A unique neuroendocrine cell model derived from the human foetal neural crest.

PURPOSE: Nowadays, no human neuroendocrine cell models derived from the neural crest are available. In this study, we present non-transformed long-term primary Neural Crest Cells (NCCs) isolated from the trunk region of the neural crest at VIII-XII gestational weeks of human foetuses obtained from voluntary legal abortion. METHODS AND RESULTS: In NCC, quantitative real-time RT PCR demonstrated the expression of neural crest specifier genes, such as Snail1, Snail2/SLUG, Sox10, FoxD3, c-Myc, and p75NTR. Moreover, these cell populations expressed stemness markers (such as Nanog and nestin), as well as markers of motility and invasion (TAGLN, MMP9, CXCR4, and CXCR7), and of neuronal/glial differentiation (MAP2, GFAP, SYP, and TAU). Functional analysis demonstrated that these cells not only possessed high migration properties, but most importantly, they expressed markers of sympatho-adrenal lineage, such as ASCL1 and tyrosine hydroxylase (TH). Moreover, the expression of TH increased after the induction with two different protocols of differentiation towards neuronal and sympatho-adrenal phenotypes. Finally, exposure to conditioned culture media from NCC induced a mature phenotype in a neuronal cell model (namely SH-SY5Y), suggesting that NCC may also act like Schwann precursors. CONCLUSION: This unique human cell model provides a solid tool for future studies addressing the bases of human neural crest-derived neuroendocrine tumours.

Evidence of DMSO-Induced Protein Aggregation in Cells.

We report on a study of protein aggregation induced on different cell samples by dimethyl sulfoxide (DMSO) addition. DMSO is the most commonly used cryoprotectant because it is supposed to readily diffuse across lipid bilayers, thus reducing water activity within cells; despite its large use, the mechanism of penetration and even the main interaction features with cell components are far from being understood. In the present work, infrared absorption spectroscopy is successfully applied to real time detection of chemical and structural changes occurring in cells during dehydration from water and water/DMSO suspensions. As a most interesting result, DMSO is observed to favor protein aggregation both in cellular model systems, as cultured lymphocytes and fibroblasts, and in human samples for clinic use, as hematopoietic stem cells from cord blood. This effect is evidenced at low water content, analogously to what is observed for protein solutions. Such tendency is not specific of the type of protein and suggests one possible origin of DMSO toxicity.

No proinflammatory signature in CD34+ hematopoietic progenitor cells in multiple sclerosis patients.

Autologous hematopoietic stem cell transplantation (aHSCT) has been used as a therapeutic approach in multiple sclerosis (MS). However, it is still unclear if the immune system that emerges from autologous CD34+ hematopoietic progenitor cells (HPC) of MS patients is pre-conditioned to re-develop the proinflammatory phenotype. The objective of this article is to compare the whole genome gene and microRNA expression signature in CD34+ HPC of MS patients and healthy donors (HD). CD34+ HPC were isolated from peripheral blood of eight MS patients and five HD and analyzed by whole genome gene expression and microRNA expression microarray. Among the differentially expressed genes (DEGs) only TNNT1 reached statistical significance (logFC=3.1, p<0.01). The microRNA expression was not significantly different between MS patients and HD. We did not find significant alterations of gene expression or microRNA profiles in CD34+ HPCs of MS patients. Our results support the use of aHSCT for treatment of MS.

Androgens and estrogens prevent rosiglitazone-induced adipogenesis in human mesenchymal stem cells.

Thiazolidinediones (TZD), a class of anti-diabetic drugs, determine bone loss and increase fractures particularly in post-menopausal women, thus suggesting a protective role of sex steroids. We have previously demonstrated that the TZD rosiglitazone (RGZ) negatively affects bone mass by inhibiting osteoblastogenesis, yet inducing adipogenesis, in bone marrow-derived human mesenchymal stem cells (hMSC). The aim of this study was to determine whether estrogens and androgens are able to revert the effects of RGZ on bone. hMSC express estrogen receptor α and ß and the androgen receptor. We found that 17ß-estradiol (10 nM), the phytoestrogen genistein (10 nM), testosterone (10 nM) and the non-aromatizable androgens dihydrotestosterone (10 nM) and methyltrienolone (10 nM) effectively counteracted the adipogenic effect of RGZ (1 µM) in hMSC induced to differentiate into adipocytes, as determined by evaluating the expression of the adipogenic marker peroxisome proliferator-activated receptor γ and the percentage of fat cells. Furthermore, when hMSC were induced to differentiate into osteoblasts, all the above-mentioned molecules and also quercetin, another phytoestrogen, significantly reverted the inhibitory effect of RGZ on the expression of the osteogenic marker osteocalcin and decreased the number of fat cells observed after RGZ exposure. Our study represents, to our knowledge, the first demonstration in hMSC that androgens, independently of their aromatization, and estrogens are able to counteract the negative effects of RGZ on bone. Our data, yet preliminary, suggest the possibility to try to prevent the negative effects of TZD on bone, using steroid receptor modulators, such as plant-derived phytoestrogens, which lack evident adverse effects.

