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ω-3 Polyunsaturated fatty acids (PUFAs) have been found to exert many actions, including neuroprotective effects. In this regard, the exact molecular mechanisms are not well understood. Parkinson’s disease (PD) is the second most common age-related neurodegenerative disease. Emerging evidence supports the hypothesis that PD is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction, and other cellular processes, such as DNA methylation. In this context, BDNF (brain-derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) have a pivotal role because they are both involved in neuron differentiation, survival, and synaptogenesis. In this study, we aimed to elucidate the potential role of two PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and their effects on BDNF and GDNF expression in the SH-SY5Y cell line. Cell viability was determined using the MTT assay, and flow cytometry analysis was used to verify the level of apoptosis. Transmission electron microscopy was performed to observe the cell ultrastructure and mitochondria morphology. BDNF and GDNF protein levels and mRNA were assayed by Western blotting and RT-PCR, respectively. Finally, methylated and hydroxymethylated DNA immunoprecipitation were performed in the BDNF and GDNF promoter regions. EPA, but not DHA, is able (i) to reduce the neurotoxic effect of neurotoxin 6-hydroxydopamine (6-OHDA) in vitro, (ii) to re-establish mitochondrial function, and (iii) to increase BNDF and GDNF expression via epigenetic mechanisms.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7sup+/supcDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

Sertoli cells (SeC) isolated from porcine testes have shown direct effects on muscle precursor cells sustaining C2C12 myoblasts proliferation and inhibiting oxidative stress and apoptosis in the early phase of the differentiation process, and stimulating myoblast fusion into myotubes and the expression of markers of myogenic differentiation in the late phase. This suggested that the cocktail of factors secreted by SeC stimulates proliferation in myoblasts without weakening their myogenic potential resulting in the formation of the critical myoblast amount necessary to rebuild the required muscle mass upon a damage. Here, we show that co-culturing C2C12 myoblasts with high doses of SeC microencapsulated in clinical grade alginate-based microcapsules (MC-SeC) for three days in differentiation medium (DM) translates into increased cell numbers and almost absence of myotube formation. However, after removal of MC-SeC, an intense fusion activity into myotubes was observed culminating in a fusion index similar to that of control after additional three days of culture in DM. These data definitely demonstrate that SeC-derived factors preserve the myogenic potential while sustaining cell proliferation in C2C12 myoblasts.

Summary Indoleamine 2,3-dioxygenases (IDOs) degrade l-tryptophan to kynurenines and drive the de novo synthesis of nicotinamide adenine dinucleotide. Unsurprisingly, various invertebrates, vertebrates, and even fungi produce IDO. In mammals, IDO1 also serves as a homeostatic regulator, modulating immune response to infection via local tryptophan deprivation, active catabolite production, and non-enzymatic cell signaling. Whether fungal Idos have pleiotropic functions that impact on host-fungal physiology is unclear. Here, we show that Aspergillus fumigatus possesses three ido genes that are expressed under conditions of hypoxia or tryptophan abundance. Loss of these genes results in increased fungal pathogenicity and inflammation in a mouse model of aspergillosis, driven by an alternative tryptophan degradation pathway to indole derivatives and the host aryl hydrocarbon receptor. Fungal tryptophan metabolic pathways thus cooperate with the host xenobiotic response to shape host-microbe interactions in local tissue microenvironments., Graphical Abstract, Highlights • Aspergillus Idos contribute to de novo NAD+ biosynthesis and fungal growth • In conditions of tryptophan abundance, Aspergillus releases indole derivatives via Aro • Activation of lung AhR enhances harmful lung inflammation during fungal infection • Pharmacological induction of Ido in Aspergillus improves infection outcome, Mammalians use the kynurenine pathway for tryptophan degradation inducing tolerogenic immune responses. Zelante et al. report that similarly, the fungus Aspergillus fumigatus uses the kynurenine pathway. Pathogenic immune response occurs when the fungus catabolizes tryptophan via the indolepyruvate pathway, which targets host AhR. Administration of Ido inducers ameliorates infection outcome.

