RESUMEN

BACKGROUND: Congenital pseudoarthrosis of the tibia (CPT) is an uncommon disease associated with failure to achieve bone union and recurrent fractures. There is evidence showing that CPT is associated with decreased osteogenesis. Based on the capacity of mesenchymal stromal cells (MSCs) to induce osteogenesis, we develop an osteogenic organoid (OstO) constituted by these cells, and other components of the bone niche, for inducing bone formation in a child diagnosed with CPT. AIM: To evaluate the capacity of an OstO to induce bone formation in a patient with CPT. METHODS: The OstO was fabricated with allogeneic bone marrow MSCs from a healthy donor, collagen microbeads (CM) and PRP clot. The CM and PRP function as extracellular matrix and scaffolds for MSC. The OstO was placed at the site of non-union. Internal and external fixation was placed in the tibia. Radiological evaluation was performed after MSCs transplantation. RESULTS: After 4 months of MSCs transplantation, radiographic imaging showed evidence of osteogenesis at the site of CPT lesion. The tibia showed bone consolidation and complete healing of the non-union CPT lesion after 6 months. Functional improvement was observed after 1 year of MSC transplantation. CONCLUSIONS: The OstO is a bone-like niche which promote osteogenesis in patients with failure in bone formation, such as CPT. To our knowledge, these results provide the first evidence showing CPT healing induced by an OstO constituted by allogeneic MSCs. Future studies incorporating a larger number of patients may confirm these results.

Asunto(s)

Osteogénesis , Seudoartrosis/congénito , Tibia , Niño , Humanos , Tibia/diagnóstico por imagen , Tibia/cirugía , Tibia/anomalías , Regeneración Ósea , Colágeno , Organoides , Diferenciación Celular

RESUMEN

BACKGROUND: Under certain clinical and experimental conditions hematopoiesis occurs in other site than bone marrow (BM), such as the liver. Here, we develop a 3D organoid that mimics several components of the hematopoietic niche present during liver extramedullary hematopoiesis. AIM: To evaluate the capacity of a 3D hematopoietic organoid (3D-HO) to function as a hematopoietic like-niche allowing for blood cell production outside of the BM. METHODS: The 3D-HO is constituted by liver sinusoidal endothelial cells (LSEC) as the stromal component, BM isolated from 5-FU treated mice (FU-BMCs), collagen microspheres and plasma clot as scaffolds. The ability of the 3D-HO to support the survival and functionality of FU-BMCs was investigated by using confocal microscopy, histology analysis, flow cytometry, and clonogenic assays. RESULTS: After 15 and 30 days, post-ectopic implantation, histological studies of the 3D-HO showed the presence of cells with myeloid and lymphoid lineage morphology. Flow cytometry analysis of these cells showed the presence of cells expressing hematopoietic stem progenitor cells (HSPC) (Sca-1+/c-Kit+), myeloid (Gr-1+) and lymphoid (B220+ and CD19+) markers. Clonogenic assays showed that cells from the 3D-HO formed hematopoietic colonies. Expression of the Sry gene by cells from the 3D-HO, implanted for 30 days in female mice, indicated that male donor cells persist in this model of extramedullary hematopoiesis. CONCLUSIONS: The 3D-HO constitutes an extramedullary hematopoietic-like niche which supports the survival and functionality of FU-BMCs. It may constitute an efficient model for investigating, in vitro and in vivo, those factors that control hematopoiesis outside BM.

