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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.

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.

Sertoli cells are located in the testes where they control several key functions in spermatogenesis. Over the past 30 years, Sertoli cells have been upgraded from a simple scaffold-like structural system to a dynamic functional system of intercellular support that delivers potent immunomodulatory and trophic factors. Since the discovery of new Sertoli cell secretory products, these cells have been utilized in experimental cell transplantation and co-transplantation protocols aimed at treating both chronic inflammatory and degenerative disorders. For these reasons, this work reviews the application of both naked and microencapsulated Sertoli cells used in cell transplantation studies of several chronic or autoimmune diseases such as diabetes mellitus, Laron dwarfism, and Duchenne muscular dystrophy and in studies aimed at the prevention of skin allograft rejection.

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.

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.

We reveal the basement membrane, a specialized connective tissue structure found in all tissue systems, as a framework in an adaptive computer aided design (CAD) strategy for the reverse engineering of 3 dimensional (3D) tissue structures. Our approach to the creation of functional 3D tissue structures is centered on our previous models of vascular supply systems which included complete and accurate replications of capillary bed systems, the circulatory interface necessary to sustain 3D tissue structures. By using the basement membrane as a guide, we seek to design models for the reverse engineering of the other extracellular connective tissue structures and their matrix elements. We demonstrate the basement membrane as a platform for the design and engineering of tissue scaffolding for vascularized alveolar systems in the lung and for the vascularized dermal skin layer. The resulting structure will support the 3D growth and differentiation of cells and their products. The biomedical industry stands to be greatly impacted by this CAD approach to the engineering of 3D tissue structures.

The 796RMB cell line is a multipotent stem cell line isolated from human fetal midbrain tissues, a region from which dopamine neurons of the substantia nigra develop. It would be useful to increase the dopaminergic characteristics of this cell line to enhance its usefulness as a cell therapy for Parkinson's disease utilizing transplantation protocols. Sertoli cells and its conditioned media isolated from the testis have been previously shown to enhance tyrosine hydroxylase expression in ventral mesencephalon neurons both in vitro and in vivo. Therefore, the present preliminary study investigated the ability of Sertoli cell pre-conditioned medium to enhance differentiation of the 796MB cell line toward the domaminergic phenotype. Results showed that secretory products derived from Sertoli cell conditioned medium increased cell proliferation and enhanced dopaminergic neuronal differentiation of the 796RMB cell line. These findings may lead to alternative therapeutic cell transplantation protocols for the treatment of Parkinson's disease.

AIM To determine the effectiveness of the sk11, sk9 and sk11 TNUA5 Sertoli cell lines in binding germ cells in vitro. METHODS The immortalized Sertoli cell lines sk9, sk11 and sk11 TNUA5 were used in co-culture experiments with germ cells in media with or without reproductive hormones and incubated for 44 h at 32 degrees . The number of germ cells bound to Sertoli cells was then determined and statistically analyzed. Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) studies were employed to investigate the presence of cell adhesion proteins and follicle stimulating hormone (FSH) receptor, respectively. RESULTS No statistical difference between the number of bound step-8 spermatids and bound pre-step 8 spermatids on Sertoli cells from any of the cell lines existed. After the addition of germ cells, Sertoli cells showed more lipid accumulation in their cytoplasm, indicating active phagocytosis. Western blot analysis in the sk11 TNUA5 line indicated the expression of N-cadherin. FSH-only and testosterone-only treatments increased N-cadherin expression, regardless of germ cell addition. The addition of germ cells to the sk11 TNUA5 Sertoli cells increased the expression of espin, as did the addition of FSH with germ cells. RT-PCR studies of the sk11 TNUA5 cells indicated that the mRNA for FSH receptor decreased with successive passages. CONCLUSION In vitro binding between isolated germ cells and sk9, sk11 or sk11 TNUA5 Sertoli cells is not feasible, and therefore these cell lines are not useful for the in vitro investigation of Sertoli-germ cell interactions and primary Sertoli cell isolates must still be used.

This chapter summarises the knowledge of Stylosanthes (stylo) anthracnose in Australia and outlines how this has contributed to improved anthracnose management. The centers of origin of C. gloeosporioides infecting stylo are believed to be in South America and the pathogen has most probably entered Australia through introduced stylo germplasm. Seed-borne inoculum is important in anthracnose epidemiology as infested seed can introduce the pathogen to new stylo pastures and also disperse new virulent forms of C. gloeosporioides. Anthracnose greatly weakens and can kill susceptible plants but the greatest impact is on the seed industry. Due to cost and operational constraints, the only viable option for anthracnose management in broadacre pastures is the utilisation of host resistance. An improved knowledge of the genetics of host resistance has to be one of the priorities of future research. Research to promote host diversity has a high priority to overcome the challenges from a variable pathogen population.