Rosiglitazone stimulates adipogenesis and decreases osteoblastogenesis in human mesenchymal stem cells.

Thiazolidinediones (TZD) are widely prescribed for the treatment of Type 2 diabetes. Increased loss of bone mass and a higher incidence of fractures have been associated with the use of this class of drugs in post-menopausal women. In vitro studies performed in rodent cell models indicated that rosiglitazone (RGZ), one of the TZD, inhibited osteoblastogenesis and induced adipogenesis in bone marrow progenitor cells. The objective of the present study was to determine for the first time the RGZ-dependent shift from osteoblastogenesis toward adipogenesis using a human cell model. To this purpose, bone marrow-derived mesenchymal stem cells were characterized and induced to differentiate along osteogenic and adipogenic lineages. We found that the exposure to RGZ potentiated adipogenic differentiation and shifted the differentiation toward an osteogenic phenotype into an adipogenic phenotype, as assessed by the appearance of lipid droplets. Accordingly, RGZ markedly increased the expression of the typical marker of adipogenesis fatty-acid binding protein 4, whereas it reduced the expression of Runx2, a marker of osteoblastogenesis. This is the first demonstration that RGZ counteracts osteoblastogenesis and induces a preferential differentiation into adipocytes in human mesenchymal stem cells.

Low extracellular sodium promotes adipogenic commitment of human mesenchymal stromal cells: a novel mechanism for chronic hyponatremia-induced bone loss.

Hyponatremia represents an independent risk factor for osteoporosis and fractures, affecting both bone density and quality. A direct stimulation of bone resorption in the presence of reduced extracellular sodium concentrations ([Na(+)]) has been shown, but the effects of low [Na(+)] on osteoblasts have not been elucidated. We investigated the effects of a chronic reduction of extracellular [Na(+)], independently of osmotic stress, on human mesenchymal stromal cells (hMSC) from bone marrow, the common progenitor for osteoblasts and adipocytes. hMSC adhesion and viability were significantly inhibited by reduced [Na(+)], but their surface antigen profile and immuno-modulatory properties were not altered. In low [Na(+)], hMSC were able to commit toward both the osteogenic and the adipogenic phenotypes, as demonstrated by differentiation markers analysis. However, the dose-dependent increase in the number of adipocytes as a function of reduced [Na(+)] suggested a preferential commitment toward the adipogenic phenotype at the expense of osteogenesis. The amplified inhibitory effect on the expression of osteoblastic markers exerted by adipocytes-derived conditioned media in low [Na(+)] further supported this observation. The analysis of cytoskeleton showed that low [Na(+)] were associated with disruption of tubulin organization in hMSC-derived osteoblasts, thus suggesting a negative effect on bone quality. Finally, hMSC-derived osteoblasts increased their expression of factors stimulating osteoclast recruitment and activity. These findings confirm that hyponatremia should be carefully taken into account because of its negative effects on bone, in addition to the known neurological effects, and indicate for the first time that impaired osteogenesis may be involved.

Synthetic peptides in the diagnosis of neurological diseases.

For several years peptides have been used to investigate many aspects of the molecular mechanisms supporting the etiopathogenesis of neurological diseases. Even if well-established and commercially-available assays for the diagnosis of neurological diseases, based on peptides, are presently not available, much research has been focused to the comprehension of the pathogenetic mechanisms of relevant neurological diseases, such as Multiple Sclerosis, Alzheimer's disease and prion diseases. Several peptides, which strongly contributed to increase in the progress, and in the understanding of the molecular mechanisms of these important diseases, have been selected as antigens to be proposed in possible alternative diagnostic methodologies. The authors focused their attention on the possible peptides to be used as diagnostics for these three characteristic neurological diseases. In fact, Multiple Sclerosis is an autoimmune disease in which peptides have been used for the characterization of both B and T immune response, while Alzheimer's disease and prion diseases can be cumulative regarded as "protein folding disorders" in which peptides can be used to identify the misfolded protein.