Asunto(s)

Hematopoyesis Extramedular , Masculino , Femenino , Ratones , Animales , Células Endoteliales , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Organoides , Células de la Médula Ósea/metabolismo

RESUMEN

Cellular therapy and platelet-rich plasma (PRP) have been used as a treatment for skin wounds. Previous evidence has shown that mesenchymal stromal cells (MSC) may improve skin wound healing. In contrast, contradictory effects have been reported by using PRP treatment on skin wound healing. However, there is evidence that PRP constitutes an excellent scaffold for tissue engineering. In this work, we aim to study the effect of MSC on skin wound healing. We used an experimental murine model of full-thickness wounds. Wounds were treated with human bone marrow-MSC contained in a PRP clot. Untreated or PRP-treated wounds were used as controls. Wound healing was evaluated by macroscopic observation and histological analysis at day 7 post-wounding. Immunohistochemical studies were performed to detect the presence of epithelial progenitor cells (EPC) and the expression of basic fibroblast growth factor (bFGF). MSC/PRP implantation induced a significant wound closure and re-epithelialization as compared with the controls. Increase of CD34+ cells and bFGF was observed in the wounds treated with MSC/PRP. Our results show that MSC included in PRP clot induce cutaneous wound repair by promoting re-epithelialization, migration of EPC and expression of bFGF. PRP alone does not exert a significant effect on wound healing. Our results support the possible clinical use of MSC in PRP scaffold as potential treatment of skin wounds.

Asunto(s)

Células Madre Mesenquimatosas , Plasma Rico en Plaquetas , Traumatismos de los Tejidos Blandos , Humanos , Ratones , Animales , Cicatrización de Heridas , Piel/patología , Plasma Rico en Plaquetas/metabolismo , Repitelización

RESUMEN

OBJECTIVE: Cartilage damage (CD) in the temporomandibular joint (TMJ) continues being a major problem in maxillofacial field. Evidence suggests that cellular therapy may be used for repairing CD in the TMJ. DESIGN: A murine model of condyle CD (CCD) was generated in the TMJ to evaluate the capacity of mesenchymal stromal cells (MSCs) to induce cartilage regeneration in CCD. A large CCD was surgically created in a condyle head of the TMJ of C57BL/6 mice. Human MSC embedded into preclotted platelet-rich plasma (PRP) were placed on the surface of CCD. As controls, untreated CCD and exposed TMJ condyle (sham) were used. After 6 weeks, animals were sacrificed, and each mandibular condyle was removed and CCD healing was assessed macroscopically and histologically. RESULTS: Macroscopic observation of CCD treated with MSC showed the presence of cartilage-like tissue in the CCD site. Histological analysis showed a complete repair of the articular surface with the presence of cartilage-like tissue and subchondral bone filling the CCD area. Chondrocytes were observed into collagen and glycosaminoglycans extracellular matrix filling the repaired tissue. There was no evidence of subchondral bone sclerosis. Untreated CCD showed denudated osteochondral lesions without signs of cartilage repair. Histological analysis showed the absence of tissue formation over the CCD. CONCLUSIONS: Transplantation of MSC induces regeneration of TMJ-CCD. These results provide strong evidence to use MSC as potential treatment in patients with cartilage lesions in the TMJ.

Asunto(s)

Cartílago Articular , Células Madre Mesenquimatosas , Animales , Cartílago Articular/patología , Condrocitos , Humanos , Ratones , Ratones Endogámicos C57BL , Articulación Temporomandibular/cirugía

RESUMEN

The hematopoietic niche is a specialized microenvironment that supports the survival, proliferation and differentiation of hematopoietic stem progenitor cells (HSPCs). Three-dimensional (3D) models mimicking hematopoiesis might allow in vitro and in vivo studies of the hematopoietic (HP) process. Here, we investigate the capacity of a 3D construct based on non-adherent murine bone marrow mononuclear cells (NA-BMMNCs), mesenchymal stromal cells (MSCs) and collagen microspheres (CMs), all embedded into plasma clot (PC) to support in vitro and in vivo hematopoiesis. Confocal analysis of the 3D hematopoietic construct (3D-HPC), cultured for 24 h, showed MSC lining the CM and the NA-BMMNCs closely associated with MSC. In vivo hematopoiesis was examined in 3D-HPC subcutaneously implanted in mice and harvested at different intervals. Hematopoiesis in the 3D-HPC was evaluated by histology, cell morphology, flow cytometry, confocal microscopy and hematopoietic colony formation assay. 3D-HPC implants were integrated and vascularized in the host tissue, after 3 months of implantation. Histological studies showed the presence of hematopoietic tissue with the presence of mature blood cells. Cells from 3D-HPC showed viability greater than 90%, expressed HSPCs markers, and formed hematopoietic colonies, in vitro. Confocal microscopy studies showed that MSCs adhered to the CM and NA-BMMNCs were scattered across the 3D-HPC area and in close association with MSC. In conclusion, the 3D-HPC mimics a hematopoietic niche supporting the survival, proliferation and differentiation of HSPCs, in vivo. 3D-HPC may allow evaluation of regulatory mechanisms involved in hematopoiesis.

