Neural crest stem cell population in craniomaxillofacial development and tissue repair.

Neural crest cells, delaminating from the neural tube during migration, undergo an epithelial-mesenchymal transition and differentiate into several cell types strongly reinforcing the mesoderm of the craniofacial body area - giving rise to bone, cartilage and other tissues and cells of this human body area. Recent studies on craniomaxillofacial neural crest-derived cells have provided evidence for the tremendous plasticity of these cells. Actually, neural crest cells can respond and adapt to the environment in which they migrate and the cranial mesoderm plays an important role toward patterning the identity of the migrating neural crest cells. In our experience, neural crest-derived stem cells, such as dental pulp stem cells, can actively proliferate, repair bone and give rise to other tissues and cytotypes, including blood vessels, smooth muscle, adipocytes and melanocytes, highlighting that their use in tissue engineering is successful. In this review, we provide an overview of the main pathways involved in neural crest formation, delamination, migration and differentiation; and, in particular, we concentrate our attention on the translatability of the latest scientific progress. Here we try to suggest new ideas and strategies that are needed to fully develop the clinical use of these cells. This effort should involve both researchers/clinicians and improvements in good manufacturing practice procedures. It is important to address studies towards clinical application or take into consideration that studies must have an effective therapeutic prospect for humans. New approaches and ideas must be concentrated also toward stem cell recruitment and activation within the human body, overcoming the classical grafting.

Fighting for territories: time-lapse analysis of dental pulp and dental follicle stem cells in co-culture reveals specific migratory capabilities.

Stem cell migration is a critical step during the repair of damaged tissues. In order to achieve appropriate cell-based therapies for tooth and periodontal ligament repair it is necessary first to understand the dynamics of tissue-specific stem cell populations such as dental pulp stem cells (DPSC) and dental follicle stem cells (DFSC). Using time-lapse imaging, we analysed migratory and proliferative capabilities of these two human stem cell lines in vitro. When cultured alone, both DPSC and DFSC exhibited low and irregular migration profiles. In co-cultures, DFSC, but not DPSC, spectacularly increased their migration activity and velocity. DFSC rapidly surrounded the DPSC, thus resembling the in vivo developmental process, where follicle cells encircle both dental epithelium and pulp. Cell morphology was dependent on the culture conditions (mono-culture or co-culture) and changed over time. Regulatory genes involved in dental cell migration and differentiation such as TWIST1, MSX1, RUNX2, SFRP1 and ADAM28, were also evaluated in co-cultures. MSX1 up-regulation indicates that DPSC and DFSC retain their odontogenic potential. However, DPSC lose their capacity to differentiate into odontoblasts in the presence of DFSC, as suggested by RUNX2 up-regulation and TWIST1 down-regulation. In contrast, the unchanged levels of SFRP1 expression suggest that DFSC retain their potential to form periodontal tissues even in the presence of DPSC. These findings demonstrate that stem cells behave differently according to their environment, retain their genetic memory, and compete with each other to acquire the appropriate territory. Understanding the mechanisms involved in stem cell migration may lead to new therapeutic approaches for tooth repair.

Dental pulp stem cells, niches, and notch signaling in tooth injury.

Stem cells guarantee tissue repair and regeneration throughout life. The decision between cell self-renewal and differentiation is influenced by a specialized microenvironment called the 'stem cell niche'. In the tooth, stem cell niches are formed at specific anatomic locations of the dental pulp. The microenvironment of these niches regulates how dental pulp stem cell populations participate in tissue maintenance, repair, and regeneration. Signaling molecules such as Notch proteins are important regulators of stem cell function, with various capacities to induce proliferation or differentiation. Dental injuries often lead to odontoblast apoptosis, which triggers activation of dental pulp stem cells followed by their proliferation, migration, and differentiation into odontoblast-like cells, which elaborate a reparative dentin. Better knowledge of the regulation of dental pulp stem cells within their niches in pathological conditions will aid in the development of novel treatments for dental tissue repair and regeneration.

Ecto-mesenchymal stem cells from dental pulp are committed to differentiate into active melanocytes.

