Xenograft of microencapsulated Sertoli cells restores glucose homeostasis in db/db mice with spontaneous diabetes mellitus.

BACKGROUND: Increased abdominal fat and chronic inflammation in the expanded adipose tissue of obesity contribute to the development of insulin resistance and type 2 diabetes mellitus (T2D). The emerging immunoregulatory and anti-inflammatory properties of Sertoli cells have prompted their application to experimental models of autoimmune/inflammatory disorders, including diabetes. The main goal of this work was to verify whether transplantation of microencapsulated prepubertal porcine Sertoli cells (MC-SC) in the subcutaneous abdominal fat depot of spontaneously diabetic and obese db/db mice (homozygous for the diabetes spontaneous mutation [Leprdb ]) would: (i) improve glucose homeostasis and (ii) modulate local and systemic immune response and adipokines profiles. METHODS: Porcine prepubertal Sertoli cells were isolated, according to previously established methods and enveloped in Barium alginate microcapsules by a mono air-jet device. MC-SC were then injected in the subcutaneous abdominal fat depot of db/db mice. RESULTS: We have preliminarily shown that graft of MC-SC restored glucose homeostasis, with normalization of glycated hemoglobin values with improvement of the intraperitoneal glucose tolerance test in 60% of the treated animals. These results were associated with consistent increase, in the adipose tissue, of uncoupling protein 1 expression, regulatory B cells, anti-inflammatory macrophages and a concomitant decrease of proinflammatory macrophages. Furthermore, the treated animals showed a reduction in inducible NOS and proinflammatory molecules and a significant increase in an anti-inflammatory cytokine such as IL-10 along with concomitant rise of circulating adiponectin levels. The anti-hyperglycemic graft effects also emerged from an increased expression of GLUT-4, in conjunction with downregulation of GLUT-2, in skeletal muscle and liver, respectively. CONCLUSIONS: Preliminarily, xenograft of MC-SC holds promises for an effective cell therapy approach for treatment of experimental T2D.

In vitro cadmium effects on ECM gene expression in human bronchial epithelial cells.

Occupational and environmental exposure to the heavy metal cadmium (Cd) and its inhalation from cigarette smoke are associated with emphysema. Many growth factors and extracellular matrix (ECM) cell signaling molecules are directly involved in the epithelial bronchial cell pathway. This study investigated the direct effects of Cd on the production of several ECM components in human bronchial epithelial cells (BEAS-2B) that were exposed in vitro for 48 h to sub-toxic and toxic concentrations of Cd. Gene expression of collagens, metalloproteases (MMPs), integrins, tenascin and vitronectin were quantified by RT-PCR. To study apoptosis cascade, annexin assay and cellular cytotoxicity by MTT assay were performed. We also investigated whether an imbalance in the TGFß/TGFß receptor (TGFßR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, ß1 and ß5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and ß3 integrins. Both Cd doses down-regulated type I collagen and up-regulated metalloproteases. Each Cd dose caused a different imbalance in the complex pattern of TGFß and its receptors. No alteration in classic apoptotic marker protein expression was observed in presence of the sub-toxic dose of Cd, suggesting this metal alters ECM production without apoptotic activation. In conclusion, all these data show even sub-toxic Cd dose exposure alters the specific gene expression of several ECM components that are crucially implicated in the mechanical properties of lung parenchyma supporting the hypothesis that the mechanism underlying Cd-induced lung disease may involve downstream changes in TGFß/TGFßR signaling.

Long-term stability, functional competence, and safety of microencapsulated specific pathogen-free neonatal porcine Sertoli cells: a potential product for cell transplant therapy.

BACKGROUND: Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti-inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen-free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. METHODS: Specific pathogen free neonatal pigs underwent testis excision under sterility. pSCs were isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function [namely, production of anti-müllerian hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGß-1)]. After microencapsulation in barium alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentrations were evaluated at 1, 24 throughout 40 h to establish pre-transplant storage conditions. RESULTS: The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior to and after microencapsulation, maintained high function and full viability up to 24 h of storage. At 40 h post-encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and was stable for 4 months in mice with no evident side effects. CONCLUSIONS: Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantation.