Asunto(s)

Células Madre Hematopoyéticas/metabolismo , Imagenología Tridimensional/métodos , Células Madre Mesenquimatosas/metabolismo , Microesferas , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Ratones , Análisis de Supervivencia

RESUMEN

BACKGROUND: Skin wounds continue to be a global health problem. Several cellular therapy protocols have been used to improve and accelerate skin wound healing. Here, we evaluated the effect of transplantation of mesenchymal stromal cells (MSC) on the wound re-epithelialization process and its possible relationship with the presence of epithelial progenitor cells (EPC) and the expression of growth factors. METHODS: An experimental wound model was developed in C57BL/6 mice. Human MSCs seeded on collagen membranes (CM) were implanted on wounds. As controls, animals with wounds without treatment or treated with CM were established. Histological and immunohistochemical (IH) studies were performed at day 3 post-treatment to detect early skin wound changes associated with the presence of EPC expressing Lgr6 and CD34 markers and the expression of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF). RESULTS: MSC transplantation enhanced skin wound re-epithelialization, as compared with controls. It was associated with an increase in Lgr6+ and CD34+ cells and the expression of KGF and bFGF in the wound bed. CONCLUSION: Our results show that cutaneous wound healing induced by MSC is associated with an increase in EPC and growth factors. These preclinical results support the possible clinical use of MSC to treat cutaneous wounds.

Asunto(s)

Trasplante de Células Madre Mesenquimatosas , Repitelización/fisiología , Piel/lesiones , Células Madre Adultas/metabolismo , Animales , Antígenos CD34/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Voluntarios Sanos , Humanos , Masculino , Ratones , Cultivo Primario de Células , Receptores Acoplados a Proteínas G/metabolismo , Piel/citología , Piel/metabolismo

RESUMEN

BACKGROUND: Deep dermal and full-thickness burns are not only difficult to treat, but they are also associated with significant morbidity and mortality. Recent reports have proposed the use of mesenchymal stromal cells (MSCs) for inducing tissue repair in burn injuries. OBJECTIVE: We aim to evaluate the effect of allogeneic MSC transplantation on full-thickness burns with delayed healing. MATERIAL AND METHODS: This study includes five patients with AB B/B burns. All patients received conservative treatments, including cleaning, debridement of necrotic tissue, and silver based dressing on the burn wounds. Cryopreserved allogeneic MSCs were thawed and rapidly expanded and used for application in burned patients. MSCs were implanted into preclotted platelet-rich plasma onto the surface of burn wounds. RESULTS: All treated burn wounds showed early granulation tissue and rapid re-epithelialization after MSC transplantation. Healing took between 1 and 5 months after MSC transplantation. Repair of burn wounds was associated with slight discoloration of the regenerated skin without hypertrophic scarring or contractures. CONCLUSION: Our results provide evidence of healing in deep- and full-thickness burns by allogeneic MSC transplantation. Rapid healing of burn patients, after MSC transplantation, improves their quality of life and reduces the length of hospitalization. Future studies incorporating a larger number of patients may confirm the results obtained in this work.