Dental pulp stem cells (DPSCs) are multipotent stem cells derived from neural crest and mesenchyme and have the capacity to differentiate into multiple cell lineages. It has already been demonstrated that DPSCs differentiate into melanocyte-like cells but only when cultivated in a specific melanocyte differentiating medium. In this study we have shown, for the first time, that DPSCs are capable of spontaneously differentiating into mature melanocytes, which display molecular and ultrastructural features of full development, including the expression of melanocyte specific markers and the presence of melanosomes up to the terminal stage of maturation. We have also compared the differentiating features of DPSCs grown in different culture conditions, following the timing of differentiation at molecular and cytochemical levels and found that in all culture conditions full development of these cells was obtained, although at different times. The spontaneous differentiating potential of these cells strongly suggests their possible applications in regenerative medicine.

Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials.

Numerous stem cell niches are present in the different tissues and organs of the adult human body. Among these tissues, dental pulp, entrapped within the 'sealed niche' of the pulp chamber, is an extremely rich site for collecting stem cells. In this study, we demonstrate that the isolation of human dental pulp stem cells by the explants culture method (hD-DPSCs) allows the recovery of a population of dental mesenchymal stem cells that exhibit an elevated proliferation potential. Moreover, we highlight that hD-DPSCs are not only capable of differentiating into osteoblasts and chondrocytes but are also able to switch their genetic programme when co-cultured with murine myoblasts. High levels of MyoD expression were detected, indicating that muscle-specific genes in dental pulp cells can be turned on through myogenic fusion, confirming thus their multipotency. A perivascular niche may be the potential source of hD-DPSCs, as suggested by the consistent Ca(2+) release from these cells in response to endothelin-1 (ET-1) treatment, which is also able to significantly increase cell proliferation. Moreover, response to ET-1 has been found to be superior in hD-DPSCs than in DPSCs, probably due to the isolation method that promotes release of stem/progenitor cells from perivascular structures. The ability to isolate, expand and direct the differentiation of hD-DPSCs into several lineages, mainly towards myogenesis, offers an opportunity for the study of events associated with cell commitment and differentiation. Therefore, hD-DPSCs display enhanced differentiation abilities when compared to DPSCs, and this might be of relevance for their use in therapy.

Human CD34+ stem cells produce bone nodules in vivo.

OBJECTIVES: The aim of this study was to select and provide enough stem cells for quick transplantation in bone engineering procedures, avoiding any in vitro expansion step. MATERIALS AND METHODS: Dental germ pulp, collected from 25 healthy subjects aged 13-20 years, were subjected to magnetic-activated cell sorting to select a CD34(+) stem cell population capable of differentiating into pre-osteoblasts. These cells were allowed to adhere to an absorbable polylactic-coglycolic acid scaffold for 30 min, without any prior expansion, and the CD34(+) cell-colonized scaffolds were then transplanted into immunocompromised rats, subcutaneously. RESULTS: After 60 days, analysis of recovered transplants revealed that they were formed of nodules of bone, of the same dimensions as the original scaffold. Bone-specific proteins were detected by immunofluorescence, within the nodules, and X-ray diffraction patterns revealed characteristic features of bone. In addition, presence of platelet endothelial cell adhesion molecule and von Willebrand factor immunoreactivity were suggestive of neo-angiogenesis and neovasculogenesis taking place within nodules. Importantly, these vessels were HLA-1(+) and, thus, clearly human in origin. CONCLUSIONS: This study indicates that CD34(+) cells obtained from dental pulp can be used for engineering bone, without the need for prior culture expanding procedures. Using autologous stem cells, this schedule could be used to provide the basis for bone regenerative surgery, with limited sacrifice of tissue, low morbidity at the collection site, and significant reduction in time needed for clinical recovery.

Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation.