The actin-based cell-cell adherens junction (AJ) between the Sertoli cell and the germ cell in the mammalian testis is important not only in mechanical adhesion of the cells, but in the morphogenesis and differentiation of the germ cells. The Sertoli ectoplasmic specialization (ES), a specialized type of AJ, is associated with Sertoli-spermatid binding and is important in cell-cell adhesion in the seminiferous epithelium. Abnormal or absent Sertoli ESs have been associated with step-8 spermatid sloughing and oligospermia in conditions associated with reduced fertility potential. The reproductive hormones, follicle stimulating hormone (FSH), and testosterone (T) have also been shown to play a role in the regulation of binding of spermatids at the Sertoli-spermatid junctional complex (STJC). Adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide] is a potential male contraceptive and is thought to exhibit its contraceptive effects by interrupting the STJC. It has been shown that this compound induces reversible germ cell loss from the seminiferous epithelium, particularly elongating/elongate/round spermatids and spermatocytes. Using a micropipette pressure transducing system (MPTS) to measure the force needed to detach step-8 spermatids from Sertoli cells, this study examined the strength of the STJC in Sertoli-spermatid cocultures in the presence of Adjudin (1 ng/mL, 50 ng/mL, 125 ng/mL, or 500 ng/mL in EtOH) and hormones [FSH (0.1 micro g/mL, NIDDK-oFSH-20, AFP7028D, 175 x NIH-FSH-S1), T (100 nM)] to optimize in vitro binding. The average forces required to detach the spermatids from the underlying Sertoli cells in the presence of 1 ng/mL, 50 ng/mL, 125 ng/mL, and 500 ng/mL Adjudin were 18.2 x 10(-10) pN, 14.3 x 10(-10) pN, 7.74 x 10(-10) pN, and 6.51 x 10(-10) pN, respectively. The average force required to detach step-8 spermatids in the presence of vehicle only (control) was 19.0 x 10(-10) pN. A significant difference for Adjudin concentrations at or above 125 ng/mL was determined by one-way ANOVA (P < .05). These data confirm that Adjudin is effective in reducing the strength of the STJC, identifying Adjudin as a potential contraceptive agent in the male by inducing spermatid sloughing and therefore oligospermia.

Cell therapy is a potentially powerful tool in the treatment of many grave disorders including leukemia, immune deficiencies, autoimmune diseases, and diabetes. However, finding matched donors is challenging and recipients may suffer from the severe complications of systemic immune suppression. Sertoli cells, when cotransplanted with both allo- and xenograft tissues, promote graft acceptance in the absence of systemic immunosuppression. How Sertoli cells do this is not, as yet, clearly defined. We have examined the ability of Sertoli cells to produce systemic immune tolerance. For this purpose, Sertoli cells were injected into an otherwise normal C57/BL6 mouse host via the lateral tail vein. No other immunosuppressive protocols were applied. Six to 8 weeks posttransplantation, blood was collected for analysis of cytokine levels. Tolerance to donor cells was determined by mixed lymphocytic culture, and production of T-cell-dependent antibody was determined by an in vitro anti-sheep red blood cell plaque-forming assay. Results showed a marked modulation of immune cytokines in the transplanted mouse host and donor-specific transplantation tolerance was achieved. Tolerant mouse lymphocytes maintained a competent humoral antibody response. Additionally, C57/BL6 mice transplanted with rat Sertoli cells tolerated rat skin grafts significantly longer than control non-Sertoli cell transplanted mice. We conclude that systemic administration of rat Sertoli cells across xenogenic barrier induces transplantation tolerance without altering systemic immune competence. These data suggest that Sertoli cells may be used as a novel and potentially powerful tool in cell transplantation therapy.

