ABSTRACT
Purpose: This study aimed to compare the degree of maturation and development of fetal pig segmental intestinal tissue with that of spheroids created by in-vitro reaggregation of dissociated fetal intestinal cells after transplantation into immunodeficient mice. Methods: Fetal pig small intestines were transplanted as segmental grafts into the omentum and subrenal capsules of immunodeficient mice or enzymatically treated to generate single cells. Spheroids made by in-vitro reaggregation of these cells were transplanted into the subrenal capsules of immunodeficient mice. The segmental grafts and spheroids were harvested four and eight weeks after transplantation, and the structural maturity and in-vivo development of these specimens were histologically evaluated. Results: The spheroids were engrafted and supplied blood vessels from the host mice, but an intestinal layered structure was not clearly observed, and there was almost no change in size. On the other hand, the segmental grafts formed deep crypts in the mucus membrane, the inner circular layer, and outer longitudinal muscles. The crypts of the transplanted grafts harvested at eight weeks were much deeper, and the smooth muscle layer and the enteric nervous system were more mature than those of grafts harvested at the fourth week, although the intestinal peristaltic wave was not observed. Conclusions: Spheroids created from fetal small intestinal cells could not form layered structures or mature sufficiently. Conversely, segmental tissues structurally matured and developed after in-vivo transplantation and are therefore potential grafts for transplantation.
Subject(s)
Animals , Mice , Swine , Transplantation, Heterologous/veterinary , Fetal Tissue Transplantation/veterinary , Fetal Organ MaturityABSTRACT
Abstract: The invention and widely use of organ allotransplantation provides effective treatment of some originally fetal diseases such as liver/kidney failure and has saved million of lives around the globe. However, the scarcity of human organs has caused many patients, who could have been treated, to die while waiting for suitable organs around the world. Pig-to human xenotransplantation provides a potential solution to solve this tough problem. Pig organs have been considered as major sources of xenotransplantation because of the sufficient number of donors, the sizes of organs, and physiologically structural similarities. However, xenotransplantation also has some problems, such as the possibility of spreading animal diseases to human, the interspecies immunological barrier, organs of animal origin challenging human nature, and potential informed consent issues. This article will discuss these potential issues and to see whether it is the suitable time to conduct clinical xenotransplantation trials in humans.
Resumen: La invención y el amplio uso de trasplantes alógenos proporciona tratamiento efectivo de algunas enfermedades de origen fetal, como la insuficiencia renal y hepática, y ha salvado a millones de pacientes en el mundo. Sin embargo, la escasez de órganos humanos ha causado que muchos pacientes en el mundo, que podrían haber sido tratados, murieran por esperar un órgano adecuado. El xenotrasplante del cerdo al humano proporciona una solución potencial para resolver este difícil problema. Los órganos de cerdo han sido considerados como fuentes mayores para xenotrasplantes debido al suficiente número de donantes, el tamaño de los órganos y estructuras fisiológicas similares. No obstante, el xenotrasplante también tiene algunos problemas, como la posibilidad de expandir enfermedades animales a humanos, la barrera inmunológica entre especies, el desafío para la naturaleza humana de tener órganos de origen animal y problemas potenciales de consentimiento informado. Este artículo discute estos temas potenciales y plantea si estamos en un momento apropiado para realizar ensayos clínicos de xenotrasplantes en humanos.
Resumo: A invenção e amplo uso de alotransplante de órgãos propicia tratamento efetivo para algumas doenças originalmente fetais tais como falência hepática/renal e tem salvo milhões de vidas em todo o globo. Entretanto, a escassez de órgãos humanos tem causado a morte de muitos pacientes que poderiam ter sido tratados - aguardando por órgãos apropriados em todo o globo. Xenotransplante porco-para-humanos propicia uma solução potencial para resolver este difícil problema. Órgãos de porco tem sido considerados como as principais fontes de xenotransplante por causa do número suficiente de doadores, do tamanho dos órgãos e de similaridades estruturais fisiológicas. Entretanto, xenotransplante também tem alguns problemas, tais como a possibilidade de disseminar doenças animais aos humanos, a barreira imunológica entre espécies, órgão de origem animal desafiando a natureza humana e aspectos potenciais de consentimento informado. Esse artigo discutirá esses aspectos potenciais e verificará se é o momento adequado para conduzir ensaios clínicos de xenotransplante em humanos.