Stromal stem cells from human dental pulp (SBP-DPSCs) were used to study osteogenic differentiation in vitro and in vivo. We previously reported that SBP-DPSCs are multipotent stem cells able to differentiate into osteoblasts, which synthesize three-dimensional woven bone tissue chips in vitro. In this study, we followed the temporal expression pattern of specific markers in SBP-DPSCs and found that, when differentiating into osteoblasts, they express, besides osteocalcin, also flk-1 (VEGF-R2). In addition, 30% of them expressed specific antigens for endothelial cells, including CD54, von-Willebrand (domain 1 and 2), CD31 (PECAM-1) and angiotensin-converting enzyme. Interestingly, we found endotheliocytes forming vessel walls, observing that stem cells synergically differentiate into osteoblasts and endotheliocytes, and that flk-1 exerts a pivotal role in coupling osteoblast and endotheliocyte differentiation. When either SBP-DPSCs or bone chips obtained in vitro were transplanted into immunocompromised rats, they generated a tissue structure with an integral blood supply similar to that of human adult bone; in fact, a large number of HLA-1+ vessels were observed either within the bone or surrounding it in a periosteal layer. This study provides direct evidence to suggest that osteogenesis and angiogenesis mediated by human SBP-DPSCs may be regulated by distinct mechanisms, leading to the organization of adult bone tissue after stem cell transplantation.

Tacrolimus, but not cyclosporine A, significantly increases expression of ICAM-1 and IFN-gamma in the NOD mouse.

We studied the alterations of cytokines and ICAM-1 expression in the NOD mouse pancreas produced by the administration of Cyclosporine A (CY) and Tacrolimus (TA), two widely used immunosuppressive drugs. Results evidenced differences in the effects of these two drugs. In fact, during treatment and after withdrawal, CY-treated animals remained euglycemic, showed good islet cell preservation and had low levels of Th1 and Th2 cytokines; ICAM-1 positivity within the islets was also found to be relatively low. On the other hand, TA-treated animals had infiltrated islets containing numerous dendritic cells, adhesion molecule overexpression, increased IFN-gamma and ICAM-1 mRNA transcripts, and interestingly, high levels of circulating ICAM-1. However, even these animals remained euglycemic. These findings lead to the thought that these drugs may exert their effects in very different ways. Moreover, in TA-treated animals, the presence of an islet infiltrate containing numerous dendritic cells coupled with maintenance of euglycemia is suggestive for the involvement of immunosurveillance mechanisms. J. Cell. Biochem. Suppl. 36: 107-116, 2001.

Sodium fusidate (fusidin) ameliorates the course of monophasic experimental allergic encephalomyelitis in the Lewis rat.

We have evaluated the effect of the immunosuppressant sodium fusidate (fusidin) on the course of acute monophasic experimental encephalomyelitis (EAE) in male Lewis rats. Prophylactic treatment with fusidin, 80 or 120 mg/kg bd wt., markedly ameliorated the course of the disease in rats immunized with myelin basic proteins in complete Freund's adjuvant, entailing delayed onset of symptoms, lower clinical scores and more rapid recovery than PBS-treated control rats. The fusidin-treated, immunized rats exhibited milder mononuclear cell infiltration of brains and spinal cords than control animals. These data provide further evidence for the anti-inflammatory effect of fusidin and suggest that this drug may be valuable for the treatment of human multiple sclerosis.

Sodium fusidate ameliorates the course of diabetes induced in mice by multiple low doses of streptozotocin.

We studied the effects of the immunosuppressant sodium fusidate (fusidin) on murine immunoinflammatory diabetes mellitus (DM) induced by multiple low doses of streptozotocin (SZ). Fusidin was given by gavage to three strains of mice (C57KsJ, C57BL/6, CD1) at doses 10 or 100 mg/kg body weight every other day. The drug was administered as an early or late prophylactic regime starting either 1 day prior to the first or after the fifth and last injection of SZ. In both situations the largest dose of fusidin successfully reduced the clinical, chemical and histological signs of DM, the treated mice having significantly lower glycaemic values and milder (often absent) insulitis compared with sham-treated animals or controls given SZ alone. The antidiabetogenic effect was long-lasting as it was maintained up to 1 month after cessation of therapy. In contrast, fusidin prophylaxis failed to prevent development of hyperglycaemia acutely induced by one single and high (160 mg/kg) dose of SZ, which is a model of DM primarily due to the toxic action of SZ on the beta cells and does not involve immunopathogenetic mechanisms. On day 14 after SZ, fusidin markedly altered the circulating cytokine profile induced in vivo by ConA, reducing the levels of IFN-gamma, IL-2 and TNF-alpha and augmenting the level of IL-6. However, only the inhibitory effect of the drug on the synthesis/release of IFN-gamma seemed to be causally related to its capacity to counteract the SZ-induced DM. In fact, the disease was prevented by a neutralizing monoclonal antibody (mAb) against IFN-gamma, but not by anti-IL-2 receptor mAb, a soluble form of TNF-receptor type 1 or recombinant human IL-6. The prevention of disease by fusidin was also partly reversed by exogenously administered recombinant mouse IFN-gamma. The data provide further in-vivo evidence for the anti-diabetogenic and immunomodulatory properties of fusidin and indicate that this drug could have a role in prevention and treatment of human type 1 DM.