Asunto(s)

Quemaduras , Trasplante de Células Madre Hematopoyéticas , Células Madre Mesenquimatosas , Quemaduras/cirugía , Humanos , Calidad de Vida , Trasplante de Piel , Cicatrización de Heridas

RESUMEN

BACKGROUND: There is evidence showing that human mesenchymal stromal cells (MSC) seeded on collagen microspheres (CM) and incorporated into platelet rich plasma (PRP) clots induce bone formation. For clinical trials it is very important to establish standardization of storage and shipment conditions to ensure the viability and functionality of cellular products. We investigate the effect of storage temperature and time on the viability and functionality of human MSC seeded on CM and included into PRP clots for using in the further clinical application for bone regeneration. METHODS: MSC/CM/PRP clots were stored at room temperature (RT), 4 °C and 37 °C for 12 h, 24 h and 48 h. At each period of time, MSC were evaluated for their viability and functionality. RESULTS: MSC from MSC/CM/PRP clots maintained at RT and 37 °C for 24 h showed a high viability (90%) and maintained their capacity of proliferation, migration and osteogenic differentiation. In contrast, MSC/CM/PRP maintained to 4 °C showed a significant reduction in their viability and migration capacity. MSC from MSC/CM/PRP clots maintained at RT for 24 h induce osteogenesis in the subcutaneous tissues of mice, after four months of transplantation. DISCUSSION: Our results show that MSC incorporated into CM/PRP clots and maintained at RT can be utilized in bone regeneration protocols during the first 24 h after their processing.

Asunto(s)

Conservación de la Sangre/métodos , Supervivencia Celular/fisiología , Células Madre Mesenquimatosas/citología , Plasma Rico en Plaquetas/fisiología , Animales , Regeneración Ósea/fisiología , Colágeno/farmacología , Femenino , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Microesferas , Modelos Animales , Osteogénesis , Temperatura , Factores de Tiempo

RESUMEN

Administration of streptozotocin (STZ) is one of the most used experimental models of diabetes (STZ-DT). STZ induces beta-cell damage in pancreatic islets. It is known that hematopoietic stem progenitor cells (HSPCs) are mobilized from bone marrow to damaged tissues. In this work, we evaluated the effects of the hematopoietic mobilizers G-CSF (250µg/kg; for five consecutive days) and AMD3100 (5mg/kg; single s.c injection) in mice treated with STZ (175mg/kg). Mice injected with STZ showed a significant reduction in the number and area of islets and in the number of beta- and alpha-cells. Concurrently, they had hyperglycemia (blood glucose over 300mg/dl) associated with very low levels of insulin in plasma. The number and area of islets from STZ-DT mice treated with G-CSF and/or AMD3100 were similar to the controls. However, these mice had neither a reduction of hyperglycemia nor an improvement in the insulin levels. Analysis of islet cellularity showed a large reduction in beta-cells with a significant expansion of alpha-cells. These results indicate that G-CSF and AMD3100 induce partial protection of islet tissues and expansion of alpha-cells in mice treated with STZ but do not protect beta-cells from the damage induced by this compound.

Asunto(s)

Células Secretoras de Glucagón/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Compuestos Heterocíclicos/administración & dosificación , Islotes Pancreáticos/efectos de los fármacos , Estreptozocina/uso terapéutico , Animales , Bencilaminas , Glucemia , Ciclamas , Diabetes Mellitus Experimental , Células Secretoras de Glucagón/fisiología , Hiperglucemia/etiología , Insulina/sangre , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Ratones , Estreptozocina/administración & dosificación