Neural transplantation is developing as a successful treatment for neurodegenerative diseases such as Parkinson's disease. The human Ntera-2/D1 (NT2) cell line is an attractive alternative to the use of human fetal neurons as a cell source for transplantation. We have explored combining NT2 cells, as a neuronal source, and Sertoli cells, which may act as a graft facilitator to enhance neuronal survival and differentiation, and ameliorate the host immune response, into a tissue construct for use in cell replacement therapy for neurodegenerative disease. This Sertoli-NT2-aggregated cell (SNAC) tissue construct is formed in the high aspect ratio vessel (HARV) bioreactor. NT2 cells differentiate to dopaminergic NT2N neurons within the SNAC tissue construct without retinoic acid. We report here that the gap junction protein connexin 43 is decreased among differentiated NT2N neurons. Inhibition of connexin 43 with 18beta glycyrrhetinic acid and carbenoxolone, a glycyrrhetinic acid derivative, during formation of the SNAC tissue constructs disrupts the differentiation of NT2 cells. Therefore, connexin 43 is important in the differentiation of NT2 cells in the SNAC tissue construct.

Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.

Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1–4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT—specific for NT2 cells) and cholera toxin subunit B (CTb—specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD.

Sertoli cells, a testes-derived cell with immunosuppressive and trophic properties, may serve as an alternative cell source for transplantation in a number of neuro degenerative diseases. However, before Sertoli cells can be considered for clinical use, safety studies must be conducted to ensure that the cells themselves produce no adverse effects when transplanted into the central nervous system. The present study assessed the behavioral effects of transplanting porcine Sertoli cells into the striatum of normal rats and provided a histological examination of the graft site and host striatum. Activity monitors revealed significant increases in nocturnal locomotor activity over time following both sham and Sertoli trnasplants. Ambulation and rearing, but not stereotypic measures, were increased compared to pre-transplant levels. Sertoli animals exhibited less behavioral alteration than sham controls. Histological examination of the striatum demonstrated surviving Sertoli cell transplants in an intact striatum. These results indicated that Sertoli cell xenografts might be a safe alternative cell source for neurotransplantation procedures requiring immune or trophic support.

Previous studies have shown that a negative relationship exists between transpiration efficiency (TE) and carbon isotope discrimination (Delta) and between TE and specific leaf area (SLA) in Stylosanthes scabra. A glasshouse experiment was conducted to confirm these relationships in an F(2) population and to study the causal nature of these relationships through quantitative trait loci (QTL) analysis. One hundred and twenty F(2) genotypes from a cross between two genotypes within S. scabra were used. Three replications for each genotype were maintained through vegetative propagation. Water stress was imposed by maintaining plants at 40% of field capacity for about 45 d. To facilitate QTL analysis, a genetic linkage map consisting of 151 RAPD markers was developed. Results from this study show that Delta was significantly and negatively correlated with TE and biomass production. Similarly, SLA showed significant negative correlation with TE and biomass production. Most of the QTL for TE and Delta were present on linkage groups 5 and 11. Similarly, QTL for SLA, transpiration and biomass productivity traits were clustered on linkage groups 13 and 24. One unlinked marker was also associated with these traits. There were several markers coincident between different traits. At all the coincident QTL, the direction of QTL effects was consistent with phenotypic data. At the coincident markers between TE and Delta, high alleles of TE were associated with low alleles of Delta. Similarly, low alleles of SLA were associated with high alleles of biomass productivity traits and transpiration. At the coincident markers between trans-4-hydroxy-N:-methyl proline (MHP) and relative water content (RWC), low alleles of MHP were associated with high alleles of RWC. This study suggests the causal nature of the relationship between TE and Delta. Phenotypic data and QTL data show that SLA was more closely associated with biomass production than with TE. This study also shows that a cause-effect relationship may exist between SLA and biomass production.

Sertoli cells (SCs) provide immune protection and nutritive support to the developing germ cells in the testis. Sertoli cells have also been shown to provide immune protection to islets transplanted outside the testes. In this study, the ability of these cells to diminish the infiltration/activation of microglia into a neural graft implanted in the lesioned striatum of a hemiparkinsonian rat was investigated. Human neuron-like cells (hNT neurons) were implanted either alone or in combination with rat SCs. Three months later, the animals were sacrificed and immunohistochemistry was performed to determine the survival of the xenografted neurons as well as microglial infiltration/activation. Cotransplantation of the SCs with the hNT neurons increased graft survival and was associated with an increase in graft size. Furthermore, there were fewer microglia present in the grafted tissue of the cotransplantation groups. These results show that SCs retain their immunosuppressive ability even within the brain. As immune responses to grafted neural tissue within the central nervous system become better understood, this ability of the SCs to provide localized immunosuppression to the transplanted tissue may become more important. This is particularly true as the search for alternative sources of neural tissue to treat neurodegenerative diseases expands to encompass other species.

