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Chui K.,Mandalmed, Inc. | Trivedi A.,Mandalmed, Inc. | Trivedi A.,University of California at San Francisco | Cherbavaz D.B.,Mandalmed, Inc. | And 6 more authors.
Cell Transplantation | Year: 2011

It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpu- berty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2′-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility, reproductive toxicology, testic- ular cancer, and spermatogenesis, and due to their unique biological properties potentially could be useful in cell therapy. © 2011 Cognizant Comm. Corp.


Dovizio M.,University of Chieti Pescara | Maier T.J.,Frankfurt University | Alberti S.,University of Chieti Pescara | Francesco L.D.,University of Chieti Pescara | And 7 more authors.
Molecular Pharmacology | Year: 2013

Cyclooxygenase (COX)-2-derived prostanoids can influence several processes that are linked to carcinogenesis. We aimed to address the hypothesis that platelets contribute to aberrant COX-2 expression in HT29 colon carcinoma cells and to reveal the role of platelet-induced COX-2 on the expression of proteins involved in malignancy and marker genes of epithelialmesenchymal transition (EMT). Human platelets cocultured with HT29 cells rapidly adhered to cancer cells and induced COX-2 mRNA expression, but not protein synthesis, which required the late release of platelet-derived growth factor and COX-2 mRNA stabilization. Platelet-induced COX-2-dependent prostaglandin E2 (PGE2) synthesis in HT29 cells was involved in the downregulation of p21WAF1/CIP1 and the upregulation of cyclinB1 since these effects were prevented by rofecoxib (a selective COX-2 inhibitor) and rescued by exogenous PGE2. Galectin-3, which is highly expressed in HT29 cells, is unique among galectins because it contains a collagen-like domain. Thus, we studied the role of galectin-3 and platelet collagen receptors in plateletinduced COX-2 overexpression. Inhibitors of galectin-3 function (b-lactose, a dominant-negative form of galectin-3, Gal-3C, and anti-galectin-3 antibody M3/38) or collagen receptor-mediated platelet adhesion (revacept, a dimeric platelet collagen receptor GPVI-Fc) prevented aberrant COX-2 expression. Inhibition of platelet-cancer cell interaction by revacept was more effective than rofecoxib in preventing platelet-induced mRNA changes of EMT markers, suggesting that direct cell-cell contact and aberrant COX-2 expression synergistically induced gene expression modifications associated with EMT. In conclusion, our findings provide the rationale for testing blockers of collagen binding sites, such as revacept, and galectin-3 inhibitors in the prevention of colon cancer metastasis in animal models, followed by studies in patients. © 2013 by The American Society for Pharmacology.


Jarvis G.A.,Center for Immunochemistry | Jarvis G.A.,University of California at San Francisco | Mirandola L.,Texas Tech University Health Sciences Center | Yuefei Y.,Texas Tech University Health Sciences Center | And 6 more authors.
ACS Symposium Series | Year: 2012

A summary is provided of the compelling data supporting galectin-3, as a target for cancer therapy, and for the clinical development of a truncated form of human galectin-3, termed galectin-3C, for cancer. Lacking the N-terminal domain of galectin-3 that facilitates its multimerization when bound to carbohydrate ligands, galectin-3C functions as an inhibitor of the galectin-3 crosslinking mediated by the N-terminal domain that can be induced by multivalent oligosaccharides and glycoconjugates intra- or extracellularly, on cell surfaces, or in extracellular matrices. Numerous studies show that galectin-3 plays a key role in tumorigenicity and metastasis and is a novel target for the development of cancer therapeutics. Data presented indicate that galectin-3C can be localized in a carbohydrate-dependent manner similar to most information reported regarding galectin-3. Exogenous galectin-3C facilitated anoikis in human breast cells, illustrating an effect it is expected to produce in vivo by reducing cell-cell or cell-ECM adhesion of metastatic cells. Based on the activity of galectin-3, we postulate that galectin-3C also inhibits the metastatic process by preventing integrin activation and focal adhesion turnover. The data described provide evidence of the important functions of galectin-3 in the processes of metastasis and tumorigenicity, and the potential therapeutic effect of inhibiting the activity of galectin-3 with galectin-3C. Galectin-3 has a complex, multi-faceted role that is critical in the relationship between the cells of various types of malignancies and the microenvironment, and further development of galectin-3C as a clinical candidate for cancer treatment is warranted. © 2012 American Chemical Society.


