The Intracellular Localization of Hormonal Activity in Transplantable Thyrotropin-Secreting Pituitary Tumors in Mice

Mouse pituitary tumors secreting almost exclusively thyroid stimulating hormone have been characterized electron microscopically. Tumors of known thyrotropin content were separated into nuclear, mitochondrial, microsomal, and soluble fractions by differential centrifugation. The hormonal activity of these fractions was correlated with that of the total homogenates and with their nitrogen and phosphorus content. Essentially all the thyrotropin of the homogenate was recovered in a particulate fraction sedimenting between 20,000 and 40,000 g. This fraction contained the RNA granules and membranous components typical of microsomal pellets, but also showed the presence of small dense bodies surrounded by smooth membranes. These bodies were also visible within the endoplasmic reticulum of intact cells, and it is postulated that these bodies may represent the sites of intracellular elaboration and/or storage of TSH. Thyrotropin is tightly associated with microsomal particles but can be brought into solution by treatment with alkaline media, deoxycholate, and certain organic solvents

Isolated Hepatocyte Transplantation for Crigler-Najjar Syndrome Type 1:

Crigler-Najjar syndrome type 1 (CN1) is an inherited disorder characterized by the absence of hepatic uridine diphosphoglucuronate glucuronosyltransferase (UDPGT), the enzyme responsible for the conjugation and excretion of bilirubin. We performed allogenic hepatocyte transplantation (AHT) in a child with CN1, aiming to improve bilirubin glucuronidation in this condition. A 9-year-old boy with CN1 was prepared with plasmapheresis and immunosuppression with prednisolone and tacrolimus. When a graft was made available, 7.5 × 10 9 hepatocytes were isolated and infused into the portal vein percutaneously. After 2 weeks phenobarbitone was added to promote the enzymatic activity of UDPGT of the transplanted hepatocytes. Nocturnal phototherapy was continued throughout the studied period. Total bilirubin was considered a reliable marker of allogenic cell function. There was no significant variation of vital signs nor complications during the infusion. Mean ± SD bilirubin level was 530 ± 38 µmol/L before and 359 ± 46 µmol/L after AHT (t-test, p < 0.001). However, the introduction of phenobarbitone was followed by a drop of tacrolimus level with increase of alanine aminotransferase (ALT) and increase of bilirubin. After standard treatment of cellular rejection bilirubin fell again but from then on it was maintained at a greater level. After discharge the patient experienced a further increase of bilirubin that returned to predischarge levels after readmission to the hospital. This was interpreted as poor compliance with phototherapy. Only partial correction of clinical jaundice and the poor tolerability to nocturnal phototherapy led the parents to refuse further hepatocyte infusions and request an orthotopic liver transplant. After 24 months the child is well, with good liver function on tacrolimus and prednisolone-based immunosuppression. Isolated AHT, though effective and safe, is not sufficient to correct CN1. Maintenance of adequate immunosuppression and family compliance are the main factors hampering the success of this procedure

The effect of mesenchymal stem cell transplantation on the recovery of bladder and hindlimb function after spinal cord contusion in rats

<p>Abstract</p> <p>Background</p> <p>Mesenchymal stem cells are widely used for transplantation into the injured spinal cord in vivo model and for safety, many human clinical trials are continuing to promote improvements of motor and sensory functions after spinal cord injury. Yet the exact mechanism for these improvements remains undefined. Neurogenic bladder following spinal cord injury is the main problem decreasing the quality of life for patients with spinal cord injury, but there are no clear data using stem cell transplantation for the improvement of neurogenic bladder for in vivo studies and the clinical setting.</p> <p>The purpose of this study was to delineate the effect of human mesenchymal stem cell (hMSCs) transplantation on the restoration of neurogenic bladder and impaired hindlimb function after spinal cord contusion of rats and the relationship between neurotrophic factors such as brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and bladder and hindlimb functions.</p> <p>Results</p> <p>Modified moderate contusion injury were performed on the thoracic spinal cord of Sprague-Dawley rats using MASCIS impactor and hMSCs, human fibroblasts or phosphate-buffered saline were transplanted into injured spinal cord 9 days after injury for hMSC and two control groups respectively. Ladder test showed more rapid restoration of hindlimb function in hMSC group than in control group, but Basso, Beattie, and Bresnahan score and coupling score were not different significantly among hMSC and two control groups. Neurogenic bladder was not improved in either group. ED1 positive macrophages were significantly reduced in hMSC group than in two control groups, but ELISA and RT-PCR studies revealed BDNF and NT-3 levels in spinal cord and bladder were not different among hMSC and two control groups regardless the experimental duration.</p> <p>Conclusion</p> <p>hMSC transplantation was effective in reducing inflammatory reaction after spinal cord contusion of rats but not sufficient to recover locomotor and bladder dysfunction. BDNF and NT-3 levels in the spinal cord and bladder were not increased 28 and 56 days after hMSC transplantation.</p

Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair

The aim of this study is to understand if human mesenchymal stem cells (hMSCs) and neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) have synergistic protective effect that promotes functional recovery in rats with severe spinal cord injury (SCI). To evaluate the effect of delayed combinatorial therapy of PACAP and hMSCs on spinal cord tissue repair, we used the immortalized hMSCs that retain their potential of neuronal differentiation under the stimulation of neurogenic factors and possess the properties for the production of several growth factors beneficial for neural cell survival. The results indicated that delayed treatment with PACAP and hMSCs at day 7 post SCI increased the remaining neuronal fibers in the injured spinal cord, leading to better locomotor functional recovery in SCI rats when compared to treatment only with PACAP or hMSCs. Western blotting also showed that the levels of antioxidant enzymes, Mn-superoxide dismutase (MnSOD) and peroxiredoxin-1/6 (Prx-1 and Prx-6), were increased at the lesion center 1 week after the delayed treatment with the combinatorial therapy when compared to that observed in the vehicle-treated control. Furthermore, in vitro studies showed that co-culture with hMSCs in the presence of PACAP not only increased a subpopulation of microglia expressing galectin-3, but also enhanced the ability of astrocytes to uptake extracellular glutamate. In summary, our in vivo and in vitro studies reveal that delayed transplantation of hMSCs combined with PACAP provides trophic molecules to promote neuronal cell survival, which also foster beneficial microenvironment for endogenous glia to increase their neuroprotective effect on the repair of injured spinal cord tissue

Restrained Th17 response and myeloid cell infiltration into the central nervous system by human decidua-derived mesenchymal stem cells during experimental autoimmune encephalomyelitis

Background: Multiple sclerosis is a widespread inflammatory demyelinating disease. Several immunomodulatory therapies are available, including interferon-β, glatiramer acetate, natalizumab, fingolimod, and mitoxantrone. Although useful to delay disease progression, they do not provide a definitive cure and are associated with some undesirable side-effects. Accordingly, the search for new therapeutic methods constitutes an active investigation field. The use of mesenchymal stem cells (MSCs) to modify the disease course is currently the subject of intense interest. Decidua-derived MSCs (DMSCs) are a cell population obtained from human placental extraembryonic membranes able to differentiate into the three germ layers. This study explores the therapeutic potential of DMSCs. Methods: We used the experimental autoimmune encephalomyelitis (EAE) animal model to evaluate the effect of DMSCs on clinical signs of the disease and on the presence of inflammatory infiltrates in the central nervous system. We also compared the inflammatory profile of spleen T cells from DMSC-treated mice with that of EAE control animals, and the influence of DMSCs on the in vitro definition of the Th17 phenotype. Furthermore, we analyzed the effects on the presence of some critical cell types in central nervous system infiltrates. Results: Preventive intraperitoneal injection of DMSCs resulted in a significant delay of external signs of EAE. In addition, treatment of animals already presenting with moderate symptoms resulted in mild EAE with reduced disease scores. Besides decreased inflammatory infiltration, diminished percentages of CD4+IL17+, CD11b+Ly6G+ and CD11b+Ly6C+ cells were found in infiltrates of treated animals. Early immune response was mitigated, with spleen cells of DMSC-treated mice displaying low proliferative response to antigen, decreased production of interleukin (IL)-17, and increased production of the anti-inflammatory cytokines IL-4 and IL-10. Moreover, lower RORγT and higher GATA-3 expression levels were detected in DMSC-treated mice. DMSCs also showed a detrimental influence on the in vitro definition of the Th17 phenotype. Conclusions: DMSCs modulated the clinical course of EAE, modified the frequency and cell composition of the central nervous system infiltrates during the disease, and mediated an impairment of Th17 phenotype establishment in favor of the Th2 subtype. These results suggest that DMSCs might provide a new cell-based therapy for the control of multiple sclerosis.This work was sponsored by grants from Acción Estratégica en Salud (PI13/00297 and PI11/00581), the Neurosciences and Aging Foundation, the Francisco Soria Melguizo Foundation, Octopharma, and Parkinson Madrid (PI2012/0032).S

Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cells secretome?

Introduction: The use of human umbilical cord Wharton Jelly-derived mesenchymal stem cells (hWJ-MSCs) has been considered a new potential source for future safe applications in regenerative medicine. Indeed, the application of hWJ-MSCs into different animal models of disease, including those from the central nervous system, has shown remarkable therapeutic benefits mostly associated with their secretome. Conventionally, hWJ-MSCs are cultured and characterized under normoxic conditions (21 % oxygen tension), although the oxygen levels within tissues are typically much lower (hypoxic) than these standard culture conditions. Therefore, oxygen tension represents an important environmental factor that may affect the performance of mesenchymal stem cells in vivo. However, the impact of hypoxic conditions on distinct mesenchymal stem cell characteristics, such as the secretome, still remains unclear. Methods: In the present study, we have examined the effects of normoxic (21 % O2) and hypoxic (5 % O2) conditions on the hWJ-MSC secretome. Subsequently, we address the impact of the distinct secretome in the neuronal cell survival and differentiation of human neural progenitor cells. Results: The present data indicate that the hWJ-MSC secretome collected from normoxic and hypoxic conditions displayed similar effects in supporting neuronal differentiation of human neural progenitor cells in vitro. However, proteomic analysis revealed that the use of hypoxic preconditioning led to the upregulation of several proteins within the hWJ-MSC secretome. Conclusions: Our results suggest that the optimization of parameters such as hypoxia may lead to the development of strategies that enhance the therapeutic effects of the secretome for future regenerative medicine studies and applications. © 2015 Teixeira et al.Portuguese Foundation for Science and Technology (FCT) (Ciência 2007 program and IF Development Grant (AJS); and pre-doctoral fellowships to FGT (SFRH/69637/ 2010) and SIA (SFRH/BD/81495/2011); Canada Research Chairs (LAB) and a SSE Postdoctoral Fellowship (KMP); The National Mass Spectrometry Network (RNEM) (REDE/1506/REM/2005); co-funded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER).info:eu-repo/semantics/publishedVersio