The 16th Meeting of the Interuniversity Institute of Myology (IIM), October 17-20, 2019, Assisi, Italy brought together scientists, pharma and patient organization representatives discussing new results on muscle research. Internationally renowned Keynote speakers presented advances on muscle development, homeostasis, metabolism, and disease. Speakers selected among submitted abstracts presented their new, unpublished data in seven scientific sessions. The remaining abstracts were showcased in two poster sessions. Young trainees where directly involved in the selection of keynote speakers, the organizing scientific sessions and roundtables discussions tailored to the interests of their peers. A broad Italian, European and North-American audience participated to the different initiatives. The meeting allowed muscle biology researchers to discuss ideas and scientific collaborations aimed at better understanding the mechanisms underlaying muscle diseases in order to develop better therapeutic strategies. The active participation of young trainees was facilitated by the friendly and inclusive atmosphere, which fostered lively discussions identifying emerging areas of myology research and stimulated scientific cross-fertilization. The meeting was a success and the IIM community will continue to bring forward significant contributions to the understanding of muscle development and function, the pathogenesis of muscular diseases and the development of novel therapeutic approaches. Here, we report abstracts of the meeting illustrating novel results of basic, translational, and clinical research, which confirms that the Myology field is strong and healthy.

Because Sertoli cells (SCs) play a central role in germ cell survival, their death may result in marked germ cell loss and infertility. SCs are the only somatic cells within the seminiferous tubules and are essential for regulating spermatogenesis. Factors that enhance or diminish the viability of SCs may have profound effects on spermatogenesis. Yet the mechanisms underlying the maintenance of SC viability remain largely unknown. Glyoxalase 1 (Glo1) detoxifies methylglyoxal (MG), a highly reactive carbonyl species mainly formed during glycolysis, which is a potent precursor of cytotoxic advanced glycation end products (AGEs). Hydroimidazolone (MG-H1) and argpyrimidine (ArgPyr) are AGEs resulting from MG-mediated post-translational modification of arginine residues in various proteins. The role of Glo1 and MG-derived AGEs in regulating the fate of SCs has never been investigated. By using gene silencing and the specific MG scavenger, aminoguanidine, the authors demonstrate that Glo1, under testosterone and follicle-stimulating hormone control, sustains viability of porcine neonatal SCs through a mechanism involving the NF-κB pathway. Glo1 knockdown induces a mitochondrial apoptotic pathway driven by the intracellular accumulation of MG-H1 and ArgPyr that desensitizes NF-κB signaling by modifying the inhibitor of NF-κB kinase, IKKß. This is the first report describing a role for Glo1 and MG-derived AGEs in SC biology, providing valuable new insights into the potential involvement of this metabolic axis into spermatogenesis.

Stem cells have high potential for cell therapy in regenerative medicine. We previously isolated stem cell types from human amniotic fluid, derived from prenatal amniocentesis. One type, characterized by a fast doubling time, was designated as fast human amniotic stem cells (fHASCs). These cells exhibited high differentiation potential and immunoregulatory properties. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that influences stem-cell pluripotency, differentiation, mobility, and regulates immune functions. In this study, we investigated the influence of S1P on fHASC migration, proliferation, differentiation and immune regulatory functions. We found that fHASC stimulation with S1P potentiated their migratory and proliferative activity in vitro. Notably, short fHASC exposure to S1P enhanced their differentiation towards multiple lineages, including adipocytes, osteocytes and endothelial cells, an effect that was associated with downregulation of the main transcription factors involved in the maintenance of a stem-cell undifferentiated state. A specific crosstalk between S1P and tumor growth factor β1 (TGF-β1) has recently been demonstrated. We found that fHASC exposure to S1P in combination with TGF-β1 promoted the expression of the immune regulatory pathway of indoleamine 2,3-dioxygenase 1 (IDO1). In addition, human peripheral blood mononuclear cells, co-cultured with fHASCs treated with S1P and TGF-β1, expanded regulatory T-cells, via a mechanism requiring IDO1. Overall, this study demonstrates that S1P potentiates several properties in fHASCs, an effect that may be critical for exploiting the therapeutic potential of fHASCs and might explain the specific effects of S1P on stem cells during pregnancy., Graphical abstract fx1