One of the major issues in neural transplantation is the low survival rate (

The efficacy of treating neurodegenerative diseases with the transplantation of fetal tissue has been demonstrated in animal models of Parkinson's disease, Huntington's disease and stroke. In the clinical setting, neural transplantation as a treatment for patients with Parkinson's disease has shown promising results. However, for this treatment method to be effective neuronal survival needs to be improved through either trophic support or localized immunoprotection. Co-transplanting Sertoli cells, which express many nutritive, regulatory, trophic and immunosuppressive factors, with fetal neural cells could provide both of these requirements. Such a strategy could enhance the recovery benefits associated with transplantation and decrease the need for, and the risks associated with, long-term systemic immunosuppression.

The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinson's disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.

Neural tissue transplantation has become an alternative treatment for Parkinson's disease (PD) and other neurodegenerative disorders. The clinical use of neural grafts as a source of dopamine for Parkinson's disease patients, although beneficial, is associated with logistical and ethical issues. Thus, alternative graft sources have been explored including polymer-encapsulated cells and nonneural cells (that is, adrenal chromaffin cells) or genetically modified cells that secrete dopamine and/or trophic factors. Although progress has been made, no current alternative graft source has ideal characteristics for transplantation. Emerging evidence suggests the importance of trophic factors in enhancing survival and regeneration of intrinsic dopaminergic neurons. It would be desirable to transplant cells that are readily available, immunologically accepted by the central nervous system and capable of producing dopamine and/or trophic factors. Sertoli cells have been shown to secrete CD-95 ligand and regulatory proteins, as well as trophic, tropic, and immunosuppressive factors that provide the testis, in part, with its "immunoprivileged" status. The present study demonstrated that transplantation of rat testis-derived Sertoli cells into adult rat brains ameliorated behavioral deficits in rats with 6-hydroxydopamine-induced hemiparkinsonism. This was associated with enhanced tyrosine hydroxylase (TH) immunoreactivity in the striatum in the area around the transplanted Sertoli cells. Furthermore, in vitro experiments demonstrated enhanced dopaminergic neuronal survival and outgrowth when embryonic neurons were cultured with medium in which rat Sertoli cells had been grown. Transplantation of Sertoli cells may provide a useful alternative treatment for PD and other neurodegenerative disorders.

The use of cyclosporine-A (CsA) immunosuppression for neural transplantation has become the treatment of choice for ameliorating host-graft rejection responses in human and animal transplant studies. However, the cytotoxic effects of CsA have warranted a search for alternative methods of protecting neural transplants. Sertoli cells produce an immunosuppressant factor, Fas ligand (Fas-L), that may provide the testis with its immunoprivileged status. Therefore, it has recently been suggested that these cells may be useful in producing localized immunosuppression for transplants. If Sertoli cells do produce localized immunosuppression, then it should be possible to successfully transplant Sertoli cells without additional immunosuppression following transplant surgery. The present study was undertaken to determine whether rat or porcine Sertoli cells transplanted into rat brain would survive for an extended period of time without CsA immunosuppression. Isolated rat or porcine Sertoli cells prelabeled with DiI were transplanted into normal rat brain. We report that both rat Sertoli cell allografts and porcine Sertoli cell xenografts survived for at least 2 months posttransplantation into the rat brain without CsA immunosuppression, indicating that these grafts were capable of producing sufficient localized immunosuppression to survive at the site of transplant without additional systemic immunosuppression.

Testis-derived Sertoli cells have been used to create an immune "privileged" site outside of the testis to facilitate cell transplantation protocols for diabetes and neurodegenerative diseases. In addition to secreting immunoprotective factors, Sertoli cells also secrete growth and trophic factors that appear to enhance the posttransplantation viability of isolated cells and, likewise, the postthaw viability of isolated, cryopreserved cells. It would be beneficial if Sertoli cells could be cryopreserved with the transplantable cell type without deleterious effects on the cells. This report describes a protocol for the cocryopreservation of rat Sertoli cells with rat ventral mesencephalic neurons, neurons from the lateral and medial ganglionic eminences and the hNT neuron cell line, and reports on the effects of Sertoli cells on the the postthaw viability of these neurons. Results of trypan blue exclusion analysis indicated that the presence of Sertoli cells did not deleteriously effect cryopreserved neurons and may improve their postthaw recoverability and viability in general. Specifically, results of the tyrosine hydroxylase immunostaining showed that Sertoli cells significantly enhance the postthaw viability of ventral mesencephalic dopaminergic cells in vitro.