Prevention of spontaneous autoimmune diabetes in NOD mice by transferring in vitro antigen-pulsed syngeneic dendritic cells.

To evaluate the effect of antigen-pulsed dendritic cell (DC) transfer on the development of diabetes, 5-week-old female NOD mice received a single iv injection of splenic syngeneic DC from euglycemic NOD mice pulsed in vitro with human y globulin (HGG). Eleven of 12 mice were protected from the development of diabetes up to the age of 25 weeks, and the insulitis score was significantly reduced. In contrast, NOD mice receiving unpulsed splenic DCs showed histological signs of insulitis and course of type 1 diabetes similar to untreated NOD mice. Treatment with HGG-pulsed DC was associated with profound modifications of cytokine secretory capacities within the islets. Thus, supernatants of islets from these mice contained increased levels of interleukin (IL)-4, IL-10, and, to a lesser extent, interferon-gamma and diminished levels of tumor necrosis factor-a compared with controls. Because exogenous IL-4 and IL-10 exert antidiabetogenic effect in NOD mice and early blockade of endogenous tumor necrosis factor-alpha prevents NOD mouse diabetes, these phenomena may be causally related to the antidiabetogenic effect of HGG-pulsed DC treatment.

Multiple low-dose and single high-dose treatments with streptozotocin do not generate nitric oxide.

Streptozotocin (STZ) is a widely used diabetogenic agent that damages pancreatic islet beta cells by activating immune mechanisms, when given in multiple low doses, and by alkylating DNA, when given at a single high dose. Actually, STZ contains a nitroso moiety. Incubation of rat islets with this compound has been found to generate nitrite; moreover, photoinduced NO production from STZ has been demonstrated. These reports have suggested that direct NO generation may be a mechanism for STZ toxicity in diabetogenesis. Several other studies have denied such a mechanism of action. This study has shown that (1) the multiple low-dose (MLDS) treatment does not stimulate NO production at the islet level; in fact, nitrite + nitrate levels and aconitase activity (also in the presence of an NO-synthase inhibitor, namely NAME) remain unmodified; RT-PCR analysis demonstrates that this treatment does not stimulate iNOS activity; (2) the high-dose (HDS) treatment does not stimulate NO production; in fact nitrite + nitrate levels remain unmodified and iNOS mRNA levels are not altered, although aconitase activity is significantly decreased. Moreover, we have confirmed that the MLDS treatment is able to decrease SOD activity by day 11 and that STZ, given in a single high dose, transiently increases superoxide dismutase (SOD) values (24 h from the administration), then dramatically lowers SOD levels. On the basis of our results, we conclude that STZ, "in vivo" is unable to generate NO, both as a MLDS or HDS treatment, thus excluding that NO exerts a role in streptozotocin-dependent diabetes mellitus.

Detection of dendritic cells in the non-obese diabetic (NOD) mouse islet pancreas infiltrate is correlated with Th2-cytokine production.

We investigate the role played by dendritic cells (DCs) in the non-obese diabetic (NOD) mouse pancreas. The early peri-islet, nondestructive infiltration phase, and intra-islet, destructive infiltration phase, which immediately precedes overt diabetes, are studied. Results show that infiltrating cells are Ia-b, ICAM-1, and, mainly, MIDC-8 immunoreactive (ir). These data from silica-treated animals and ultrastructural observations strongly support the hypothesis that DCs are both Ia-b-ir and ICAM-1-ir and that they exert a pivotal role during the period of early infiltration. This is a novel finding for NOD mice and increases the interest for this protective cell type during the rather complex islet infiltration process. Moreover, the cytokine profile demonstrates that Th2 protective cytokines are specific for peri-islet infiltrate. Disappearance of DCs from the infiltrate is concomitant with both the formation of intra-islet infiltration and the increase in proinflammatory Th1 cytokine levels. This further supports the hypothesis that DCs may exert a protective role against diabetes development.