Mirandola L.,Texas Tech University Health Sciences Center | Yu Y.,Texas Tech University Health Sciences Center | Jenkins M.R.,Texas Tech University Health Sciences Center | Chiaramonte R.,Texas Tech University Health Sciences Center | And 4 more authors.
BMC Cancer | Year: 2011

Background: Multiple myeloma (MM) is a fatal malignancy ranking second in prevalence among hematological tumors. Continuous efforts are being made to develop innovative and more effective treatments. The preclinical evaluation of new therapies relies on the use of murine models of the disease.Methods: Here we describe a new MM animal model in NOD-Rag1null IL2rgnull (NRG) mice that supports the engraftment of cell lines and primary MM cells that can be tracked with the tumor antigen, AKAP-4.Results: Human MM cell lines, U266 and H929, and primary MM cells were successfully engrafted in NRG mice after intravenous administration, and were found in the bone marrow, blood and spleen of tumor-challenged animals. The AKAP-4 expression pattern was similar to that of known MM markers, such as paraproteins, CD38 and CD45.Conclusions: We developed for the first time a murine model allowing for the growth of both MM cell lines and primary cells in multifocal sites, thus mimicking the disease seen in patients. Additionally, we validated the use of AKAP-4 antigen to track tumor growth in vivo and to specifically identify MM cells in mouse tissues. We expect that our model will significantly improve the pre-clinical evaluation of new anti-myeloma therapies. © 2011 Mirandola et al; licensee BioMed Central Ltd.


Mirandola L.,University of Milan | Mirandola L.,Texas Tech University | Mirandola L.,Laura W Bush Institute For Womens Health | Yu Y.,Texas Tech University | And 7 more authors.
ACS Symposium Series | Year: 2012

After decades of research, multiple myeloma is still a fatal disease accounting for almost one out of five cases of all hematological malignancies. Recent evidences indicate that novel pharmacological agents, such as thalidomide, lenalidomide and bortezomib, improve the response rate and extend the overall survival. Yet, prolonged therapies are required to prevent or control recurrence, ultimately resulting in treatment-related toxicities seriously affecting the patients' quality of life. The urgent need of innovative therapeutic approaches may be answered by molecules disrupting the interactions between myeloma cells and the local microenvironment. Specifically, Galectin-3 plays key roles in the connections linking tumor cells with bone marrow stroma and the extracellular matrix. We evaluate here the multifaceted outcomes and potential applications of treatments based on Galectin-3C, a truncated dominant negative inhibitor of endogenous Galectin-3. Additionally, we hypothesize the molecular mechanism of action of Galectin-3C and present the future challenges and unique opportunities that a Galectin-3-tailored anti-myeloma therapy may offer in the near future. © 2012 American Chemical Society.


Mirandola L.,Texas Tech University Health Sciences Center | Yu Y.,Texas Tech University Health Sciences Center | Chui K.,Mandalmed, Inc. | Jenkins M.R.,Texas Tech University Health Sciences Center | And 3 more authors.
PLoS ONE | Year: 2011

Galectin-3 is a human lectin involved in many cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Galectin-3 was expressed at varying levels by all 9 human MM cell lines tested. In vitro galectin-3C exhibited modest anti-proliferative effects on MM cells and inhibited chemotaxis and invasion of U266 MM cells induced by stromal cell-derived factor (SDF)-1α. Galectin-3C facilitated the anticancer activity of bortezomib, a proteasome inhibitor approved by the FDA for MM treatment. Galectin-3C and bortezomib also synergistically inhibited MM-induced angiogenesis activity in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. The average tumor volume of bortezomib-treated animals was 19.6% and of galectin-3C treated animals was 13.5% of the average volume of the untreated controls at day 35. The maximal effect was obtained with the combination of galectin-3C with bortezomib that afforded a reduction of 94% in the mean tumor volume compared to the untreated controls at day 35. In conclusion, this is the first study to show that inhibition of galectin-3 is efficacious in a murine model of human MM. Our results demonstrated that galectin-3C alone was efficacious in a xenograft mouse model of human MM, and that it enhanced the anti-tumor activity of bortezomib in vitro and in vivo. These data provide the rationale for continued testing of galectin-3C towards initiation of clinical trials for treatment of MM. © 2011 Mirandola et al.