Smoke components, such as nicotine and its major metabolites, cross the blood-testis barrier and are detectable in the seminal plasma of both active smokers and individuals exposed to cigarette smoke. In vivo studies in a rat model have further demonstrated that nicotine exposure reduces the weight of the testis, as well as the number of spermatocytes and spermatids, and affects the ultrastructure of Sertoli cells (SC) - which serve as sentinels of spermatogenesis - causing intense germ cell sloughing in the tubular lumen that compromises offspring fertility. This study sought to determine the effects of nicotine on the viability and function of purified pig pre-pubertal SC. Nicotine exposure reduced the mRNA expression and protein levels of anti-Mullerian hormone (AMH) and inhibin B and impaired FSH-r sensitivity via the downregulation of FSH-r and aromatase gene expression compared to untreated SC. Overall, our study suggests that nicotine can significantly alter extracellular matrix and tight junction protein gene expression (e.g., laminin, integrin, and occludin), thus compromising cross-talk between the interstitial and tubular compartments and enhancing blood-testis barrier (BTB) permeability via downregulation of the mitogen-activated protein kinase (MAPK) pathway. These findings further elucidate a potential mechanism of action underlying nicotine exposure's detrimental effects on SC function in vivo.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration leading to impaired locomotion, respiratory failure and premature death. In DMD patients, inflammatory events secondary to dystrophin mutation play a major role in the progression of the pathology. Sertoli cells (SeC) have been largely used to protect xenogeneic engraftments or induce trophic effects thanks to their ability to secrete trophic, antiinflammatory, and immunomodulatory factors. Here we have purified SeC from specific pathogen-free (SPF)-certified neonatal pigs, and embedded them into clinical grade alginate microcapsules. We show that a single intraperitoneal injection of microencapsulated SPF SeC (SeC-MC) in an experimental model of DMD can rescue muscle morphology and performance in the absence of pharmacologic immunosuppressive treatments. Once i.p. injected, SeC-MC act as a drug delivery system that modulates the inflammatory response in muscle tissue, and upregulates the expression of the dystrophin paralogue, utrophin in muscles through systemic release of heregulin-β1, thus promoting sarcolemma stability. Analyses performed five months after single injection show high biocompatibility and long-term efficacy of SeC-MC. Our results might open new avenues for the treatment of patients with DMD and related diseases.

Alginate represents one of the most appealing biopolymers for pharmaceutical and biomedical applications. Alginate as a biomaterial for clinical use has been established, although not free from issues. Here we provide a critical review on some of the main recent advances in alginate research in drug delivery and its prominent role in cell microencapsulation for the treatment of diseases, such as type 1 diabetes mellitus. A brief description of the basic properties of the polymer will be provided as well. Based on our experience and contributions, as well as wide research in the field, the correlation between physicochemical and biological properties of alginate systems and clinical outcomes will be investigated and discussed to address the actual future clinical impact of alginate-based delivery strategies.

Background: Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune dis- eases as they deliver molecules associated with trophic and anti-inflam- matory 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-trans- plant 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 immunofluores- cence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function (namely, production of anti-m€ hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGb-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 stor- age 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 transpor- tation and that Ba-MCpSCs could be potentially employable for xeno- transplantation.

