. The panel recommended that the article should be externally peer-reviewed. Following a peer-review process managed by The Lancet editorial team, all five external reviewers recommended rejection, as a result of which the expert panel recommended permanent withdrawal. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Mesenchymal cells inhibit expansion but not cytotoxicity exerted by gamma-delta T cells.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) exert a relevant immunosuppressive activity by inhibiting T- and B-lymphocytes, natural killer (NK) cells and dendritic cell expansion. Nevertheless, a possible activity on gamma/delta T cells has still not been evaluated. Gamma-delta T lymphocytes play an important role in the control of cancer and they have been shown to be implicated in graft-vs.-host disease. Thus, modulation of activation and proliferation of these cells could be relevant for therapeutic purposes. MATERIALS AND METHODS: Peripheral blood mononuclear cells from 21 healthy donors were used as source for gamma-delta T cells, expanded in presence of 10 IU mL(-1) interleukin-2 (IL-2) and 1 microM zoledronate. MSCs were recovered from patients undergoing routine total hip replacement surgery, and characterised by flow cytometry. Cytotoxicity on multiple myeloma and melanoma cell lines was assessed by measuring dilution of the carboxyfluorescein diacetate succinimydylester dye (CFSE). Gamma-delta T cells were then incubated with MSCs in contact cultures, and with addition of MSC-conditioned medium. RESULTS: In this article we confirmed that (1) in vitro expanded gamma-delta T cells play a significant anti-proliferative effect on multiple myeloma and melanoma cells and (2) multipotent mesenchymal stromal cells effectively suppress the ex vivo expansion of T cells carrying a specific T-cell receptor gene (TCR) rearrangement, Vgamma9/Vdelta2, induced by the combination of IL-2 and zoledronate, without interfering with their cytotoxic activity. DISCUSSION: These findings contribute to explain the activity of ex vivo expanded mesenchymal cells, suggesting that MSCs would interact with gamma-delta T lymphocytes. CONCLUSION: This effect could be relevant in separating graft-vs.-host from the graft-vs.-tumour effect, especially considering the possibility of modulating T-lymphocytes activity by the immunomodulating drugs now available.

PS-341 (Bortezomib) inhibits proliferation and induces apoptosis of megakaryoblastic MO7-e cells.

PS-341 (Bortezomib) is a dipeptide boronic acid proteasome inhibitor with antitumor activity that induces apoptosis in different human cancer cell lines. We investigated effects of PS-341 (Bortezomib) on cell proliferation, cell cycle progression, induction of apoptosis and differentiation in a megakaryoblastic (MO7-e) cell line. PS-341 was able to retain NF-kappaB in the cytoplasm and inhibit cell growth (IC(50)=22.5 nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines (KG1a, K562/R7, HL60/DNR) were unaffected by the PS-341 treatment. Moreover, PS-341 in MO7-e induced a significant pro-apoptotic effect from 10 nM concentration (40% versus 12% in the control, p<0.05). On the other hand, at lower concentration (5 nM), Bortezomib blocked cell cycle in the G2 phase. Finally, this compound was able to down-regulate WT1 expression. No significant effects on cell differentiation were found. Because a spontaneous NF-kappaB activation has been reported in megakaryocytes from patients affected by myeloproliferative disorders, Bortezomib would so be an attractive therapeutic tool for these malignancies, including essential thrombocythemia or idiopathic myelofibrosis. Preliminary data show an inhibiting activity of Bortezomib in the megakaryocytic colonies formation. Finally, also down-regulation of the WT1 gene Bortezomib-driven could be relevant, because of the role that this gene would play in the pathogenesis of acute and chronic myeloproliferative disorders.

p66SHC: the apoptotic side of Shc proteins.

Initially identified as components of the signaling pathways triggered by receptor tyrosine kinases and leading to Ras activation, Shc proteins have been more recently implicated in the regulation of signals controlling not only cell proliferation, but also cell survival and apoptosis. Here we briefly review the current understanding of Shc proteins as promoters of apoptosis. Specifically, we focus on the 66 kDa isoform of ShcA, whose paramount importance in the regulation of oxidative stress responses leading to cell apoptosis and ageing has been by now firmly established.

Interstitital cells of Cajal in the human stomach: distribution and relationship with enteric innervation.