Sub-toxic nicotine concentrations affect extracellular matrix and growth factor signaling gene expressions in human osteoblasts.

Exposure to nicotine and other compounds contained in cigarette smoking affects human health. This study examined the effects of exposure to a single or multiple sub-toxic nicotine concentrations on human osteoblasts. Cell growth and expression of genes involved in bone differentiation, extracellular matrix (ECM) metabolism, and growth factor signaling pathways were investigated in nicotine-treated cells compared to untreated cells. Depending on osteoblast concentration and maturation stages, nicotine differently regulated cell growth. Real-time PCR showed regulated expressions of genes expressed by nicotine-treated osteoblasts compared to untreated cells. Among ECM genes, type I collagen was down-regulated and osteonectin was up-regulated in nicotine-treated osteoblasts; similarly, fibroblast growth factor-1 (FGF1) and fibroblast growth factor-2 (FGF2), two members of FGF signaling system, were discordantly modulated; genes involved in osteoblast maturation and differentiation such as alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2), and bone sialoprotein (BSP) were over-expressed after drug treatment. Our results show a positive association between nicotine exposure and osteoblast phenotype and illustrate for the first time a mechanism whereby acute or chronic exposure to sub-toxic nicotine concentrations may affect bone formation through the impairment of growth factor signaling system and ECM metabolism.

Melatonin as a Repairing Agent in Cadmium- and Free Fatty Acid-Induced Lipotoxicity.

(1) Background: Cadmium (Cd) is a potentially toxic element with a long half-life in the human body (20-40 years). Cytotoxicity mechanisms of Cd include increased levels of oxidative stress and apoptotic signaling, and recent studies have suggested that these aspects of Cd toxicity contribute a role in the pathobiology of non-alcoholic fatty liver disease (NAFLD), a highly prevalent ailment associated with hepatic lipotoxicity and an increased generation of reactive oxygen species (ROS). In this study, Cd toxicity and its interplay with fatty acid (FA)-induced lipotoxicity have been studied in intestinal epithelium and liver cells; the cytoprotective function of melatonin (MLT) has been also evaluated. (2) Methods: human liver cells (HepaRG), primary murine hepatocytes and Caco-2 intestinal epithelial cells were exposed to CdCl2 before and after induction of lipotoxicity with oleic acid (OA) and/or palmitic acid (PA), and in some experiments, FA was combined with MLT (50 nM) treatment. (3) Results: CdCl2 toxicity was associated with ROS induction and reduced cell viability in both the hepatic and intestinal cells. Cd and FA synergized to induce lipid droplet formation and ROS production; the latter was higher for PA compared to OA in liver cells, resulting in a higher reduction in cell viability, especially in HepaRG and primary hepatocytes, whereas CACO-2 cells showed higher resistance to Cd/PA-induced lipotoxicity compared to liver cells. MLT showed significant protection against Cd toxicity either considered alone or combined with FFA-induced lipotoxicity in primary liver cells. (4) Conclusions: Cd and PA combine their pro-oxidant activity to induce lipotoxicity in cellular populations of the gut-liver axis. MLT can be used to lessen the synergistic effect of Cd-PA on cellular ROS formation.

Zinc restores functionality in porcine prepubertal Sertoli cells exposed to subtoxic cadmium concentration via regulating the Nrf2 signaling pathway.