Xiao X.,Mary M Wohlford Laboratory For Male Contraceptive Research | Xiao X.,Zhejiang Academy of Medical science | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research | Tang E.I.,Mary M Wohlford Laboratory For Male Contraceptive Research | And 6 more authors.
Human Reproduction | Year: 2014

STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood-testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5-20 μM) and bisphenol A (0.4-200 μM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by mislocalization of actin filament barbed end capping and bundling protein Eps8, and branched actin polymerization protein Arp3. Besides impeding actin dynamics, endocytic vesicle-mediated trafficking and the proper localization of actin regulatory proteins c-Src and annexin II in Sertoli cells were also affected. Results of statistical analysis demonstrate that these findings were not obtained by chance. LIMITATIONS, REASONS FOR CAUTION (i) This study was done in vitro and might not extrapolate to the in vivo state, (ii) conclusions are based on the use of Sertoli cell samples from three men and (iii) it is uncertain if the concentrations of toxicants used in the experiments are reached in vivo. WIDER IMPLICATIONS OF THE FINDINGS Human Sertoli cells cultured in vitro provide a robust model to monitor environmental toxicant-mediated disruption of Sertoli cell BTB function and to study the mechanism(s) of toxicant-induced testicular dysfunction. © The Author 2014.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.73K | Year: 2016

PROJECT SUMMARY ABSTRACT The overall goal of this project is to develop a human protein that is an inhibitor of galectin as a biologic to aid in the prevention and treatment of harmful remodeling after myocardial infarction MI heart attack and thereby improve cardiac function and reduce mortality from subsequent heart failure MI is the most common cause of cardiac morbidity and mortality in the Western world The incidence in the United States is new attacks and recurrent attacks annually approximately one every seconds Fibrosis is triggered by the physiological response to injury or infection and leads to the deposition of extracellular matrix and for mation of new connective tissue Excessive or dysregulated fibrosis from insults can dramatically reduce the functioning of the heart and other organs In the heart excessive interstitial fibrosis reduces contractility elas ticity and distensibility exacerbating processes that lead to heart failure Although there are therapeutic agents currently used after MI that are efficacious such as the mineralocorticoid receptor antagonists MRAs angiotensin converting enzyme ACE inhibitors and angiotensin II receptor blockers ARBs and that have shown anti fibrotic effects in animal studies the health burden from MI remains significant Fibrosis is regulated by a number of inflammatory cytokines and growth factors and galectin has recently been implicated as a major and novel mediator of organ fibrosis Increased serum levels of galectin have been approved in the United States and the European Union as prognostic indicators of risk of death from progressive heart failure supporting the hypothesis that galectin is a target for drug development Based on its mechanism of action and structure the protein is a unique inhibitor of galectin with properties that convey therapeutic advantage Our preliminary studies in a rat ischemia reperfusion I R injury model of MI showed very promising efficacy The Specific Aims for this Fast Track Phase I II project are the following Phase I Aim is to determine efficacy of Gal C therapy in animal models Determine antifibrotic potential of Gal C in the context of greater injury caused by permanent ligation of the coronary artery and evaluate efficacy of Gal C therapy relative to ARB and antifibrotic control drugs in a rat I R MI model Phase II Aim is to better understand efficacy of Gal C therapy in animal models Determine efficacy and optimal dosage of Gal C in rat I R MI determine efficacy of Gal C in comparison to a MRA and in combination with an ARB in rat I R MI model and determine efficacy of Gal C in miniswine I R model of MI Aim is to develop GLP GMP production methods and a formulation for Gal C Aim is to perform pharmacokinetic studies and acute subacute toxicology in rodents Achievement of these aims is expected to position MandalMed to complete pre clinical development in the near term and to subsequently file an Investigational New Drug IND application PROJECT NARRATIVE The overall goal of this project is to develop a protein inhibitor of galectin termed galectin C as a biologic to prevent and treat harmful remodeling after myocardial infarction and thereby improve cardiac function and reduce mortality from subsequent heart failure Myocardial infarction is the most common cause of cardiac morbidity and mortality in the western world The annual incidence in the United States is new attacks and recurrent attacks Because current standard practice of minimizing time from onset of myocardial infarction to re opening of the blocked artery has greatly reduced the incidence of death from acute myocardial infarction heart failure subsequent to myocardial infarction has become the main mortality associated with coronary events