Nicotinamide decreases MHC class II but not MHC class I expression and increases intercellular adhesion molecule-1 structures in non-obese diabetic mouse pancreas.

Pancreases of untreated and nicotinamide (NIC)-treated pre-diabetic (10-week-old) and overtly diabetic (25-week-old) female NOD (non-obese diabetic) mice and of NON (non-obese non-diabetic) control mice were studied, with the following results. (1) Islets and ducts of overtly diabetic untreated NOD mice (25-week-old) were found to express low levels of MHC class I and II molecules, like NON controls, and high levels of adhesive molecules. (2) NIC was able to slightly affect glycaemia and insulitis, slowing down diabetes progression. Moreover it significantly decreased MHC class II expression (but not class I) in vivo by week 10, and significantly enhanced intercellular adhesion molecule-1 (ICAM-1) expression, mainly by week 25, within the pancreas, where 5-bromo-2'-deoxyuridine positive nuclei and insulin positive cells were present, demonstrating that a stimulation of endocrine cell proliferation occurs. (3) In addition, NIC partly counteracted the fall of superoxide dismutase levels, observed in untreated diabetic NOD animals. (4) In vitro studies demonstrated that NIC: (i) was able to significantly reduce nitrite accumulation and to increase NAD+NADH content significantly, and (ii) was able to increase the levels of interleukin-4, a T helper 2 lymphocyte (Th2) protective cytokine, and of interferon-alpha (IFN-alpha), which is known to be able to induce MHC class I and ICAM-1 but not MHC class II expression, as well as IFN-gamma, which is also known to be able to induce MHC class I and ICAM-1 expression. The latter, although known to be a proinflammatory Th1 cytokine, has also recently been found to exert an anti-diabetogenic role. This study therefore clearly shows that adhesive mechanisms are ongoing during the later periods of diabetes in pancreatic ducts of NOD mice, and suggests they may be involved in a persistence of the immune mechanisms of recognition, adhesion and cytolysis and/or endocrine regeneration or differentiation processes, as both NIC-increased ICAM-1 expression and 5-bromo-2'-deoxyuridine positivity imply. The effects of NIC on MHC class II (i.e. a reduction) but not class I, and, mainly, on ICAM-1 expression (i.e. an increase), together with the increase in Th2 protective cytokine levels are very interesting, and could help to explain its mechanism of action and the reasons for alternate success or failure in protecting against type 1 diabetes development.

Controlled clinical trial of pefloxacin versus imipenem in severe acute pancreatitis.

BACKGROUND & AIMS: Antibiotic prophylaxis in severe pancreatitis has recently yielded promising clinical results, with imipenem significantly reducing the incidence of infected necrosis compared with an untreated control group. On the bases of pefloxacin's spectrum of action and pancreatic penetration, we investigated whether such drugs represent a valid alternative to imipenem. METHODS: In a multicenter study, 60 patients with severe acute pancreatitis with necrosis affecting at least 50% of the pancreas were randomly allocated to receive intravenous treatment for 2 weeks with pefloxacin, 400 mg twice daily (30 patients), or imipenem, 500 mg three times daily (30 patients), within 120 hours of onset of symptoms. Age, sex, body weight, Ranson and Apache II scores, C-reactive protein, etiology, and time from onset of symptoms to treatment were well matched in the two groups. RESULTS: The incidences of infected necrosis and extrapancreatic infections were 34% and 44%, respectively, in the pefloxacin group and 10% and 20% in the imipenem group. Imipenem proved significantly more effective in prevention of pancreatic infections (P

Macrophages and antioxidant status in the NOD mouse pancreas.