RESUMEN

PURPOSE: There is evidence showing that mesenchymal stromal cells (MSC) may constitute a potential therapeutic strategy to induce bone regeneration. In this work, we investigate the capacity of autologous bone marrow (BM) MSC loaded on collagen microspheres (CM) and included into autologous platelet-rich plasma (PRP) clots (MSC/CM/PRP) to induce bone formation in patients with nonunion lesions. METHODS: MSC were isolated from BM cells of patients with nonunion lesions. Phenotypical (marker expression) and functional studies (osteogenic differentiation) were performed. MSC were seeded on CM and included into autologous PRP clot (MSC/CM/PRP). The capacity of MSC/CM/PRP to induce bone formation was evaluated in three patients diagnosed with nonunion. RESULTS: MSC loaded on CM/PRP clots maintain their biological functions, in vitro. After three months, post-MSC transplantation, all patients showed evidence of osteogenesis at the site of nonunion. After one year, all patients showed a complete healing of the nonunion. CONCLUSIONS: Our results support the use of autologous MSC transplanted as MSC/CM/PRP for the treatment of nonunion fractures. Future studies incorporating a larger number of patients may confirm the results obtained in this work.

Asunto(s)

Regeneración Ósea/efectos de los fármacos , Fracturas no Consolidadas/tratamiento farmacológico , Trasplante de Células Madre Mesenquimatosas/métodos , Cicatrización de Heridas/efectos de los fármacos , Adulto , Anciano de 80 o más Años , Colágeno/metabolismo , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Microesferas , Osteogénesis/efectos de los fármacos , Plasma Rico en Plaquetas/efectos de los fármacos

RESUMEN

Congenital pseudoarthrosis of the tibia (CPT) is an uncommon disease whose etiology and pathogenesis is unknown. Several evidences suggest that decreased osteogenic capacities, impaired local vascularization, and microenvironment alterations may play a role in the pathogenesis of CPT. Additionally, it is not clear if the pathogenesis of this disease is related to the absence of cells with osteogenic capacity of differentiation. In this work, a two-year-old patient diagnosed with CPT underwent an orthopedic surgery to promote bone union in a pseudoarthrosis lesion. Tissue from CPT lesion was excised, and histological evaluation and tissue culture were performed. Histologic analysis of the soft CPT lesion showed the presence of highly cellular fibrous tissue, vascularization, and abundant extracellular matrix. Fusiform cells of mesenchymal appearance were observed but osteoblasts, osteoclasts, chondrocytes, and adipose cells were not found. There was no evidence of osteogenesis. CPT tissue cultured as explants showed, after one month of culture, evidence of osteogenesis, chondrogenesis, and adipogenesis. Cells isolated from explants of CPT tissue showed a fibroblast-like morphology and expressed the mesenchymal stromal cell (MSC) markers: CD105, CD73, and CD90 (CPT-MSC). Functional analysis showed that CPT-MSC differentiate, in vitro, into osteogenic, chondrogenic, and adipocytic cells. CPT-MSC expressed osteocalcin and agrecan. CPT-MSC produced collagen in the presence of ascorbic acid. MSC from BM of normal individuals were used as control. In summary, our results indicate that CPT tissue contains MSC with osteogenic capacity of differentiation. It is possible that CPT microenvironment may contribute to impair the osteogenic capacity of differentiation of CPT-MSC.

Asunto(s)

Células Madre Mesenquimatosas/fisiología , Seudoartrosis/congénito , Tibia/citología , Tibia/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Preescolar , Humanos , Masculino , Seudoartrosis/diagnóstico por imagen , Seudoartrosis/cirugía , Radiografía , Tibia/diagnóstico por imagen