PubMed | Mandalmed, Inc.
Type: Journal Article | Journal: Cell transplantation | Year: 2011

It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpuberty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility, reproductive toxicology, testicular cancer, and spermatogenesis, and due to their unique biological properties potentially could be useful in cell therapy.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 250.00K | Year: 2011

DESCRIPTION (provided by applicant): More than 87,000 chemicals have been developed and distributed over the past 50 years. The vast majority of these have not been tested for potential toxic effects in humans or animals. Most systematic toxicology research is conducted in animals and is very expensive. Although animal studies contribute important information, they may not closely approximate the exposure or the exposure effects in humans. There is an unmet need for cost-effective bioassay systems that mimic and assess human exposures and their reproductive effects for systematic, prospective investigation of potentially toxic substances. The overall goal of the research is to develop a functional in vitro human cell-based testis model of spermatogenesis asa clinically relevant product for reproductive toxicology. Development of the first generation product is enabled by our capability to isolate and propagate primary Sertoli cells from adult human donors (hSertCs) and to manipulate primordial germ cells (PGCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs) in vitro to produce haploid cells. The hSertCs have been available to researchers worldwide through a partnership with Lonza (Walkersville, MD) since May 2009, and have been used to create a model of the blood-testis barrier (BTB) in coated inserts of transwell plates. By silencing and overexpressing genes that encode germ-cell-specific cytoplasmic RNA-binding proteins, we have been able to modulate human germ-cell formationand developmental progression, to promote later stages of meiosis and development of haploid gametes. In Phase I, we aim to establish a model using primary human Sertoli, Leydig, and peritubular myoid cells to form a functional (BTB) in a closed hollow fiber system mimicking physiologic shear stress conditions, and to perform analytical and toxicological studies of the cells. There are three Specific Aims for Phase I. 1. Develop an in vitro human Sertoli cell-based, 3-dimensional (3-D) model of the testis. 2. Develop methods to detect and quantify the cells within the testis model. 3. Demonstrate the effect of reproductive toxins on the viability of human testicular cells. In Phase II, we intend to drive the differentiation of induced pluripotent stem cells (iPSCs) and spermatogonial stem cells (SSCs) to the haploid state within the model, and test the effect of toxins on this process. From this effort, we expect to identify reducible, reliable, and relevant endpoints for reproductive toxicant testing, andthen to establish outcome markers and assays enabling for F.D.A. approval of the model in conjunction with other toxicology testing required for all New Drug Applications. The need for more relevant and improved reproductive toxicology testing represents asignificant commercial opportunity, especially in the pharmaceutical industry. The bioassay product would be provided to industry on a fee-for-service basis initially. This effort potentially could lead to the capability to replicate normal human spermatogenesis in vitro and generate de novo mature sperm for severely infertile, azoospermic men, including cancer survivors. PUBLIC HEALTH RELEVANCE: More than 87,000 chemicals have been developed and distributed discarded over the past 50 years. The vast majority of these have not been tested for potential toxic effects in humans or animals. There is an unmet need for more cost-effective bioassay systems that will mimic and assess human exposures and their reproductive effects for systematic, prospectiveinvestigation into potentially toxic substances. The overall goal of the research is to develop a functional in vitro human cell-based testis model of spermatogenesis as a clinically relevant product for reproductive toxicology. Better identification of reproductive toxins could help protect developing fetuses from unwanted toxic exposures, reduce the incidence of male infertility, potentially lower rates of testis cancer, and create safer drugs for the marketplace. Also, part of our interest in developinga testis model is develop the capability to replicate normal human spermatogenesis in vitro to potentially generate de novo mature sperm for severely infertile, azoospermic men, including cancer survivors.

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