Neural transplantation therapy as a possible alternative treatment for neurological movement disorders, such as in Parkinson's disease (PD), has accentuated research interest on the immune status of the central nervous system (CNS). Most animal studies concerned with neural transplantation for the treatment of PD have utilized dopamine (DA) neurons from tissues of the embryonic ventral mesencephalon. Rat embryonic DA neurons, grafted either as solid blocks or dissociated into a cell suspension and stereotaxically injected intraparenchymally into a rat lesion model of PD, have been shown to survive and form connections with the host brain, and ameliorate the behavioral deficits of PD. Similarly, studies on nonhuman primate models of PD provide considerable support for neural transplantation of DA neurons as an experimental clinical procedure for the treatment of PD. To this end, experimental clinical trials have been centered upon transplantation of the embryonic ventral mesencephalic cells for PD patients. Although not conclusive, the findings from clinical studies have provided some evidence that most patients with marked increases in fluorodopa uptake (indicating graft survival) have been immunosuppressed. Furthermore, immune reactions have been observed in rats xenografted with human embryonic tissue. Of note, embryonic ventral mesencephalic tissues compared to adult tissues produce better morphological and long-lasting behavioral amelioration of the neurobehavioral deficits of PD, thus advocating the use of grafts from young donors (embryo) to circumvent the CNS immune rejection. The possible graft rejection due to CNS immune reactions, coupled with the social and ethical problems surrounding the use of embryonic neural tissue, and the logistical problems concerning tissue availability have prompted the development of alternative sources of DA-secreting cells. To circumvent these obstacles, several methods have been suggested including the use of immunosuppressants such as Cyclosporine-A, transplantation of autografts, polymer-encapsulated DA-secreting cells, co-culturing and co-transplantation of DA-secreting cells with microcarrier beads, with Sertoli cells, or with fragments of a monoclonal antibody that can mask the MHC class I antigens, and genetically modifying cells that can withstand CNS immune reactions. Some of these techniques allow transplantation of allograft (same species transplantation), or even xenograft (cross species transplantation) without immunosuppression of the recipient. We discuss recent CNS immunosuppression techniques that pose some promise for enhanced survival of neural grafts. When possible, advantages and disadvantages of each method are presented. Hopefully, such critical analysis of different immunosuppression techniques will produce innovated ideas that will lead to a better understanding of CNS immune response and its modulatory function on graft rejection and survival.

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.

Specialized junctional binding of step 8 spermatids to Sertoli cells is an important spermiogenic event. In the hypophysectomized (Hypox) rat, the daily sperm production (DSP) is reduced and Sertoli cells become binding incompetent. Delayed replacement of testosterone (DT-Hypox) does not restore the normal DSP and Sertoli cells remain binding incompetent. In this study, DT-Hypox rats received FSH daily for 2 days to 3 wk concurrent with delayed testosterone replacement and were killed 8 wk after the initiation of hormone treatment. The DT-Hypox rat treated with FSH for 2 days to 2 wk had a significantly reduced DSP. Sertoli cells remained binding incompetent as evidenced by structurally abnormal ectoplasmic specializations and an abnormal pattern of f-actin and vinculin immunostaining. The DT-Hypox rat treated with FSH for 3 wk had a normal DSP. Sertoli cells were binding-competent as evidenced by structurally intact ectoplasmic specializations and the normal pattern of f-actin and vinculin immunostaining. The results indicate that the "priming" effect of FSH necessary to restore normal spermiogenesis is associated with restoration of the junction-related Sertoli cell cytoskeleton (i.e., FSH induction of binding competency) expressed as intact ectoplasmic specializations and peripheral distribution of f-actin and vinculin.

Using current methodologies, drug delivery to small airways, terminal bronchioles, and alveoli (deep lung) is inefficient, especially to the lower lungs. Urgent lung pathologies such as acute respiratory distress syndrome (ARDS) and post-lung transplantation complications are difficult to treat, in part due to the methodological limitations in targeting the deep lung with high efficiency drug distribution to the site of pathology. To overcome drug delivery limitations inhibiting the optimization of deep lung therapy, isolated rat Sertoli cells preloaded with chitosan nanoparticles were use to obtain a high-density distribution and concentration (92%) of the nanoparticles in the lungs of mice by way of the peripheral venous vasculature rather than the more commonly used pulmonary route. Additionally, Sertoli cells were preloaded with chitosan nanoparticles coupled with the anti-inflammatory compound curcumin and then injected intravenously into control or experimental mice with deep lung inflammation. By 24 h postinjection, most of the curcumin load (~90%) delivered in the injected Sertoli cells was present and distributed throughout the lungs, including the perialveloar sac area in the lower lungs. This was based on the high-density, positive quantification of both nanoparticles and curcumin in the lungs. There was a marked positive therapeutic effect achieved 24 h following curcumin treatment delivered by this Sertoli cell nanoparticle protocol (SNAP). Results identify a novel and efficient protocol for targeted delivery of drugs to the deep lung mediated by extratesticular Sertoli cells. Utilization of SNAP delivery may optimize drug therapy for conditions such as ARDS, status asthmaticus, pulmonary hypertension, lung cancer, and complications following lung transplantation where the use of high concentrations of anti-inflammatory drugs is desirable, but often limited by risks of systemic drug toxicity.