RESUMEN

Patients with mild cognitive impairment (MCI) or Alzheimer's disease (AD) might develop olfactory dysfunction that correlates with progression of disease. Alteration of olfactory neuroepithelium associated with MCI may be useful as predictor of cognitive decline. Biomarkers with higher sensitivity and specificity would allow to understand the biological progression of the pathology in association with the clinical course of the disease. In this study, magnetic resonance images, apolipoprotein E (ApoE) load, Olfactory Connecticut test and Montreal Cognitive Assessment (MoCA) indices were obtained from noncognitive impaired (NCI), MCI and AD patients. We established a culture of patient-derived olfactory stromal cells from biopsies of olfactory mucosa (OM) to test whether biological properties of mesenchymal stromal cells (MSC) are concurrent with MCI and AD psychophysical pathology. We determined the expression of amyloid Aß peptides in the neuroepithelium of tissue sections from MCI and AD, as well as in cultured cells of OM. Reduced migration and proliferation of stromal (CD90(+) ) cells in MCI and AD with respect to NCI patients was determined. A higher proportion of anosmic MCI and AD cases were concurrent with the ApoE ε4 allele. In summary, dysmetabolism of amyloid was concurrent with migration and proliferation impairment of patient-derived stem cells.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Disfunción Cognitiva/metabolismo , Células Madre Mesenquimatosas/metabolismo , Trastornos del Olfato/complicaciones , Mucosa Olfatoria/metabolismo , Adulto , Anciano , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Movimiento Celular , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/genética , Disfunción Cognitiva/fisiopatología , Femenino , Hipocampo/patología , Humanos , Masculino , Células Madre Mesenquimatosas/fisiología , Persona de Mediana Edad

RESUMEN

Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors and CD34(+) cells were found, at 43 days of co-culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines.

Asunto(s)

Diferenciación Celular , Proliferación Celular , Células Madre Hematopoyéticas/citología , Células Madre Mesenquimatosas/metabolismo , Mucosa Olfatoria/citología , Antígenos CD34/genética , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Antígeno CD56/genética , Antígeno CD56/metabolismo , Recuento de Células , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular , Células Cultivadas , Microambiente Celular , Técnicas de Cocultivo/métodos , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Leucocitos Mononucleares/metabolismo , Linfopoyesis , Células Madre Mesenquimatosas/citología , Mielopoyesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Células Madre/genética , Factor de Células Madre/metabolismo , Factores de Tiempo

RESUMEN

Although there is evidence suggesting that statins may exert an endothelial protecting effect, recent in vitro data have shown that these compounds may induce endothelial cells (EC) apoptosis. We previously reported that the Fas-death receptor may induce apoptosis of the liver sinusoid endothelial cells (LSEC), and that TNF-alpha increases the susceptibility of these cells to suffer Fas-mediated apoptosis. Based on this evidence, in this study, we investigated the effect of simvastatin on Fas-mediated LSEC apoptosis. Simvastatin induced a significant reduction in LSEC viability, in a dose dependent manner, under serum-containing or serum-free conditions. This effect was prevented by mevalonate and GGPP, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. The simvastatin effect on LSEC death was not associated with increased activation of caspase-3. We found that simvastatin increased the susceptibility of LSEC death mediated by Fas. Further, simvastatin increased LSEC-apoptosis induced by Fas and TNF-alpha. Mevalonate and GGPP partially prevented simvastatin-induced sensitization to LSEC death mediated by Jo2 and TNF-alpha, but not Jo2 alone. Simvastatin did not induce up-regulation of the Fas on the LSEC. Our results provide evidence of simvastatin in modulating Fas-mediated apoptosis in endothelial cells. These results may have clinical implications in those clinical conditions associated with high levels of FasL and TNF-alpha.

Asunto(s)

Anticolesterolemiantes/farmacología , Apoptosis/efectos de los fármacos , Células Endoteliales , Hígado/citología , Simvastatina/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/metabolismo , Animales , Anticuerpos Antinucleares/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular , Células Cultivadas , Cicloheximida/metabolismo , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Activación Enzimática/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Humanos , Ácido Mevalónico/metabolismo , Ratones , Fosfatos de Poliisoprenilo/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo

RESUMEN

Although the bone marrow (BM) microenvironment is the main inducer niche of early B lymphopoiesis during the adult life, other extramedullar microenvironments, such as the liver, may also have potential for supporting B-cell development. Previously, we reported that murine liver sinusoidal endothelial cells (LSECs) support in vitro and in vivo hematopoietic stem cell (HSC) proliferation and myeloid differentiation. In the present study, we investigated the capacity of LSEC to promote B lymphopoiesis from BM progenitor lineage-negative (Lin(-)) cells. Murine BM Lin(-) cells were co-cultured with LSEC, in the absence of exogenous cytokines. B cells were characterized by flow cytometry and cytokine expression by RT-PCR. We show that BM Lin(-) cells differentiated to early B-lymphoid progenitors (B220(+)) and subsequently to mature (CD19(+)) B cells. Functional studies showed the presence of a high number of non-adherent cells (NACs), collected from lipopolysaccharide (LPS)-treated Lin(-)/LSEC co-cultures, expressing IgM on their surface (sIgM). Colony formation from NAC was observed in the presence of IL-7 (CFU-IL-7). LSEC constitutively express IL-7, Flt-3L, and SCF at the mRNA level, and VCAM-1 on their surface, which may explain the capacity of these cells to promote B lymphopoiesis. These data demonstrate that LSEC promote all stages of B lymphopoiesis. To our knowledge, this is the first report that LSEC constitute an in vitro microenvironment for B lymphopoiesis. Further studies will establish whether LSEC can serve in vivo as a B-lymphopoietic niche under physiological or pathological condition, or when HSC are mobilized.

Asunto(s)

Linfocitos B/citología , Células de la Médula Ósea/citología , Diferenciación Celular , Hígado/citología , Animales , Antígenos CD19/análisis , Linfocitos B/metabolismo , Células de la Médula Ósea/metabolismo , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Endotelio/citología , Endotelio/metabolismo , Femenino , Citometría de Flujo , Expresión Génica , Interleucina-7/genética , Antígenos Comunes de Leucocito/análisis , Hígado/metabolismo , Linfopoyesis , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Molécula 1 de Adhesión Celular Vascular/genética

RESUMEN

Because statins and ajoene inhibit the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, we evaluated the hypothesis that the cytotoxic effect of these compounds may be potentiated when both are used in combination on tumor cells. We showed that cotreatment of the murine melanoma B16F10 cell with statins (atorvastatin and pravastatin) and ajoene, all at nontoxic doses, dramatically increased their cytotoxicity. B16F10 cell death induced by statins, but not by ajoene, was prevented by mevalonate and geranylgeranylpyrophosphate. To our knowledge, this is the first report that the combination of statins and ajoene, which alters the mevalonate pathway, might potentiate their cytotoxic effects on tumor cells.

Asunto(s)

Disulfuros/farmacología , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Melanocitos/efectos de los fármacos , Melanoma Experimental/patología , Pravastatina/farmacología , Pirroles/farmacología , Animales , Apoptosis/efectos de los fármacos , Atorvastatina , Línea Celular Tumoral/efectos de los fármacos , Disulfuros/antagonistas & inhibidores , Disulfuros/farmacocinética , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Citometría de Flujo , Ácidos Heptanoicos/antagonistas & inhibidores , Ácidos Heptanoicos/farmacocinética , Ácido Mevalónico/farmacología , Ratones , Fosfatos de Poliisoprenilo/farmacología , Pravastatina/antagonistas & inhibidores , Pravastatina/farmacocinética , Pirroles/antagonistas & inhibidores , Pirroles/farmacocinética , Sulfóxidos , Terpenos/metabolismo