Introduction: Among substances released into the environment by anthropogenic activities, the heavy metal cadmium (Cd) is known to induce severe testicular injury causing male subfertility/infertility. Zinc (Zn) is another heavy metal that, unlike Cd, is physiologically present in the testis, being essential for spermatogenesis. We aimed to examine the possibility that 50 µM ZnCl2 could counteract the toxic effects induced by Cd in an in vitro model of porcine prepubertal Sertoli cells (SCs) exposed to both subtoxic (5 µM) and toxic (10 µM) concentrations of CdCl2 for 48 h. Materials and Methods: Apoptosis, cell cycle, and cell functionality were assessed. The gene expression of Nrf2 and its downstream antioxidant enzymes, ERK1/2, and AKT kinase signaling pathways were evaluated. Materials and Results: We found that Zn, in co-treatment with subtoxic and toxic Cd concentration, increased the number of metabolically active SCs compared to Cd exposure alone but restored SC functionality only in co-treatment with subtoxic Cd concentration with respect to subtoxic Cd alone. Exposure of Cd disrupted cell cycle in SCs, and Zn co-treatment was not able to counteract this effect. Cd alone induced SC death through apoptosis and necrosis in a dose-dependent manner, and co-treatment with Zn increased the pro-apoptotic effect of Cd. Subtoxic and toxic Cd exposures activated the Nrf2 signaling pathway by increasing gene expression of Nrf2 and its downstream genes (SOD, HO-1, and GSHPx). Zn co-treatment with subtoxic Cd attenuated upregulation on the Nrf2 system, while with toxic Cd, the effect was more erratic. Studying ERK1/2 and AKT pathways as a target, we found that the phosphorylation ratio of p-ERK1/2 and p-AKT was upregulated by both subtoxic and toxic Cd exposure alone and in co-treatment with Zn. Discussion: Our results suggest that Zn could counteract Cd effects by increasing the number of metabolically active SCs, fully or partially restoring their functionality by modulating Nrf2, ERK1/2, and AKT pathways. Our SC model could be useful to study the effects of early Cd exposure on immature testis, evaluating the possible protective effects of Zn.

Nickel oxide nanoparticles exposure as a risk factor for male infertility: "In vitro" effects on porcine pre-pubertal Sertoli cells.

Lately, nickel oxide nanoparticles (NiO NPs) have been employed in different industrial and biomedical fields. Several studies have reported that NiO NPs may affect the development of reproductive organs inducing oxidative stress and, resulting in male infertility. We investigated the in vitro effects of NiO NPs on porcine pre-pubertal Sertoli cells (SCs) which undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposure at two subtoxic doses of NiO NPs of 1 µg/ml and 5 µg/ml. After NiO NPs exposure we performed the following analysis: (a) SCs morphological analysis (Light Microscopy); (b) ROS production and oxidative DNA damage, gene expression of antioxidant enzymes (c) SCs functionality (AMH, inhibin B Real-time PCR analysis and ELISA test); (d) apoptosis (WB analysis); (e) pro-inflammatory cytokines (Real-time PCR analysis), and (f) MAPK kinase signaling pathway (WB analysis). We found that the SCs exposed to both subtoxic doses of NiO NPs didn't sustain substantial morphological changes. NiO NPs exposure, at each concentration, reported a marked increase of intracellular ROS at the third week of treatment and DNA damage at all exposure times. We demonstrated, un up-regulation of SOD and HO-1 gene expression, at both concentrations tested. The both subtoxic doses of NiO NPs detected a down-regulation of AMH and inhibin B gene expression and secreted proteins. Only the 5 µg/ml dose induced the activation of caspase-3 at the third week. At the two subtoxic doses of NiO NPs a clear pro-inflammatory response was resulted in an up-regulation of TNF-α and IL-6 in terms of mRNA. Finally, an increased phosphorylation ratio of p-ERK1/2, p-38 and p-AKT was observed up to the third week, at both concentrations. Our results show the negative impact of subtoxic doses NiO NPs chronic exposure on porcine SCs functionality and viability.

Effect of EPA on Neonatal Pig Sertoli Cells "In Vitro": A Possible Treatment to Help Maintain Fertility in Pre-Pubertal Boys Undergoing Treatment With Gonado-Toxic Therapies.