Testosterone is the principal hormone necessary for insuring the completion of normal spermatogenesis. However, its precise role in spermatid maturation is not clear. In hypophysectomized rats, testosterone can maintain spermiogenesis if replaced soon after surgery. Delaying treatment results in a reduction in the number of mature spermatids. In culture, testosterone and FSH are required to maximize spermatid attachment to binding-competent Sertoli cells. This binding event is an essential step in the process of spermiogenesis and is dependent on components of the Sertoli cell cytoskeleton. The present study was undertaken to determine the binding competency of Sertoli cells and their junctional interaction with spermatids in the hypophysectomized rat after immediate testosterone replacement and after delayed testosterone replacement. Hypophysectomized rats treated with immediate testosterone replacement had peripheral distribution of Sertoli cell f-actin and vinculin, structurally intact Sertoli ectoplasmic specializations facing step 8 spermatids, and daily sperm production similar to these parameters as observed in intact controls. In the delayed treatment group, these parameters were abnormal and were similar to those observed in the untreated hypophysectomized animals. The results suggest that testosterone can maintain binding competency of the Sertoli cell and normal Sertoli-spermatid junctional interaction but cannot restore them.

Prolonged survival of Islet- allo- and xenografts can be induced following implantation of the islets into the abdominal testis of diabetic rats. We previously showed that a factor released by Sertoli cells appears to be responsible for the protection of the intratesticular islet allo- and xenografts against rejection. The aim of this study was to examine whether an immunologically privileged site can be established in an organ site in vivo, other than the testis, such as the renal, subcapsular space, to make feasible the grafting of female recipients as well. A total of 36 male and 21 female, diabetic, PVG rats were divided into six different treatment groups: 1) Six male rats were grafted with islets from Sprague-Dawley (S-D) donor rats only. 2) Ten male rats were grafted with islets from (S-D) donors and were then given a short course of cyclosporine (CsA) posttransplantation. 3) Ten male rats were grafted with islets from (S-D) donors and with Sertoli cell-enriched fractions (SEF) from PVG donors but without CsA. 4) Ten male rats were grafted with a combination of islets from (S-D) and SEF from (PVG), donors, respectively, and CsA. 5) Ten female rats were given an identical combination of cells and CsA as depicted for group 5.6) Ten female rats were grafted with a combination of islets and SEF, both cell types from S-D donors, and CsA. The results showed that 70% to 100% of the grafted rats in groups 1, 2, and 3 remained hyperglycemic. Prolonged normoglycemia in excess of 100 days was induced in more than 75% of the grafted rats only in groups 4,5, and 6, or in those animals who were grafted with a combination of islets and SEF and who were given a short course of CsA as well. Electron microscopic examination of the grafted tissues showed the presence of intact beta cells and of cells with features characteristic of Sertoli cells. Our results suggest that 1) the protection of islet allografts in nonimmunologically privileged site can be achieved in male and female rats by means of the simultaneous transplantation of Sertoli cells. 2) Sertoli cells apparently maintain the capacity to secrete an immune inhibitor in organ sites other than the testis. We conclude that it is feasible to create an immunologically privileged site for the transplantation of isolated islets in male and female diabetic recipients without the need for sustained immunosuppression.

To determine if the pubertal testosterone rise plays a role in the termination of the peripubertal follicle-stimulating hormone (FSH) increase, male rats were injected with ethylene dimethanesulfonate (EDS) at 40 days of age to eradicate the Leydig cells just before the onset of the testosterone rise. Rats were decapitated at weekly intervals from age 26 to 96 days. Compared with vehicle-injected controls, EDS treatment resulted in a delay in the peripubertal increase in the relative weights of prostates and seminal vesicles of approximately 2 wk. Serum testosterone remained at pretreatment levels for 1 wk postinjection. Testicular interstitial fluid testosterone remained at pretreatment concentrations for considerably longer and was significantly lower than controls for 2 wk postinjection. EDS treatment resulted in serum FSH levels that were elevated by 1 wk postinjection. They remained significantly higher than controls until 96 days of age. Compared with controls, serum alpha-inhibin was elevated after EDS as was serum luteinizing hormone. These results suggest that the pubertal testosterone increase plays an important role in terminating the peripubertal FSH rise.