Palmitoyl derivatives of GpMBP epitopes: T-cell response and peptidases susceptibility.

We report for the first time the immunoadjuvant effects of lipoconjugation of peptide antigens in an in vitro system by using CD4+ T-cells. The lipopeptides obtained by conjugating a palmitoyl moiety at the N(alpha)-terminal of Gln(74) or at the epsilon-NH(2) of Lys(75) of GpMBP(74-85) induced increased T-cell responsiveness compared to the native nonlipidated peptide. On the other hand, lipoderivatives of GpMBP(82-98) did not increase the T-cell response, demonstrating that the superagonist inducing effect of lipoconjugation is epitope-specific. Digestion of the two native peptides with cathepsin D and L, both implicated in antigen processing, and with a complete lysosomal fraction of a EBV-transformed B cell line shows that GpMBP(74-85) is resistant to cellular proteases, while GpMBP(82-98) is easily digested by these enzymes. These results suggest that the first prerequisite for increasing the T-cell response by lipoconjugation is the stability of the native peptide to peptidases, providing an important insight into the understanding of the immunoadjuvant effect of lipoderivative antigens.

Short-term dynamics of circulating T cell receptor V beta repertoire in relapsing-remitting MS.

To understand the short-term dynamics of the circulating T cell receptor V beta (TCRBV) repertoire in relapsing-remitting multiple sclerosis (MS), we monitored the TCRBV profiles of untreated MS patients and healthy controls. Expansions of TCRBV genes in MS patients were significantly more frequent than in controls (P<0.001), were predominantly oligoclonal (80%) and were significantly correlated with immune responses to myelin basic protein (MBP) (P<0.02) and with inflammatory disease activity detected by magnetic resonance imaging (MRI) (P<0.05). Autoreactive T cell responses against myelin antigens may be implicated in perturbations of TCR repertoire in untreated MS patients, detectable even in the absence of clinically evident manifestations.

IL-7-enhanced T-cell response to myelin proteins in multiple sclerosis.

In this study, we investigated the in vitro proliferative response of peripheral blood T lymphocytes from MS patients and controls to MBP and MOG either in the absence or in the presence of the conditioning factor IL-7. In the absence of IL-7, T-cell reactivity to MOG and MBP was similar in MS patients and controls even if an increased MBP response was found in a subgroup of patients with active disease. In the presence of IL-7, increased T-cell reactivity to MBP was observed in MS patients suggesting that their MBP-specific T cells are in a different functional state.

Pixantrone (BBR2778) reduces the severity of experimental allergic encephalomyelitis.

Pixantrone is less cardiotoxic and is similarly effective to mitoxantrone (MTX) as an antineoplastic drug. In our study, pixantrone reduced the severity of acute and decreased the relapse rate of chronic relapsing experimental allergic encephalomyelitis (EAE) in rats. A marked and long-lasting decrease in CD3+, CD4+, CD8+ and CD45RA+ blood cells and reduced anti-MBP titers were observed with both pixantrone and MTX. In vitro mitogen- and antigen-induced T-cell proliferation tests of human and rodents cells evidenced that pixantrone was effective at concentrations which can be effectively obtained after i.v. administration in humans. Cardiotoxicity was present only in MTX-treated rats. The effectiveness and the favorable safety profile makes pixantrone a most promising immunosuppressant agent for clinical use in multiple sclerosis (MS).

Epidemiological study of myasthenia gravis in the province of Ferrara, Italy.

The only prevalence rate of myasthenia gravis (MG) so far estimated in Italy by an epidemiological study carried out in Pavia, North Italy, indicates a prevalence quite similar to that observed in other countries. The purpose of the survey was to verify the frequency of the disease in a geographically well-defined and previously surveyed community. On the basis of 39 cases, on 31 December 1987 the prevalence per million was 105.3 (102.3 if standardized for the Italian population). This is the highest prevalence figure yet found, indicating a value similar to that established in Northern Europe. According to Kurtzke this high prevalence rate reflects the high standard of the local public health service, which permitted a more intensive search for affected subjects.