Interstitial cells of Cajal (ICC) are distributed throughout the gastrointestinal muscle coat with a region-specific location, and are considered to be pace-maker and/or mediators of neurotransmission. Little is known about their shape, size, distribution and relationships with excitatory and inhibitory nerves in human stomach. With this aim, we labeled the ICC, using c-Kit immunohistochemistry, followed by a quantitative analysis to evaluate the distribution and area occupied by these cells in the circular and longitudinal muscle layers and at the myenteric plexus level in the human fundus, corpus and antrum. Furthermore, by NADPH-d histochemistry and substance P (SP) immunohistochemistry, we labeled and quantified nitric oxide (NO)-producing and SP-containing nerves and evidenced their relationships with the ICC in these three gastric regions. In the fundus, the ICC appeared as bipolar cells and in the corpus and antrum they mainly appeared as multipolar cells, with highly ramified processes. The networks formed by ICC differed in the three gastric regions. The ICC number was significantly higher and cell area smaller in the fundus compared to the corpus and antrum. The area occupied by the ICC was significantly higher at the myenteric plexus level compared with circular and longitudinal muscle layers. Everywhere, NADPH-d-positive nerves were more numerous than SP-positive ones. Both kinds of fibers were closely apposed to the ICC in the corpus and antrum. In conclusion, in the human stomach, the ICC have region-specific shape, size and distribution and in the corpus and antrum have close contact with both inhibitory and excitatory nerves. Presumably, as suggested for laboratory mammals, these differences are in relationship with the motor activities peculiar to each gastric area.

Modification of plasma glycosaminoglycans in long distance runners.

BACKGROUND: It is well documented that exercise reduces the risk of thromboembolic disease, possibly by increasing the plasma concentration of anticoagulant-antithrombotic compounds. OBJECTIVES: As plasma glycosaminoglycans (GAGs) play a role in the anticoagulant-antithrombotic potential of plasma, to examine the concentration and profile of these compounds in well trained, long distance runners and sedentary subjects. METHODS: Plasma GAGs were measured in 10 male, long distance runners and 10 sedentary counterparts before and after ergometric tests. GAGs were extracted, purified, and identified by electrophoretic and enzymatic methods, and measured as hexosamine. RESULTS: Plasma GAGs found in sedentary subjects were slow migrating heparan sulphates I and II, keratan sulphate I, and chondroitin 4-6-sulphate. Those found in trained athletes were slow migrating heparan sulphate I, chondroitin 4-6-sulphate (or keratan sulphate I), and fast migrating heparan sulphate. Total plasma concentrations of GAGs were higher in athletes than in sedentary subjects at rest. In sedentary subjects, plasma GAGs did not change after cycle ergometric exercise at 80% of their anaerobic threshold. However, the appearance of a novel band of heparan sulphate migrating faster than fast migrating heparan sulphate was observed in athletes after exercise. CONCLUSIONS: Exercise changes the amount and profile of plasma GAGs; these changes may play a role in protecting subjects who practise aerobic sports against developing cardiovascular disease.

Effect of simvastatin treatment on bone mineral density and bone turnover in hypercholesterolemic postmenopausal women: a 1-year longitudinal study.

Although several studies have reported a lower risk of osteoporotic fracture in hypercholesterolemic patients treated with statins, so far longitudinal studies on the effects of statins on bone are lacking. The aim of the present study was to evaluate bone mineral density (BMD) and bone turnover changes induced by 1-year simvastatin treatment on postmenopausal women. Thirty consecutive postmenopausal hypercholesterolemic women (61.2 +/- 4.9 years) were treated for 12 months with 40 mg/day simvastatin and 30 normocholesterolemic age-matched postmenopausal women provided control data. In all subjects, at baseline and at 3-month intervals, serum lipids, calcium, phosphate, total and bone alkaline phosphatase (Bone-ALP), and carboxy-terminal fragment of type I collagen (CTx) were measured in a fasting blood sample. At baseline and after 6 and 12 months BMD was measured at lumbar spine (BMD-LS) and at femur (BMD-Ftot) and at femoral neck (BMD-Fn) by DXA. In the simvastatin-treated group Bone-ALP showed a significant increase (P < 0.05) with respect to baseline from the sixth month, whereas serum CTx showed a weak and nonsignificant increase over the study period. In treated women BMD-LS, BMD-Fn, and BMD-Ftot increased respectively by 1.1, 0.9, and 0.4% at Month 6; and by 2.8, 1.0, and 0.8% at Month 12. In controls BMD-LS, BMD-Fn, and BMD-Ftot at the end of the study period decreased by 1.6, 1.4, and 1.2%, respectively. The difference between controls and simvastatin-treated patients was significant (P < 0.05) for both BMD-LS and BMD-Fn only at Month 12. In conclusion our results, although obtained from a small sample of postmenopausal hypercholesterolemic women, suggest a probable positive effect of simvastatin on bone formation and BMD.

Changes in serum HDL and LDL cholesterol in patients with Paget's bone disease treated with pamidronate.