This study showed that citiolone (CIT), a free radical scavenger, significantly increased superoxide dismutase (P < 0.001 vs. untreated NOD, NMMA-treated, and silica-treated animals), catalase (P < 0.01 vs. untreated NOD), and glutathione peroxidase (P < 0.001 vs. untreated NOD and C57BL6/J) values. Silica treatment was capable of counteracting the plasma antioxidant capacity (TRAP) decrease observed in untreated NOD mice, although it did not block the blood glucose rise and insulitis progression in type 1 diabetes significantly. Conversely, early silica administration was able to deplete macrophages (as demonstrated by immunocytochemistry) and to block the rise in blood glucose levels and insulitis progression significantly. Silica-treated animals in this study showed the highest TRAP levels, demonstrating that depletion of macrophages also was able to improve the antioxidant status. This study suggested that macrophages are essential for type 1 diabetes development and showed that they also are involved when the antioxidant status is affected. The reported findings are significant in view of previous studies indicating that oxygen and/or nitrogen free radicals contribute to the islet beta-cell destruction in type 1 diabetes animal models.

Adhesion molecules and microvascular changes in the nonobese diabetic (NOD) mouse pancreas. An NO-inhibitor (L-NAME) is unable to block adhesion inflammation-induced activation.

The aim of the present study was to investigate the immunoreactivity of pancreatic microvasculature with emphasis on the adhesion molecule expression in NOD mice at a very early stage and after the start of infiltration, before the onset of the diabetic disease. Immunoreactivity for Ia-b, BM8 (mouse macrophages) and inter-cellular-adhesion-molecule-1 (ICAM-1) molecules in untreated control mice and in animals treated using an inhibitor of nitric oxide (NO) formation (L-arginine analogue), as well as islet culture, nitrite assay and ultrastructural studies were performed. Results showed that Ia-b and ICAM-1 immunoreactivities on endothelia are a very early phenomenon and that pancreatic blood vessels and, in particular, some peri-islet venules, as well as several venules of the exocrine parenchyma, undergo significant morphological changes. Several endothelial cells of both peri-islet and extra-islet compartments, often showed Ia-b and ICAM-1 immunoreactivities, demonstrating that these cells are important for the adhesion processes taking place during early autoimmune inflammation. Inhibition of NO formation does not significantly affect ICAM-1 and Ia-b immunoreactivity both in vivo and in vitro, BM8 immunoreactive cells were considerably less in number although these were detected either around islets or along pancreatic septa, but rarely within the epithelial layer.

Effects of butylated hydroxytoluene (BHT) enriched diet on serum antioxidant activity in pre-and overtly diabetic nod mice.

Preventive (antioxidant activity) and chain-breaking (total peroxyl radical-trapping parameter) antioxidants in the serum of controls and butylated hydroxytoluene (BHT)-diet enriched nonobese diabetic (NOD) and C57B16/J mice from 5 to 25 weeks of age are measured in this study. A significant decrease in the overall potency of both antioxidant types is demonstrated in NOD untreated controls but not in animals whose diet was BHT-enriched. Therefore, we show that alterations of the antioxidant status in NOD mice is efficaciously counteracted by BHT.

Prognostic role of cyclin D1 in non small cell lung cancer: an immunohistochemical analysis.

Lung cancer is a worldwide problem and in many countires it is the most lethal malignancy. Because relapse is frequent after resection of non small cell lung cancer, an urgent need exists to define prognostic factors which could help in choosing the best therapeutic approach. We performed immunohistochemistry on 60 formalin-fixed paraffin-embedded non small cell lung cancer specimens in order to evaluate the frequency of cyclin D1 overexpression, and to relate it to the degree of malignancy of these tumors and to the overall survival time of the patients. All specimens were positive for cyclin D1 immunostaining. We found cyclin D1 overexpression in 30 (50%) of our specimens, with no significant difference among the different histological types. Cyclin D1 overexpression correlates in a statistical manner with short-term patient survival. Mantel-Cox analysis of these data generated a significant P value = 0.003. The mean survival time and the five-year survival rate also differed statistically. We did not find any statistically significant correlation between cyclin D1 overexpression and histological grading, tumor stage or TNM status. We concluded that cyclin D1 overexpression in 30 patients is a frequent event in non small cell lung cancer pathogenesis and may have prognostic relevance.