Treatment of experimental esophagogastric myotomy with bone marrow mesenchymal stem cells in a rat model.

BACKGROUND: Over the last 15 years, many studies demonstrated the myogenic regenerative potential of bone marrow mesenchymal stem cells (BM-MSC), making them an attractive tool for the regeneration of damaged tissues. In this study, we have developed an animal model of esophagogastric myotomy (MY) aimed at determining the role of autologous MSC in the regeneration of the lower esophageal sphincter (LES) after surgery. METHODS: Syngeneic BM-MSC were locally injected at the site of MY. Histological and functional analysis were performed to evaluate muscle regeneration, contractive capacity, and the presence of green fluorescent protein-positive BM-MSC (BM-MSC-GFP(+) ) in the damaged area at different time points from implantation. KEY RESULTS: Treatment with syngeneic BM-MSC improved muscle regeneration and increased contractile function of damaged LES. Transplanted BM-MSC-GFP(+) remained on site up to 30 days post injection. Immunohistochemical analysis demonstrated that MSC maintain their phenotype and no differentiation toward smooth or striated muscle was shown at any time point. CONCLUSIONS & INFERENCES: Our data support the use of autologous BM-MSC to both improve sphincter regeneration of LES and to control the gastro-esophageal reflux after MY.

Human de-epidermized dermis as a stem cell carrier.

Recently several types of skin equivalents have been developed, consisting of differentiated keratinocytes cultured on various dermal substitutes. Different models of reconstructed human skin have been proposed, such as human and animal de-epidermized dermis, inert filters, collagen matrices, lyophilized collagen membranes populated with fibroblasts, and other models populated with melanocytes and/or Langerhans cells. These skin equivalents mimic native skin in vivo. They have provided information about dermal-epidermal interactions, cell-cell, and cell-matrix interactions; responses of dermal and epithelial cells to biological signals and pharmacological agents; as well as effects of drugs and growth factors on wound healing. Human allodermis from tissue banks has been used for clinical purposes, namely, as support for autologous keratinocyte cultures and as a potentially ideal scaffold for dermal replacement. This bioproduct is considered to be the most suitable clinical carrier for autologous fibroblasts and keratinocytes, as well as an useful experimental model to study angiogenesis and to stimulate vascularization in reconstructed human skin. Because it is human-derived, it is in our opinion the safest of all available types of skin equivalent. Having epidermal and dermal structures, it can be used in one-stage grafting procedures for wound closure.

Transforming growth factor-beta 1 inhibits the proliferation of rat astrocytes induced by serum and growth factors.

A number of cytokines and growth factors may affect astrocyte proliferation and functions. Transforming growth factor-beta 1 (TGF-beta 1) is a pleiotropic cytokine which exerts multiple effects on growth and differentiation of different cell types. TGF-beta 1 is present in low amounts in the normal brain. TGF-beta 1 gene expression, however, is increased in the central nervous system (CNS) in several pathological conditions. In this study we examined the in vitro effects of TGF-beta 1 on the proliferative response of rat astrocytes to serum and growth factors. Astrocyte cultures were established from the cerebellum and cortex of newborn Lewis rats. The proliferative response of these cultures to serum and growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), IGF-2, interleukin 1 (IL-1)] was studied by [3H]-thymidine incorporation test in the presence or absence of TGF-beta 1. TGF-beta 1 significantly inhibited the proliferative response of astrocyte cultures to both autologous and heterologous serum. In addition, a strong inhibition of bFGF-, EGF-, and PDGF-induced proliferation was observed. The effect of TGF-beta 1 on the proliferative response to IL-1 was less evident but still significant. No effect was observed when TGF-beta 1 was added to IGF-1 and IGF-2 stimulated cultures. These data confirm previous reports showing a down-regulating activity of TGF-beta on astrocyte proliferation and suggest that this cytokine may play physiological and pharmacological roles in the regulation of reactive astrocytosis in the CNS.

Comparative study on the prevalence of myasthenia gravis in the provinces of Bologna and Ferrara, Italy.