Two-dimensional micro-computed tomography (micro-CT) slices can be reconstructed into three-dimensional (3D) models that demonstrate capillary beds. This study focused on the acquisition of data necessary to create scaffolding that directly mimics the unique structural patterns of a microvascular tree system. The Microfil vascular contrasting method was compared to the Baston's methylmethacrylate corrosion casting (BMCC) method to determine which provided the most accurate and high-resolution results for 3D micro-CT reconstruction derived from the two-dimensional micro-CT slices of the capillary beds. It was determined that the BMCC, a method traditionally used in the scanning electron microscopic analysis of the microvasculature, was the best method for representing capillary lumina for micro-CT scanning. The removal of tissues from the BMCC cast resulted in samples that eliminated background material, thus increasing the X-ray contrast levels of the CT images. This provided for a more complete and more distinguishable high-resolution image of the represented capillary lumina. Images created with this BMCC method were reconstructed in a stereolithography file format as 3D mesh structure for later importing into computer-aided design (CAD) software. The resulting Bio-CAD, then, can be used to guide the more accurate fabrication of the microvascular scaffolding and then serve as the framework for tissue engineering of microvascular structures. Results from this study clearly indicated that the BMCC method is superior to the Microfil method for accurate and complete high-resolution imaging of capillary beds.

Rats with short-term diabetes show a greater than 50% reduction of serum testosterone and increased lipid in Leydig cells but normal testicular structure. The purpose of this study was to determine the extent of testicular pathology (morphology index), integrity of the blood-testis barrier, daily sperm production (DSP), number of Leydig cells per testis (LC/T), and total trunk testosterone (TTT) in diabetic rats (BBWORdp) with long-term hyperglycemia (300-350 mg/dl for greater than 180 days) and to evaluate its effects on fertility potential. Results were compared with similarly aged normoglycemic rats (BBWORdr) and normal control Wistar rats. After 6 mo of diabetes, testis weights, DSPs, TTTs, and the morphology index were significantly reduced. The LC/T was not different from BBWORdr rats. The blood-testis barrier appeared intact, although structural abnormalities were noted in Sertoli-Sertoli junction complexes. There was a significant reduction in the number of pregnancies per rat and implantations per pregnancy in matings utilizing the diabetic BBWORdp rat and control Wistar female rats. Results indicate that long-term diabetes with sustained hyperglycemia leads to significant testicular dysfunction associated with decreased fertility potential.

Pancreatic islet allo- and xenografts are not rejected and exhibit long-term beta-cell function if transplanted into the abdominal testis of the diabetic host. Successful transplantation appears dependent on local factors unique to the abdominal testis. Because Leydig cells remain viable in abdominal testes, which also retain high levels of testosterone, the following question was addressed: do Leydig cells and/or their secretory products influence islet transplantability in the successful islet/abdominal testis transplantation model? Streptozotocin-induced diabetic rats (Sprague-Dawley) were injected with 75 mg/kg ethane dimethanesulfonate (EDS) to selectively eliminate Leydig cells prior to or following transplantation with islets isolated from the BBWORdr rat. Subcutaneous silastic tubes packed with estradiol prevented Leydig cell repopulation in the EDS-treated recipient. Grafted diabetic animals, including the EDS-treated rats with serum testosterone at castration levels, became nornoglycemic following islet transplantation and remained so far for up to ten months. Leydig cells were not observed in testes of the EDS- or EDS/estradiol-treated rats, whereas the transplanted islets within these testes appeared structurally normal and highly vascularized. Islets resided within the testicular interstitial compartment and contained alpha-, beta and delta-cells, as identified by electron microscopy. Beta cells were most prominent, contained secretory granules and exhibited a close structural and functional relationship with adjacent intraislet capillaries. We conclude that Leydig cells and Leydig cell secretory products, including testosterone, are not necessary for protecting islets against rejection and they do not play an obligatory role in the success of the islet/abdominal testis transplantation protocol. Leydig cells and Leydig cell secretory products do not promote long-term beta-cell function and are not required for the return to and maintenance of normoglycemia in the grafted diabetic rat.