Although human amniotic fluid does contain different populations of foetal-derived stem cells, scanty information is available on the stemness and the potential immunomodulatory activity of in vitro expanded, amniotic fluid stem cells. By means of a methodology unrequiring immune selection, we isolated and characterized different stem cell types from second-trimester human amniotic fluid samples (human amniotic fluid stem cells, HASCs). Of those populations, one was characterized by a fast doubling time, and cells were thus designated as fHASCs. Cells maintained their original phenotype under prolonged in vitro passaging, and they were able to originate embryoid bodies. Moreover, fHASCs exhibited regulatory properties when treated with interferon (IFN)-γ, including induction of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). On coculture with human peripheral blood mononuclear cells, IFN-γ-treated fHASCs caused significantly decreased T-cell proliferation and increased frequency in CD4(+) CD25(+) FOXP3(+) regulatory T cells. Both effects required an intact IDO1 function and were cell contact-independent. An unprecedented finding in our study was that purified vesicles from IFN-γ-treated fHASCs abundantly expressed the functional IDO1 protein, and those vesicles were endowed with an fHASC-like regulatory function. In vivo, fHASCs were capable of immunoregulatory function, promoting allograft survival in a mouse model of allogeneic skin transplantation. This was concurrent with the expansion of CD4(+) CD25(+) Foxp3(+) T cells in graft-draining lymph nodes from recipient mice. Thus fHASCs, or vesicles thereof, may represent a novel opportunity for immunoregulatory maneuvers both in vitro and in vivo.

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.

Environmental pollution is one of the main factors responsible for reducing fertility in males. Lead is one of the major heavy metal contaminants that impairs several organs; it preferentially accumulates in male reproductive organs and alters sperm quality both in vivo and in vitro. However, the underlying mechanisms remain unclear. Sertoli cells (SCs) provide structural and physiological support to spermatogenic cells within seminiferous tubules. Therefore, changes in SCs affect the developing germ cells and alter spermatogenesis. This study aimed to assess whether exposure to subtoxic doses of adversely affects SC functioning in higher mammals. Purified and functional porcine neonatal SCs were exposed to lead acetate at three different concentrations. Lead exposure decreased the mRNA expression and protein levels of inhibin B and anti-Mullerian hormone (AMH) compared to control, indicating loss of FSH-r integrity in terms of 17-β-oestradiol production under FSH stimulation. In addition, we observed an increase in the mRNA levels of Akt and mTOR, and the phosphorylation of p38 and Akt in SCs exposed to lead at all concentrations compared to unexposed control SCs. In conclusion, lead is toxic to SCs, even at low concentrations, and is expected to alter spermatogenesis.

Neonatal porcine Sertoli cells (NPSC) are immune privileged cells showing innate phagocytic and antibacterial activities. NPSC have been shown capable of immunoaltering the body's response and possess lung homing capacity. These properties encourage investigation of NPSC as functional components of cell-based therapeutic protocols to treat lung infections and related complications. In this work, for the first time, NPSC were tailored to carry an antibiotic drug loaded into poly( d , l lactic) acid microparticles (MP). A loading protocol was developed, which afforded 30% drug uptake and high stability over time, with little or no effects on NPSC viability, morphology, reactive oxygen species production and DNA integrity. FSH receptor integrity, and TGFβ (transforming growth factor β) and AMH (anti-Mullerian hormone) expressions were unchanged after 1 month of cryopreservation. Protein tyrosine kinase activation due to phagocytosis may have had resulted in changes in inhibin B expression. The activity of MP-loaded or NPSC alone against Pseudomonas aeruginosa was maintained throughout 1 month of storage. NPSC couple an innate antibacterial activity with the capacity to embody drug loaded MP. We showed for the first time that engineered NPSC can be cryopreserved with no loss of their basic properties, thereby possibly representing a novel approach for cell-based therapeutic and drug delivery system.

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.

Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon-γ is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor-β (TGF-β) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-β-driven tolerance in noninflammatory contexts.

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.

Summary Introduction Immune dysfunction, promoted by pro-inflammatory cytokines, plays a pivotal role in neurodegeneration associated with Huntington's disease. Aims The aim of this study was to investigate the emerging immunoregulatory and antiinflammatory properties of Sertoli cells in Huntington's disease. Methods The experimental R6/2 mouse model of Huntington's disease was treated by a single intraperitoneal injection of microencapsulated prepubertal porcine Sertoli cells and lifespan, motor performance and striatal inflammatory pattern have been evaluated. Results The results of this study demonstrated that a single intraperitoneal injection of microencapsulated prepubertal porcine Sertoli cells uniquely improved performances and extended the life expectancy of R6/2 Huntington's disease mice, by immune dysfunction modulation in brain. Conclusions This study highlights the immunomodulatory and trophic role of Sertoli cells that could be of help in the treatment of neurodegenerative disorders.