This cooperative study was performed in the provinces of Bologna and Ferrara to estimate the prevalence rate of Myasthenia Gravis (MG). Two independent groups used the same case collection method and diagnostic criteria. The study was performed from January 1, 1987 to March 1, 1988. Cases were collected through: 1) review of clinical files from neurology clinics in Bologna and Ferrara, and from the main North-Italian centers for MG (neurology clinics of Milan and Turin; thoracic surgery unit, Turin); 2) thoracic surgery and intensive care units in Bologna and Ferrara; 3) physicians prescribing anticholinesterase drugs. Prevalence rates of generalized MG per million population were 60.9 in the city and 41.0 in the remaining province for Bologna and 118.9 in the city and 87.9 in the remaining province for Ferrara. The difference in prevalence rates between the two areas was statistically significant. Our survey shows that prevalence studies on MG may be biased by factors that cannot be controlled and that most previous studies have probably underestimated the prevalence of MG.

Decrypting the spectrum of antigen-specific T-cell responses: the avidity repertoire of MBP-specific T-cells.

Myelin basic protein (MBP) is a well-characterized autoantigen potentially involved in the pathogenesis of the most common human demyelinating disease of the central nervous system (CNS), multiple sclerosis (MS). It is known that MBP-specific T-cell responses differ widely among different individuals and also within a single donor in terms of fine specificity and functional characteristics including the avidity in antigen recognition. In this report, we demonstrate that the in vitro selection of MBP-reactive T-cell repertoire is strictly dependent upon the antigen dose used in the primary cultures. MBP-specific T-cell lines (TCLs) were generated from MS patients and healthy donors using different antigen concentration in cultures (0.1 to 50 microg/ml). In both MS patients and controls, the number of obtained T-cell lines was affected by the antigen concentration. In addition, low and high antigen concentrations selected in vitro different T-cell populations in terms of peptide specificity patterns and different functional avidities in antigen recognition. Low concentrations of MBP in the primary cultures yielded a small number of TCLs recognizing the specific antigen with higher avidity whereas high antigen concentrations allowed the in vitro expansion of a higher numbers of T-cells recognizing MBP with lower avidity. The use of different antigen concentrations in the primary cultures can be applied as a simple experimental system to investigate the overall avidity repertoire of antigen-specific T-cell response in humans.

Short-term evolution of autoreactive T cell repertoire in multiple sclerosis.

T cells reactive to self-antigens are present in the peripheral blood of patients with autoimmune diseases as well as in healthy subjects. Although T cell-response to the self-myelin antigen myelin basic protein (MBP) has been widely investigated in multiple sclerosis (MS) patients, very little is known about the evolution over time of this response and its correlation with the disease activity. In recent years magnetic resonance imaging (MRI) techniques have provided new tools for following the inflammatory activity in the central nervous system (CNS) of MS patients. In the present study the T cell response to MBP was longitudinally investigated in terms of frequency, epitope specificity, and cytokine production profile in four patients with relapsing-remitting MS enrolled in a gadolinium-enhanced MRI serial study. In spite of different profiles of inflammatory activity within the CNS, all the patients examined showed major changes in their reactivity to MBP during the follow-up period in terms of both frequency and epitope specificity. Episodic expansions of MBP-specific T cell populations were observed in each patient, and overall they did not correlate with disease activity. In these patients the expansions: 1) occurred in the context of a steady level of disease activity, 2) correlated with a burst of CNS inflammation, 3) followed the appearance of a new active lesion, and 4) were observed even in the absence of detectable signs of CNS inflammation during the entire follow-up period. These results suggest that the evolution over time of the T cell response to a self-antigen such as MBP is more complex than previously expected. The short-term repertoire dynamics of autoreactive T cells in MS underscore the importance of longitudinal studies for evaluating autoreactivity to myelin antigens and probably to any self-antigen in other autoimmune diseases.

A synthetic glycopeptide of human myelin oligodendrocyte glycoprotein to detect antibody responses in multiple sclerosis and other neurological diseases.

Glycopeptides of hMOG(30-50) containing a glucosyl moiety on the side-chains of Asn, Ser or Hyp at position 31 were synthesised. Antibody titres to hMOG(30-50) and to its glucoderivatives were measured by ELISA in sera of patients affected by different neurological diseases. Anti-hMOG(30-50) antibodies were detected only using the glycopeptide [Asn31(N-Glc)]hMOG(30-50).