The incidence of cancer in pre-pubertal boys has significantly increased and, it has been recognized that the gonado-toxic effect of the cancer treatments may lead to infertility. Here, we have evaluated the effects on porcine neonatal Sertoli cells (SCs) of three commonly used chemotherapy drugs; cisplatin, 4-Hydroperoxycyclophosphamide and doxorubicin. All three drugs induced a statistical reduction of 5-hydroxymethylcytosine in comparison with the control group, performed by Immunofluorescence Analysis. The gene and protein expression levels of GDNF, were significantly down-regulated after treatment to all three chemotherapy drugs comparison with the control group. Specifically, differences in the mRNA levels of GDNF were: 0,8200 ± 0,0440, 0,6400 ± 0,0140, 0,4400 ± 0,0130 fold change at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at 0.33 and 1.66 µM vs SCs and ***p < 0.001 at 3.33µM vs SCs); 0,6000 ± 0,0340, 0,4200 ± 0,0130 fold change at 50 and 100 µM of 4-Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7000 ± 0,0340, 0,6200 ± 0,0240, 0,4000 ± 0,0230 fold change at 0.1, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Differences in the protein expression levels of GDNF were: 0,7400 ± 0,0340, 0,2000 ± 0,0240, 0,0400 ± 0,0230 A.U. at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7300 ± 0,0340, 0,4000 ± 0,0130 A.U. at 50 and 100 µM of 4- Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,6200 ± 0,0340, 0,4000 ± 0,0240, 0,3800 ± 0,0230 A.U. at 0.l, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Furthermore, we have demonstrated the protective effect of eicosapentaenoic acid on SCs only at the highest concentration of cisplatin, resulting in an increase in both gene and protein expression levels of GDNF (1,3400 ± 0,0280 fold change; **p < 0.01 vs SCs); and of AMH and inhibin B that were significantly recovered with values comparable to the control group. Results from this study, offers the opportunity to develop future therapeutic strategies for male fertility management, especially in pre-pubertal boys.

Effects of Titanium Dioxide Nanoparticles on Porcine Prepubertal Sertoli Cells: An "In Vitro" Study.

The increasing use of nanomaterials in a variety of industrial, commercial, medical products, and their environmental spreading has raised concerns regarding their potential toxicity on human health. Titanium dioxide nanoparticles (TiO2 NPs) represent one of the most commonly used nanoparticles. Emerging evidence suggested that exposure to TiO2 NPs induced reproductive toxicity in male animals. In this in vitro study, porcine prepubertal Sertoli cells (SCs) have undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposures at both subtoxic (5 µg/ml) and toxic (100 µg/ml) doses of TiO2 NPs. After performing synthesis and characterization of nanoparticles, we focused on SCs morphological/ultrastructural analysis, apoptosis, and functionality (AMH, inhibin B), ROS production and oxidative DNA damage, gene expression of antioxidant enzymes, proinflammatory/immunomodulatory cytokines, and MAPK kinase signaling pathway. We found that 5 µg/ml TiO2 NPs did not induce substantial morphological changes overtime, but ultrastructural alterations appeared at the third week. Conversely, SCs exposed to 100 µg/ml TiO2 NPs throughout the whole experiment showed morphological and ultrastructural modifications. TiO2 NPs exposure, at each concentration, induced the activation of caspase-3 at the first and second week. AMH and inhibin B gene expression significantly decreased up to the third week at both concentrations of nanoparticles. The toxic dose of TiO2 NPs induced a marked increase of intracellular ROS and DNA damage at all exposure times. At both concentrations, the increased gene expression of antioxidant enzymes such as SOD and HO-1 was observed whereas, at the toxic dose, a clear proinflammatory stress was evaluated along with the steady increase in the gene expression of IL-1α and IL-6. At both concentrations, an increased phosphorylation ratio of p-ERK1/2 was observed up to the second week followed by the increased phosphorylation ratio of p-NF-kB in the chronic exposure. Although in vitro, this pilot study highlights the adverse effects even of subtoxic dose of TiO2 NPs on porcine prepubertal SCs functionality and viability and, more importantly, set the basis for further in vivo studies, especially in chronic exposure at subtoxic dose of TiO2 NPs, a condition closer to the human exposure to this nanoagent.

Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism.

Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine-treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age-matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real-time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF-beta, retinoic acid (RA), and GABA-ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin alpha2, and MMP13 genes were concordantly modulated, while integrin beta5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition.