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild—yet chronic—neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1—but not pertussis toxin, which affects Gi protein-dependent responses—abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis., Highlights • ADX88178, a selective mGluR4 PAM, exerts long-term therapeutic effects in RR-EAE. • ADX88178 activates a noncanonical mGluR4 signaling in DCs. • ADX88178 induces a tolerogenic functional phenotype in DCs via immunoregulatory IDO1. • Highly selective mGluR4 PAMs may represent novel drugs in chronic neuroinflammation.

Reactivation of latent human cytomegalovirus (HCMV) following allogeneic transplantation is a major cause of morbidity and mortality and predisposes to severe complications, including superinfection by Aspergillus species (spp). Antimicrobial polypeptides, including defensins and mannan-binding lectin, are known to block viral fusion by cross-linking sugars on cell surface. Pentraxin 3 (PTX3), a member of the long pentraxin family, successfully restored antifungal immunity in experimental hematopoietic transplantation. We assessed here whether PTX3 binds HCMV and murine virus (MCMV) and the impact on viral infectivity and superinfection in vivo. We found that PTX3 bound both viruses, reduced viral entry and infectivity in vitro, and protected from MCMV primary infection and reactivation as well as Aspergillus superinfection. This occurred through the activation of interferon (IFN) regulatory factor 3 (IRF3) in dendritic cells via the TLR9/MyD88-independent viral recognition sensing and the promotion of the interleukin-12 (IL-12)/IFN-γ–dependent effector pathway.

To improve the functional performance of microencapsulated islets, we examined the effects of putative cellular support systems, consisting of rat purified Sertoli cells (SC) and astrocytes (AA), on coenveloped allogeneic islets. Coincubation of islets with SC but not AA, resulted in significant stimulation of beta cell mitogenesis, coupled with a significant increase in in vitro glucose-stimulated insulin release. Preliminarily, the xenotransplantation of coencapsulated rat islets and homologous SC significantly prolonged remission of hyperglycemia in diabetic mice.

Normal bone tissue is characterised by a balancing of osteoblast and osteoclast activity. The activity and differentiation of these cells are regulated by vitamins, hormones and cytokines. The action of these factors on bone tissue cells depends on the composition and mineralisation of extracellular bone matrix. In particular, transforming growth factor beta 1 (TGFbeta1) acts on collagen fibres, glycosaminoglycan secretion and on the enzymes correlated to the turnover of glycosaminoglycans. The normal functions of bone tissue also depend on its mineralisation, which is highly altered in the process of uraemia.In this study, we analysed in vitro the effect of transforming growth factor beta on osteoblast proliferation, collagen synthesis and glycosaminoglycan secretion with 3H-thymidine, 3H-proline or 3H-glucosamine incorporation, and on enzymes, such as beta-N-acetyl-D-glucosaminidase and beta-glucuronidase, involved in extracellular matrix turnover. Moreover, phosphatase alkaline activity and osteocalcin related to mineralisation of extracellular matrix were determined.Our data show that TGFbeta1 significantly decreases 3H-thymidine and 3H-proline incorporation and increases (por = 0.01) extracellular sulphated glycosaminoglycan synthesis. It also increases osteocalcin levels, phosphatase alkaline, beta-N-acetyl-D-glucosaminidase and beta-glucoronidase activities.TGFbeta1 changes the synthesis of extracellular matrix components by osteoblasts. These variations favour the action of cytokine and osteoclasts. Since the TGFbeta1 accumulates in bone tissue and increases during uraemia, with due limitations this action leads to an imbalance between synthesis and degradation and could explain bone alterations in uraemic patients.

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 TGFβ/TGFβ receptor (TGFβR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, β1 and β5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and β3 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 TGFβ 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 TGFβ/TGFβR signaling.