Subject(s)
Humans , Animals , Swine , Transplantation, Heterologous/ethics , Clinical Trials as Topic , Transplantation, Heterologous/adverse effects , Transplantation, Heterologous/psychology , Zoonoses/etiology , Genetic Engineering , Informed ConsentSubject(s)
Humans , Male , Middle Aged , Transplantation, Heterologous , Heart Transplantation , Graft RejectionABSTRACT
Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.
Subject(s)
Animals , Humans , Animals, Genetically Modified , Gene Knockout Techniques , HLA Antigens , Nuclear Transfer Techniques , Swine , Transplantation, HeterologousABSTRACT
ABSTRACT Are presented results of experimental pig kidney xenotransplantation in Brazil, which aims to reduce the waiting list mortality due to shortage of organs. Recent clinical results obtained abroad are commented.
RESUMO Apresentam-se resultados de xenotransplante suíno de rim experimental no Brasil que visa reduzir as listas de espera nas quais falecem muitos inscritos à espera do transplante. Comentam-se os recentes resultados clínicos obtidos no exterior.
Subject(s)
Animals , Kidney Transplantation , Swine , Transplantation, Heterologous/methods , Brazil , Waiting Lists , KidneyABSTRACT
An Andean fox was transferred to the Wildlife Hospital of the Universidad San Francisco de Quito for evaluation of injuries caused by a run over. Clinical signs of hypovolemic shock were detected. Radiographies showed multiple pelvic fractures and free fluid in retroperitoneal cavity. The presumptive diagnosis was hemorrhagic shock caused by blood loss secondary to a pelvis fracture. An emergency xenotransfusion using blood from a domestic dog was performed without acute transfusion reactions observed. This is the first report of successful xenotransfusion between a domestic dog and an Andean fox presenting a procedure that could be applied in emergency situations.(AU)
Uma raposa andina foi levada ao Hospital da Vida Selvagem da Universidad San Francisco de Quito para avaliar os ferimentos causados por um atropelamento. Sinais clínicos de choque hipovolêmico foram detectados. Radiografias mostraram múltiplas fraturas pélvicas e fluido livre na cavidade retroperitoneal. O diagnóstico presuntivo foi um choque hemorrágico causado por perda sanguínea secundária a uma fratura pélvica. Uma xenotransfusão de emergência foi realizada com o sangue de um cão doméstico sem reações agudas transfusionais. Este é o primeiro relato bem sucedido de xenotransfusão entre um cão doméstico e uma raposa andina, demonstrando que é um procedimento que poderá ser considerado em situações de emergência.(AU)
Subject(s)
Animals , Dogs , Dogs/blood , Foxes/blood , Shock , Transplantation, Heterologous , Blood Transfusion/veterinaryABSTRACT
Abstract Purpose To evaluate the use of demineralized bone matrix of caprine origin in experimental bone defects of the tibia in New Zealand rabbits. Methods Fragments of the tibia diaphysis were collected aseptically from clinically healthy goats. The bones were sectioned into 1 cm fragments and stored at -20°C for subsequent hydrochloric acid (HCL) demineralization. A 70 mg portion of DBMc was used to fill the experimental bone defects. Twenty-four female adult New Zealand rabbits were divided into 2 groups: the MG (matrix group, left tibia) and CG (control group, right tibia). Additionally, they were separated into 4 groups with 6 animals, according to the period of analysis (15, 30, 60 and 90 days postoperatively). Using microCT, volumetric parameters were evaluated: bone volume, relationship between bone volume and total volume, bone surface area, relationship between bone surface area and total volume, number of trabeculae, trabecular thickness and trabecular separation. Results There was a statistically significant difference (P<0.05) between groups considering bone volume (BV) and bone:total volume (BV/TV), on 15, 30 and 90 days postoperatively. Control group showed a statistically significant superiority (P < 0.05) considering the mean of the variables bone surface (BS), number of trabeculae (Tb.N) and between bone surface and total volume (BS/TV) at 15 and 90 days. Conclusions Caprine demineralized bone matrix was safe and tolerable. No signs of material rejection were seen macroscopically. It is an alternative for the treatment of bone defects when autologous graft is not available or in insufficient quantities.