RESUMEN

El desarrollo de reactivos hemoclasificadores mediante la aplicación de la tecnología para la producción de anticuerpos monoclonales (AcMo) ha sido exitoso y ello ha permitido reducir los costos asociados a su producción. En Venezuela el consumo de estos reactivos depende principalmente de la importación, con el consecuente gasto de divisas. Con el propósito de ayudar a solventar esta situación el presente trabajo se planteó como. 1) Generar hibridomas productores de AcMo con especificidad anti-A y anti-B, 2) Caracterizar y producir a mediana escala los AcMo obtenidos, 3) Realizar estudios de campo, con el fin de lograr su certificación como reactivos hemoclasificadores. El producto de este trabajo fue la obtención de 22 hibridomas, 11 productores de AcMo anti-A y 11 productores de anti-B. Cuatro AcMo fueron caracterizados y estudiados: Au18Kt3F, MG3 (ambos IgM anti-A), SS4.5 (IgG anti-B) y BB2-3 (IgM anti-B). Para la producción de estos AcMo a mayor escala se emplearon los bio-reactores comerciales “miniPerm” y “Tecnomouse”, lográndose una concentración elevada de los mismos. Los valores de parámetros funcionales como avidez, potencia y especificidad de los AcMo producidos resultaron aceptables al compararse con hemoclasificadores comerciales, lo que hace viable su utilización como reactivos hemoclasificadores

Asunto(s)

Masculino , Femenino , Humanos , Sistema del Grupo Sanguíneo ABO , Anticuerpos Monoclonales , Hibridomas , Indicadores y Reactivos , Medicina , Venezuela

RESUMEN

The Monoclonal Antibody (MoAb) technology has been successfully applied to develop reagents for human blood group classification. There is no production of this kind of reagents in Venezuela, and the local demand (blood banks and clinical laboratories) is mainly supplied with imported material. Considering this we decided to apply MoAb techniques to generate murine hybridomas secreting anti-A or anti-B specific MoAb. MoAb obtained were characterized and produced in enough quantity to perform validation studies as blood typing reagents. Out of 22 hybridomas that were initially selected, 11 were anti-A secretors and 11 were anti-B secretors. Four MoAb were further characterized: Au18Kt3F, MG3 (both IgM anti-A), SS4.5 (IgG1 anti-B) and BB2-3 (IgM anti-B). Conditions were also established for growing the hybridomas Au18Kt3F and BB2-3 in the bioreactors "miniPerm" and "Tecnomouse", allowing for scale-up production of these MoAb. Avidity and specificity were estimated for each one, and the results were comparable to those obtained from commercially available reagents, making feasible its use as blood typing reagents.

Asunto(s)

Sistema del Grupo Sanguíneo ABO/clasificación , Anticuerpos Monoclonales/biosíntesis , Sistema del Grupo Sanguíneo ABO/inmunología , Animales , Células Cultivadas , Técnicas Inmunológicas , Ratones , Ratones Endogámicos BALB C

RESUMEN

The hyperacute rejection observed in models of pig-to-human xenotransplantation is mainly because of the presence of natural antibodies in human blood with specificity for the Galalpha(1-3)Gal (Gal) carbohydrate moiety present on the surface of porcine endothelial cells. Human monoclonal anti-Gal antibodies could be of use both in the study of the basic mechanisms of hyperacute rejection as well as in its clinical prevention. In the present study we prepared 42 heterohybridomas (human-mouse) secreting antibodies with specificity for the Gal epitope. All of the antibodies produced were of the IgM isotype, according to a dot-blot assay. Twenty-seven antibodies were further characterized, and shown to be specific for Gal by different methods, including an enzyme-linked immunosorbent assay, in which the plates were sensitized with mouse laminin as a source of Gal. Specificity was also confirmed using purified Gal carbohydrate in a hemagglutination inhibition assay. The antibodies were shown to mediate lysis of Gal-expressing rabbit erythrocytes in the presence of complement. However, the heterohybridomas themselves were shown to express Gal, a result of the mouse P3x63Ag8.653 hybridoma cells used during hybridoma generation. The presence of this epitope on the surface of anti-Gal-producing cells, and on the antibody itself, represents a limitation to the production of high affinity anti-Gal antibodies.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Disacáridos/inmunología , Animales , Especificidad de Anticuerpos , Pruebas de Fijación del Complemento , Rechazo de Injerto/inmunología , Humanos , Hibridomas/inmunología , Inmunoglobulina M/biosíntesis , Ratones , Conejos , Porcinos , Trasplante Heterólogo