Cyclosporin A is a powerful immunosuppressive drug that has had a major impact on transplant therapy. It apparently links to different enzymatic pathways, and affects multiple enzymatic systems. Transforming growth factor β induces the deposition of glycosaminoglycans, proteoglycans, and collagen fibers in the extracellular matrix. The aim of this study of normal and hypertrophic human gingival fibroblast cultures was to evaluate the cytoskeletal and extracellular changes in glycosaminoglycan secretion due to the presence of cyclosporin A and transforming growth factor β. The results showed that there is an increase in total and individual classes of extracellular glycosaminoglycans in the presence of cyclosporin A and transforming growth factor β, but the action of the latter was significantly greater. Immunohistochemical analysis of the cytoskeleton did not reveal any morphological differences between treated and control cells. Our data suggest that the biochemical changes in the extracellular matrix are caused more by cytokine, and that cyclosporin A does not induce any morphological changes in fibroblast cultures derived from hypertrophic and normal gingiva.

The present study provides the first evidence that fibroblasts obtained from patients affected by Crouzon syndrome, a rare craniosynostosis, despite mutations in the high-affinity bFGF receptor retain their capacity to respond to bFGF. The growth factor reduces IL-1 secretion, downregulates biglycan and procollagen α1(I), and increases betaglycan expression. Since betaglycan is a co-receptor for bFGF signalling, an alternative signal transduction pathway is suggested in Crouzon fibroblasts, to explain the documented changes in ECM macromolecule production.

Insulin resistance in type 2 diabetes mellitus (T2DM) may be due to a chronic inflammation of the visceral adipose tissue (VAT) leading to local and systemic increases in proinflammatory cytokines. Microencapsulated porcine Sertoli cells (MC-pSC), by provision of immunomodulatory and trophic factors, have been successfully used to reduce such inflammation in rodent animal models of type 1 diabetes with no complications or deleterious side effects. Herein, we have begun to investigate this novel and safe therapeutic approach in the spontaneously obese nonhuman primate with spontaneous, insulin-dependent T2DM. After MC-pSC intraperitoneal injection we have evaluated, throughout a 6-month follow-up period, daily ad libitum fed glucose levels, daily exogenous insulin supplementation, biweekly body weight measurements, periodic fasting blood glucose concentrations, glycated hemoglobin (HbA1c) levels, glucose tolerance tests (GTT), and fluorescence-activated cell sorting cytometry (FACS) assessment of peripheral blood mononuclear cells. Very preliminarily, we have observed a slight reduction in fasting (FPG) and mean nonfasting (NF) plasma glucose levels. We found minimal changes, only in 1 animal, in daily exogenous insulin requirements and HbA1c levels. Flow cytometric analysis was associated with decrease in CD8 + cells only in 1 recipient with a reduction in mean regulatory T Cells (Treg), whereas interestingly, decrease of B lymphocytes was observed in both animals. These results may suggest that this novel MC-SC–based transplantation protocol might possibly impact the metabolic status of T2DM in higher mammals that are close to humans.

The terahertz dynamics of human cells of the U937 line and their chromatin has been investigated by high-resolution inelastic X-ray scattering. To highlight its dynamical features in situ, nuclear DNA has been stained by uranyl-acetate salt. The general behavior of the collective dynamics of the whole cell is quite similar to that of bulk water, with a nearly wavevector-independent branch located at about 5 meV and a propagating mode with a linear trend corresponding to a speed of sound of 2900 +/- 100 m/s. We provide the first experimental evidence for the existence of two branches also in the dispersion curves of chromatin. The high-energy mode displays an acoustic-like behavior with a sound velocity similar to unstained cells, but in this case the branch likely originates from the superposition of intramolecular DNA optic modes. A low-energy optic-like branch, distinctive of the chromatin moiety, is found at about 2.5 meV.