Amino bisphosphonates represent one of the most important advances in the management of Paget's and other metabolic bone diseases. Although their mechanism of action has not yet been completely clarified, they seem to inhibit the mevalonate pathway and so they could interfere with cholesterol synthesis. The present study aimed to evaluate cholesterol and lipoprotein serum levels in patients with Paget's bone disease treated with intravenous pamidronate. The study included 20 consecutive patients (mean age, 67.6 +/- 11.0 years) with Paget's bone disease for at least 1 year, who needed intravenous amino bisphosphonate treatment; 12 patients with inactive Paget's bone disease served as controls. The patients with active Paget's bone disease underwent three cycles (every 3 months) of treatment with 60 mg of intravenous pamidronate. Controls were given a saline infusion following the same administration schedule. In all subjects total alkaline phosphatase (total ALP), bone alkaline phosphatase (bone ALP), total cholesterol (TC), tryglycerides (TG), and high- and low-density lipoprotein cholesterol (HDL-C and LDL-C, respectively) were measured before infusions (pamidronate or saline) at baseline and at 3-month intervals up to 9 months. In the control group no significant changes were observed through the study period for any of the biochemical parameters. In the pamidronate-treated patients, both bone ALP and total ALP significantly fell at the end of the study. In patients with active treatment, at the end of the study period HDL-C significantly (P < 0.05) increased by 10.3%, whereas LDL-C significantly (P < 0.05) decreased by 5.5%. In these patients TC showed a negative trend without reaching statistical significance, whereas the HDL-C/LDL-C ratio rose 16.2% above the basal value and TC/HDL-C decreased by 12.5%. In conclusion, pamidronate given intravenously seems to be able to induce a prolonged shifting in circulating cholesterol from the LDL-C to the HDL-C from associated with a weak decrease in total cholesterol, thus producing a possible improvement in the atherosclerotic risk index.

A method for separation of heparin species from biological samples by ethanol precipitation of compounds solubilized in guanidine hydrochloride.

In this paper we describe a procedure to determine glycosaminoglycan and oligosaccharide composition of biological samples such as cell cultures or tissue explants. We demonstrate that heparin species of different molecular mass can be easily fractionated by sequential ethanol precipitation in 4.0 M guanidine hydrochloride. We studied by gradient polyacrylamide gel electrophoresis fractionation of standard heparin and heparin-derived oligosaccharides by anion-exchange chromatography on DEAE-Sephacel resin eluted by increasing concentration of guanidine hydrochloride. The use of guanidine salts followed by sequential precipitation by increasing ethanol concentration allowed recovery of heparin and heparin-derived oligosaccharides.

Dual X-ray absorptiometry and bone ultrasonography in patients with Rett syndrome.

This study evaluated bone status and bone turnover in 82 females (ages 2-21 years) with the Rett Syndrome (RS) and 82 age-matched controls. Bone mineral density (BMD) by dual X-ray absorptiometry (DXA) at the ultradistal and proximal radius and ultrasonographic (QUS) parameters at the calcaneus [speed of sound(SOS), broadband ultrasound attenuation(BUA), and stiffness] and at the phalanxes (amplitude dependent speed of sound: AD-SOS) were measured. We also measured serum calcium, phosphate, 25-hydroxyvitamin D, and biochemical markers of bone turnover. DXA and QUS parameters were significantly lower in patients with RS compared with controls and, among RS alone, in those treated with anticonvulsants and in those who are nonambulatory. Ambulatory RS patients showed QUS and DXA parameters significantly greater than nonambulatory patients but significantly lower than controls. Patients with RS treated with anticonvulsants presented QUS and DXA parameters lower than those of other RS. In RS patients, walking significantly influences BMD-UD, BMD-P, SOS. BUA. and Stiffness. Serum 25-hydroxyvitamin D was significantly lower in RS than in controls. These results suggest that ambulatory status, to a major extent, and anticonvulsant therapy certainly play an important role in the reduction of bone mass and bone quality, but they cannot completely explain the altered bone status. Whatever the cause, girls with RS present abnormal bone status with an increase in the risk of fracture.

Temporally regulated assembly of a dynamic signaling complex associated with the activated TCR.

TCR triggering promotes multiple tyrosine kinase-dependent interactions involving proteins with one or more protein binding modules. Reported interactions mostly exceed the binding potential of these proteins. A solution to this paradox is the temporally regulated recruitment of alternative ligands. We have tested this hypothesis by analyzing the time course of protein/protein interactions triggered by TCR engagement. We show that a short-lived and dynamic multimolecular complex is assembled on tyrosine-phosphorylated CD3zeta. Specific components of this complex are recruited and shed in a temporal sequence distinct for each of the proteins analyzed. The temporally regulated assembly of a higher order structure at the activated TCR is likely to be crucial in achieving both signal longevity and signal specificity.