The chronic use of dichloroacetate (DCA) for diabetes mellitus or hyperlipoproteinemias has been compromised by neurologic and other forms of toxicity. DCA is metabolized to glyoxylate, which is converted to oxalate and, in the presence of adequate thiamine levels, to other metabolites. DCA stimulates the thiamine-dependent enzymes pyruvate dehydrogenase and alpha-ketoacid dehydrogenase. We postulated that the neurotoxicity from chronic DCA administration could result from depletion of body thiamine stores and abnormal metabolism of oxalate, a known neurotoxin. For 7 weeks, rats were fed ad lib. Purina chow and water or chow plus sodium DCA (50 mg/kg or 1.1 g/kg) in water. A portion of the DCA-treated animals also received intraperitoneal injections of 600 micrograms thiamine three times weekly or 600 micrograms thiamine daily by mouth. Thiamine status was assessed by determining red cell transketolase activity and, in a blinded manner, by recording the development of clinical signs known to be associated with thiamine deficiency. At the 50 mg/kg dose, chronic administration of DCA showed no clinical toxicity or effect on transketolase activity. At the 1.1 g/kg dose, however, DCA markedly increased the frequency and severity of toxicity and decreased transketolase activity 25%, compared to controls. Coadministration of thiamine substantially reduced evidence of thiamine deficiency and normalized transketolase activity. Inhibition of transketolase by DCA in vivo was not due to a direct action on the enzyme, however, since DCA, glyoxylate, or oxalate had no appreciable effects on transketolase activity in vitro. After 7 weeks, plasma DCA concentrations were similar in rats receiving DCA alone or DCA plus thiamine, while urinary oxalate was 86% above control in DCA-treated rats but only 28% above control in DCA plus thiamine-treated animals. No light microscopic changes were seen in peripheral nerve, lens, testis, or kidney morphology in either DCA-treated group, nor was there disruption of normal sperm production in the DCA-treated group. We conclude that stimulation by DCA of thiamine-requiring enzymes may lead to depletion of total body thiamine stores and to both a fall in transketolase activity and an increase in oxalate accumulation in vivo. DCA neurotoxicity may thus be due, at least in part, to thiamine deficiency and may be preventable with thiamine treatment.

NTRACEREBRAL transplantation therapy as a treatment for movement disorders, such as Parkinson’s disease, has accentuated research interest in the possibility of restoring CNS circuits to achieve some functional recovery following transplantation of fetal tissue.‘,’ The possible graft rejection due to CNS immune reactions have prompted investigations to find alternative methods for immunosuppression.3 Moreover, the logistical problems concerning tissue availability has focused research on the possibility of adding trophic factors to enhance the yield of tissue, as well as finding new sources for obtaining tissue for intracerebral transplantation. Recently, we demonstrated that cotransplantation of adrenal chromaffin cells with testis-derived Sertoli cells into the striatum of hemiparkinsonian rats produced localized immunosuppression and enhanced survival of the xenograft and, when transplanted alone, resulted in behavioral recovery.43”

Background Cortactin, an actin binding protein, has been associated with Sertoli cell ectoplasmic specializations in vivo, based on its immunolocalization around the heads of elongated spermatids, but not previously identified in isolated Sertoli cells. In an in vitro model of Sertoli cell-spermatid binding, cortactin was identified around debris and dead germ cells. Based on this observation, we hypothesized that this actin binding protein may be associated with a non-junction-related physiological function, such as phagocytosis. The purpose of this study was to identify the presence and distribution of cortactin in isolated rat Sertoli cells active in phagocytic activity following the addition of 0.8 μm latex beads. Results Sertoli cell monocultures were incubated with or without follicle stimulating hormone (FSH; 0.1 μg/ml) in the presence or absence of cytochalasin D (2 μM), as an actin disrupter. Cortactin was identified by standard immunostaining with anti-cortactin, clone 4F11 (Upstate) after incubation times of 15 min, 2 hr, and 24 hr with or without beads. Cells exposed to no hormone and no beads appeared to have a ubiquitous distribution of cortactin throughout the cytoplasm. In the presence of cytochalasin D, cortactin immunostaining was punctate and distributed in a pattern similar to that reported for actin in cells exposed to cytochalasin D. Sertoli cells not exposed to FSH, but activated with beads, did not show cortactin immunostaining around the phagocytized beads at any of the time periods. FSH exposure did not alter the distribution of cortactin within Sertoli cells, even when phagocytic activity was upregulated by the presence of beads. Conclusion Results of this study suggest cortactin is not associated with peripheralized actin at junctional or phagocytic sites. Further studies are necessary to clarify the role of cortactin in Sertoli cells.