Human gingival fibroblasts were cultured in vitro using as substrates an extracellular matrix (matrix) and polytetrafluoride (PTFE) membranes, which are used in guided tissue regeneration. To test the degree of biocompatibility of these membranes, the cellular proliferation and the accumulation of extracellular matrix (ECM) macromolecules were considered as parameters. The fibroblasts were cultured in vitro for 24 and 48 hours without serum on plastic, matrix, and PTFE membranes in the presence of 3H-thymidine, 3H-glucosamine, and 3H-proline to study the neo-synthesis of DNA, glycosaminoglycans (GAG), and collagen proteins, respectively. Studies on cell proliferation showed that fibroblasts grown on matrix membrane significantly increased 3H-thymidine incorporation, while fibroblasts grown on PTFE membrane decreased 3H-thymidine incorporation, compared to plastic used as a control. Moreover, the PTFE membrane induced a marked decrease of collagen and GAG accumulation both in the cellular and extracellular pool, while the matrix membrane provoked a decrease of the two macromolecules in the cellular pool and an increase in the extracellular one, compared to the control. The data we obtained demonstrate that matrix membranes are the most suitable to stimulate both cellular proliferation and ECM macromolecule accumulation.

Mesenchymal stem cells (MSCs) are self-renewable, multipotent progenitor cells able to differentiate into various mesodermal lineages.1 MSCs are present in basically all tissues, and have a pivotal role in tissue repair and in local control of inflammation.

The aim of this work was to improve an aqueous two-phase system methodology for fabrication of coherent microcapsules. Simulated microgravity was investigated as tool to improve the cell cluster morphology in order to increase the overall quality of conformal polymer coatings, while the application of two concentric alginate layers and the use of barium instead of calcium as gelling ion was evaluated. Simulated microgravity enabled improvement of neonatal porcine cell cluster sphericity however the freely floating cells, originated during incubation and often found on the capsule surface, raised immunological concerns. Overall, these technical changes translated into improving quality of microcapsules, in terms of either morphologic aspects or the membrane's functional performance. Preparation procedure did not seem to adversely affect viability of the embodied cells. Moreover, the employed alginates high biocompatibility, per se, would promote a good encapsulated cell engraftment. Minimization of last generation microcapsule's size, made of highly purified alginates, represents a further advance on the new horizons of cell therapy for the treatment of a wide variety of chronic disorders, including insulin-dependent diabetes mellitus. (c) 2012 Elsevier B.V. All rights reserved.

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.

Background Spermatogenesis is a complex process where spermatogonial germ cells become spermatozoa with the indispensable support of Sertoli cells (SCs), which provide 'ad hoc' structural and nutritional support. Unfortunately, for most sperm dysfunctions, no therapies are yet available except assisted reproductive technologies (ART) that are based on the use of different culture media to preserve sperm in vitro. However, sperm culture is only possible for short periods of time, since long-term culture would invariably and irreversibly damage the cells with negative impact on their fertilization potential. Methods Fresh sperm cells (5 ml of 20 × 10(6)/ml) were co-cultured with SCs layers, derived from prepubertal pig testes or incubated in cell free SC medium or BWW (Biggers, Whitten and Whittingham) medium for 2, 4 or 7 days. Sperm viability, motility, mitochondrial status, DNA fragmentation, chromatin integrity, intracellular calcium and acrosome status were assessed after every co-culture or incubation time, but capacitation and induction of acrosome reaction (AR) with progesterone was only evaluated after 7 days. Results SCs layers derived from prepubertal pig testes (co-culture of sperm and SC feeder, CCSCF) were able to preserve normal sperm viability, motility and normal mitochondrial function, after 7 days of culture; CCSCF did not induce AR or hyperactivation of spermatozoa, keeping the sperm in a quiescent state for 7 days of culture. Nevertheless, the sperm were readily able to initiate AR after stimulation with progesterone. Conclusions CCSCF maintained good sperm viability and motility for 7 days. This approach could improve retention of sperm viability and motility during ART procedures and maintain sperm viability, during transfer between two distant Centres, avoiding the need for cryopreservation.