Defective recruitment and activation of ZAP-70 in common variable immunodeficiency patients with T cell defects.

We have previously identified a subset of common variable immunodeficiency (CVID) patients with defective T cell function associated with impaired activation of the TCR-dependent tyrosine phosphorylation cascade. Here we have assessed the structural and functional integrity of the principal components involved in coupling the TCR/CD3 complex to intracellular tyrosine kinases in two of these patients. We show that ZAP-70 fails to bind the signaling-competent CD3zeta tyrosine phosphorylation isoform and to become activated following TCR engagement, suggesting that defective recruitment of ZAP-70 might underlie the TCR signaling dysfunction in these patients. Determination of the nucleotide sequences encoding the intracellular domains of the CD3/zeta subunits and ZAP-70 did not reveal any mutation. Furthermore, ZAP-70 from these patients could interact in vitro with recombinant phospho-zeta, ruling out genetic defects at the immunoreceptor tyrosine-based activation motif/SH2 domain interface responsible for ZAP-70 recruitment to the activated TCR. No defect was found in expression, activity or subcellular localization of Lck, which is thought to be primarily responsible for CD3zeta phosphorylation. Hence, while the T cell defect in these CVID patients can be pinpointed to the interaction between ZAP-70 and CD3zeta, the integrity in the components of the signaling machinery involved in this process suggests that additional components might be required for completion of this step.

Quantitative ultrasound and bone mineral density in patients with primary hyperparathyroidism before and after surgical treatment.

The aim of this study was to assess the pattern of ultrasound (QUS) parameters and bone mineral density at different skeletal sites in patients with primary hyperparathyroidism (PHPT) before and after surgical treatment. In 22 patients (age range 28-74 years) with PHPT we measured speed of sound (SOS), attenuation (BUA) and Stiffness at the calcaneus, amplitude-dependent speed of sound (AD-SoS) at proximal phalanges, and bone mineral density at lumbar spine (BMD-LS) and at the mid-radius (BMD-MR) and ultra-distal radius (BMD-UDR) before, 1 and 2 years after surgical operation. Twenty-two age- and sex-matched healthy subjects provided control data. Before surgery, all parameters apart from SOS were significantly lower in PHPT patients than in controls. At the end of the study period, BMD-LS increased by 7.0%, BMD-UDR by 7.4% and BMD-MR by 11.0%. The changes in ultrasound parameters after surgery were lower (0.44% for SOS, 2.2% for BUA, 3.3% for Stiffness and 2.6% for AD-SoS); however, the increase was statistically significant (p < 0.05 and p < 0.01, respectively) only for Stiffness and AD-SoS. Our results indicate that parathyroidectomy increases both axial and appendicular BMD and influences QUS parameters differently at the calcaneus and at the phalanges. The combined use of BMD and QUS could improve the assessment of skeletal status in patients with PHPT before and after surgery.

Constitutive activation of the Ras/MAP kinase pathway and enhanced TCR signaling by targeting the Shc adaptor to membrane rafts.

The Shc adaptor is responsible for coupling receptor tyrosine kinases and tyrosine kinase-associated receptors to the Ras/MAP kinase pathway. Shc is believed to be regulated by a change in subcellular localization from the cytosol to the plasma membrane, where it recruits Grb-2/Sos complexes and hence permits juxtaposition of the guanine nucleotide exchange factor Sos to Ras, resulting in GDP/GTP exchange and Ras activation. Shc has been recently shown to inducibly colocalize in detergent-resistant membrane rafts together with the activated TCR and associated signaling molecules. To understand whether Shc localization in membrane rafts is sufficient to regulate Shc function, we constructed a Shc chimera containing the Ras membrane localization motif at the C-terminus. We show that membrane targeted Shc was constitutively localized in the plasma membrane of T-cells, and was mostly compartmentalized in lipid rafts. Membrane targeted Shc was phosphorylated on tyrosine residues and bound Grb-2/Sos in the absence of TCR engagement. Furthermore, expression of membrane targeted Shc resulted in constitutive downstream signaling, including Erk2 activation and enhancement of TCR dependent activation of the TCR responsive transcription factor NF-AT. Hence localization of Shc in membrane rafts is sufficient for Shc to acquire a signaling competent state. Interestingly, a membrane targeted Shc mutant lacking both Grb-2 binding sites was not only incapable of signaling in the absence of TCR triggering, but transdominantly inhibited endogenous Shc, supporting a non redundant role for Shc in the activation of the Ras/MAP kinase pathway in T-cells.