In "Vitro" Lps-Stimulated Sertoli Cells Pre-Loaded With Microparticles: Intracellular Activation Pathways.

Sertoli cells (SC) are immune privileged cells with the capacity of modulating the immune response by expressing several immune-regulatory factors. SC have the capacity to respond to external stimuli through innate phagocytic and antibacterial activities. This evidence evoked a potential role of SC as drug carriers and therapeutic agents. Such stimuli drive SC towards a still unknown evolution, the clinical relevance of which as yet remains undisclosed. This study sought to investigate the effects of external stimuli in the form of polymeric microparticles (MP) and bacteria derived endotoxins, such as lipopolysaccharides (LPS), in order to identify the pathways potentially involved in cell phenotype modifications. Compared to single stimulation, when combined, MP and LPS provoked a significant increase in the gene expression of IDO, PD-L1, FAS-L, TLR-3, TLR-4, MHC-II, ICAM-1, TFGß1, BDF123, BDF129, BDF3 and pEP2C. Western Blotting analysis demonstrated up-regulation of the ERK 1-2 and NF-kB p65 phosphorylation ratios. Our study, showing the exponential increase of these mediators upon combined MP and LPS stimulation, suggests a "switch" of SC function from typical cells of the blood-testicular barrier to nonprofessional tolerogenic antigen-presenting cells. Further studies should target the clinical and technological implications of such stimuli-induced SC transformation.

On the Origin of Testicular Germ Cell Tumors: From Gonocytes to Testicular Cancer.

Human primordial germ cells (PGCs) have been described in the yolk sac wall around the beginning of the third week. From week 4 to 5, they migrate under control of SCF/c-KIT signaling pathway to the genital ridge, where they become gonocytes. PGCs and gonocytes express classic pluripotency markers, such as KIT, NANOG, and OCT3/4 that, during spermatogonia differentiation, are gradually suppressed, and substituted by the expression of some germ cell specific genes, such as VASA, SOX17, and TSPY. These genes, during normal development of germ cells, are tightly regulated by epigenetic modification, in terms of microRNA expression and DNA methylation. In adolescents and young adults, testicular germ cell tumors (TGCT) have a common precursor, the germ cell neoplasia in situ (GCNIS); the hypothesis of their origin from PGCs or gonocytes, whose maturation is altered, is widely accepted. The origin of TGCT, probably starting at early stages of embryogenesis, seems to be a part of the Testicular Dysgenesis Syndrome (TDS) where some early PGC/gonocytes, for still unclear reasons, are blocked in their differentiation, retaining their early marker profile. In this paper, current knowledge on the combination of epidemiological and genomic factors, involved in the development of testicular germ cell tumors, is reviewed.

Role of Sertoli Cell Proteins in Immunomodulation.

BACKGROUND: Sertoli cell, over the past 30 years, have been elevated from simple mechanical elements to the rank of a "sentinel" in spermatogenesis. By delivering potent immunomodulatory and trophic proteins, Sertoli cells are unique cell type with a pivotal role in maintaining testis immune privilege and the immune-protection of the antigenic germ cells. CONCLUSIONS: The findings from SC transplantation studies utilizing experimental animal models of disease, demonstrate the presence of the same immuno-modulation properties and mechanisms at tissue and organ sites far from testis. The complex pathways that generate and maintain the immune tolerance involve the production of several immunomodulatory or immune-related proteins such as cytokines, chemokines, growth factors, mediators of the inflammation, complement inhibitors or adhesion molecules. A better definition and understanding of these Sertoli cell proteins and the mechanisms of immunoprotection should help to elucidate their role in the spermatogenic process. The demonstration of their capabilities in transplantation experiments suggests that Sertoli cells may be good candidates in cell therapy for a number of cell-mediated chronic diseases.

Testosterone and FSH modulate Sertoli cell extracellular secretion: Proteomic analysis.

Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and by juxtacrine testicular cross-talk. Sertoli cells (SC) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors, but many of the molecular pathways of SC action remain controversial and unclear. Over the last two decades, research has focused on extracellular vesicles as an important mechanism of intercellular communication. The aim of this study was to investigate the presence of extracellular vesicles (EVs) in SC and the modulation of their content in SC after FSH and testosterone stimulation. Highly purified porcine pre-pubertal Sertoli cells were isolated according to previously established methods. After 48 h of culture with FSH or FSH + testosterone stimulation, we identified sertolian EVs containing specific mRNAs. Proteomic analysis of EVs content identified 29 proteins under non-stimulatory conditions, most of which were related to receptor binding activity. FSH stimulation induced increases in inhibin-alpha, inhibin-beta, plakoglobin, haptoglobin, D-3-phosphoglycerate dehydrogenase and sodium/potassium-transporting ATPase in sertolian EVs. Testosterone stimulation enhanced the abundance of inhibin-alpha, inhibin-beta, tissue-type plasminogen activator, epidermal growth factor-like protein 8, elongating factor 1-gamma and D-3-phosphoglycerate dehydrogenase. These results are likely to help determine the unknown molecular secretion of Sertoli cells.

Accelerated functional maturation of isolated neonatal porcine cell clusters: in vitro and in vivo results in NOD mice.

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/ differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the beta-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from < 3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were determined. Double fluorescence immunolabeling showed increase in beta-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional beta-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.

Basic fibroblast growth factor: effects on matrix remodeling, receptor expression, and transduction pathway in human periosteal fibroblasts with FGFR2 gene mutation.

The Crouzon syndrome, which is associated with fibroblast growth factor receptor (FGFR2) mutations, is characterized by premature fusion of cranial sutures. We used an in vitro model of cultured periosteal fibroblasts from normal subjects and from Crouzon patients with FGFR2 mutation. We analyzed the matrix turnover rate and the effects of adding FGF2 by evaluating fibronectin synthesis and the activity of some proteolytic enzymes. To assess the role of some FGF signaling molecules involved in FGFR2 regulation, we studied Grb2 tyrosine phosphorylation and the phosphotyrosine proteins associated with Grb2. The iodinate FGF binding assay was performed to quantify FGFR expression. Compared with normal fibroblasts, fibronectin synthesis was decreased in Crouzon fibroblasts, and protease activities in cells and medium were enhanced, suggesting that excess fibronectin catabolism is present. Differences were more marked when FGF2 was added. Very few phosphoproteins were visible in anti-Grb2 immunoprecipitations from Crouzon fibroblasts, which showed a significant increase in the number of high-affinity and low-affinity FGF2 receptors. These results suggest that the abnormal genotype and the Crouzon cellular phenotype are related. To compensate the low levels of tyrosine phosphorylation, Crouzon cells might increase the numbers of FGFR2, thus increasing the cell surface binding sites for FGF2.

Mitogenic effects of Brazilian arthropod venom on isolated islet beta cells: in vitro morphologic ultrastructural and functional studies.

BACKGROUND: One of the major pitfalls associated with use of isolated adult islets of Langerhans' cells is their minimal mitotic capacity. Consequently, maintenance of a steady viable islet cell mass is very difficult. To explore how to enhance beta-cell mitogenesis, we have examined the effects of venom fractions extracted from a Brazilian scorpion on morphologic and functional beta-cell patterns. The venom was previously known to induce nesidioblastosis-like effects with chronic hypoglycemia and pancreatitis in animal models. METHODS: Venom fractions purified from Tityus bahiensis were incubated with batches of isolated rat islets, while a morphologic examination, glucose-stimulated insulin release, insulin content, and insulin messenger ribonucleic acid (mRNA) were carried out early during incubation. On fixation and double fluorescence immunolabeling (rhodamine for anti-insulin monoclonal antibodies; fluorescein for anti-5-bromodeoxyuridine), the preparations were imaged by confocal laser microscopy (CLM) for morphometric quantification of the mitoses. Insulin recovery and mRNA were also assessed at 21 days of culture. RESULTS: Under CLM examination, the beta-cell mitotic rate significantly rose from 1 to 12.8% for the venom-exposed islets. At day 7, insulin release and content were significantly lower for the venom-exposed than the control islets. However, at day 21 of culture, insulin release in response to static incubation with glucose and insulin mRNA from the venom-exposed islets was higher than controls (p < .05). CONCLUSIONS: Incubation with the scorpion venom induced a rapid and significant increase in the beta-cell proliferation not associated with a short-term increase in insulin secretion. The latter fully resumed and overcame controls later in culture, possibly after completion of the beta-cell expansion process.