Since occupational and environmental exposure to the heavy metal Cadmium (Cd) affects human health this study investigated the effects of exposure to a single, or multiple, sub-toxic Cd concentrations on sub-confluent and confluent human osteoblast growth and expression of specific bone differentiation markers. RT-PCR quantified gene expression of type I collagen, metalloprotease (MMP13), runt-related transcription factor-2 (RUNX2), osterix, osteocalcin, osteonectin, alkaline phosphatase, integrins and bone sialoprotein (BSP). Expression of fibroblast growth factors 1 and 2 (FGF1, FGF2), transforming growth factor-β3 (TGFβ3) and bone morphogenetic protein-2 (BMP2) were also evaluated to determine whether Cd-related effects were mediated by an imbalance in expression. Depending on osteoblast concentration and maturation stages, Cd inhibited or stimulated cell growth, decreased type I collagen, increased MMP13, FGF1 and BMP2 gene expression and stimulated the mineralization process only in continuously exposed cultures. These results suggest that in vivo, acute or chronic exposure to sub-toxic Cd concentrations may affect bone formation differently and support the hypothesis that Cd-induced bone disorders may involve downstream changes in growth factor expression. The results are of interest in forensic and occupational medicine in establishing preventive measures to reduce professional exposure risks.

To assess whether prevention of unexpected in vivo adverse inflammatory and immune responses to biohybrid organ grafts for the treatment of Type I Diabetes Mellitus (T1DM) is possible by superoxide dismutase and ketoprofen controlled release.Superoxide dismutase and ketoprofen-loaded polyester microspheres were prepared by W/O/W and O/W methods, embodied into purified alginate-poly-L-ornithine-alginate microcapsules and intraperitoneally implanted into CD1 mice. The microspheres were characterized for morphology, size, encapsulation efficiency, enzyme activity and in vitro release. Purified alginate contaminants were assayed, and the obtained microcapsules were investigated for size and morphology before and after implantation over 30 days. Cell pericapsular overgrowth and expression were evaluated by optical microscopy and flow cytometry.Superoxide dismutase and ketoprofen sustained release reduced cell pericapsular overgrowth in comparison to the control. Superoxide dismutase release allowed preserving the microcapsules over 30 days. Ketoprofen-loaded microspheres showed some effect in the immediate post-grafting period. A higher macrophage and T-cell expression was observed for the control group.Microspheres containing superoxide dismutase and ketoprofen may represent novel tools to limit or prevent unpredictable adverse in vivo response to alginate, thus contributing to improve cell transplantation success rates in T1DM treatment.

The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM). Starting from isolated neonatal porcine pancreatic islets (NPIs), we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs) for different time periods (7, 14, 21 days). To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation ofβ-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIsβ-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immatureβ-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM.

Human mesenchymal stem cells (hMSCs) are primary cells capable of differentiating to osteocytic lineage when stimulated under appropriate conditions. This study examined changes in hMSC morphology, proliferation, and gene expression after growth on machined or dual acid-etched (AE) titanium surfaces.hMSCs, isolated from adult human bone marrow, were cultured on titanium surfaces. The two specimens of titanium surfaces in this study included machined and AE titanium disks. Cell morphology was evaluated by scanning electron microscopy, and cell proliferation and collagen synthesis were estimated by measuring the amount of 3H-thymidine incorporation into DNA and 3H-proline incorporation into collagen fibers. Alkaline phosphatase (ALP) activity was determined by measuring the release of p-nitrophenol from disodium p-nitrophenyl phosphate. Changes in gene expression for bone morphogenetic protein-2 (BMP-2), Runx2 type II, Osterix (Osx), osteopontin, type I collagen, ALP, osteocalcin, and bone sialoprotein were determined by reverse-transcriptase polymerase chain reaction after 22 days of in vitro culture in osteogenic medium.The two substrates had no significant effects on cell adhesion and proliferation. Morphologic characteristics were observed by scanning electron microscopy. hMSCs on the machined surface spread more and were flatter than cells cultured on the AE surface. Osteopontin mRNA expression was similar on all surfaces, and the other mRNA transcripts were increased in hMSC cultured on AE surface. In particular, BMP-2, Runx2, and Osx, three osteogenic factors that induce the progressive differentiation of multipotent mesenchymal cells into osteoblasts, were expressed more on AE titanium than on machined titanium. Collagen and ALP assays confirmed the highest level of mRNA transcripts correlated with increases in these proteins.These results showed that an AE titanium surface stimulated the expression of markers of osteoblastic phenotype more than a machined titanium surface.