Cross-talk between interleukin-6 and transforming growth factor-beta3 regulates extracellular matrix production by human fibroblasts from subjects with non-syndromic cleft lip and palate.

BACKGROUND: Transforming growth factor-beta (TGF-beta) interference with interleukin 6 (IL-6) activity and the role of the latter in early human embryonic development prompted us to examine the effects IL-6 on matrix synthesis and the effects of TGF-beta3 on IL-6 expression human cleft lip and palate (CLP) fibroblasts. METHODS: Collagen and glycosaminoglycan (GAG) synthesis were determined by radiolabeled precursors and biglycan expression by Northern blotting before and after adding IL-6. The effects of TGF-beta3 on IL-6 production were assayed by evaluating IL-6 transcript by Northern blotting and IL-6 protein secretion by enzyme-linked immunosorbent assay. RESULTS: The results showed that IL-6 elicited an inhibitory effect on collagen and GAG levels in CLP fibroblasts by lowering hyaluronan and dermatan sulfate secretion. IL-6 up-regulated biglycan expression, but less strongly than TGF-beta3. TGF-beta3 significantly down-regulated IL-6 transcript and secretion in CLP fibroblasts. CONCLUSIONS: These data suggest the increase in matrix components that characterize the CLP fibroblast phenotype might be due to a concerted TGF-beta3-IL-6 action. We hypothesize changes in cross-talk between TGF-beta3 and IL-6 signal transduction pathways are involved in the induction of cleft palate.

Reversal of experimental Laron Syndrome by xenotransplantation of microencapsulated porcine Sertoli cells.

Recombinant human IGF-1 currently represents the only available treatment option for the Laron Syndrome, a rare human disorder caused by defects in the gene encoding growth hormone receptor, resulting in irreversibly retarded growth. Unfortunately, this treatment therapy, poorly impacts longitudinal growth (13% in females and 19% in males), while burdening the patients with severe side effects, including hypoglycemia, in association with the unfair chore of taking multiple daily injections that cause local intense pain. In this study, we have demonstrated that a single intraperitoneal graft of microencapsulated pig Sertoli cells, producing pig insulin-like growth factor-1, successfully promoted significant proportional growth in the Laron mouse, a unique animal model of the human Laron Syndrome. These findings indicate a novel, simply, safe and successful method for the cell therapy-based cure of the Laron Syndrome, potentially applicable to humans.

Prolongation of skin allograft survival in rats by the transplantation of microencapsulated xenogeneic neonatal porcine Sertoli cells.

Skin rejection remains a major hurdle in skin reconstructive transplantation surgery. In fact, 85% of the grafted patients experience at least one episode of acute skin rejection in the first year. It has been observed that Sertoli cells (SC), when co-transplanted with allo- or xenogeneic cell/tissues, can induce graft acceptance in the absence of systemic immunosuppression. A method aimed at significantly prolonging skin allografts in rats transplanted with barium alginate-based microencapsulated xenogeneic porcine SC (SC-MCs) is described. Results demonstrated that intraperitoneal (IP) transplantation of SC-MCs with high cellular viability and function can significantly prolong allogeneic skin grafts when compared to transplantation controls receiving only empty alginate capsules (E-MCs). Lymphocytic infiltration at the skin graft site was not observed in 80% of the SC-MCs transplanted rats and these recipient animals showed a significant increased expression of T regulatory (Tregs) cells when compared to E-MCs transplantation controls. The findings of this report further substantiate the positive therapeutic effects of SC on transplantation technology mediated by Sertoli cell-induced alterations of the host's immune system and indicate new perspectives and new strategies for successful skin tissue allografts.