Subject(s)
Goats , Bone Transplantation , Rabbits , Tibia , Transplantation, Heterologous , Bone Matrix , HeterograftsABSTRACT
OBJECTIVE@#To investigate the effect of β-arrestin1 overexpression on tumor progression in a NCG mouse model bearing T-cell acute lymphocytic leukemia (T-ALL) Molt-4 cell xenograft.@*METHODS@#Molt-4 cells were tagged with firefly-luciferase (F-Luc) by lentiviral infection, and fluorescence intensity of the cells was detected using a luminescence detector. Molt-4 cell lines with β-arrestin1 overexpression or knockdown were constructed by lentivirus infection and injected the tail vein in sub-lethal irradiated NCG mice. Body weight changes and survival time of the xenografted mice were observed, and the progression of T-ALL in the mice was evaluated using an fluorescence imaging system. Sixteen days after xenografting, the mice were euthanatized and tumor cell infiltration was observed in the slices of the liver and spleen.@*RESULTS@#We successfully tagged Molt-4 cells with F-Luc and overexpressed or knocked down β-arrestin1 in the tagged cells. Bioluminescent imaging showed obvious luminescence catalyzed by F-Luc in Molt-4 cells. After injection of Molt-4-Luc cells into irradiated NCG mice, a gradual enhancement of luminescence in the xenografted mice was observed over time, while the body weight of the mice decreased. Compared with the control mice, the mice xenografted with β-arrestin1-overexpressing Molt-4 cells had significantly prolonged survival time ( < 0.001), while the survival time of the mice xenografted with Molt-4 cells with β- arrestin1 knockdown was significantly shortened ( < 0.001). Histological examination revealed fewer infiltrating tumor cells in the liver and spleen of the mice xenografted with β-arrestin1-overexpressing Molt-4 cells in comparison with the mice bearing parental Molt-4 cell xenografts.@*CONCLUSIONS@#β-arrestin1 overexpression suppresses tumor progression in mice bearing Molt-4 cell xenograft.
Subject(s)
Animals , Humans , Mice , Disease Progression , Heterografts , T-Lymphocytes , Transplantation, Heterologous , beta-Arrestin 1ABSTRACT
Objetivo: A obtenção do número adequado de órgãos cadavéricos para transplantes é um item de política pública importante. Modelos econômicos tradicionais supõem que a solução pode ser obtida por incentivos financeiros. Críticos dessa abordagem insistem que o mercado não resolve o problema, mas a intervenção estatal sim. Métodos: Este artigo apresenta o modelo do homo economicus maturus de Frey (1997), cujo principal mérito é mostrar que ambas as críticas estão incorretamente colocadas, pois indivíduos respondem não apenas a incentivos monetários de forma positiva, como demonstram os modelos tradicionais, mas também podem apresentar reações psicológicas adversas. Esse modelo é aplicado ao dilema da doação de órgãos com a inclusão do fator tecnológico, por meio da substituição de órgãos humanos por órgãos não humanos (xenotransplantes). Resultados: O principal resultado do artigo é mostrar como um avanço tecnológico pode melhorar o bem-estar sem alterar o dilema moral dos indivíduos. Conclusões: A extensão do modelo econômico tradicional permite uma análise com novas possibilidades acerca de mudanças tecnológicas e dos dilemas morais que elas podem trazer.
Objective: Obtaining the adequate number of cadaveric organs for transplantation is an important public policy item. Traditional economic models assume that solution can be obtained by financial incentives. Critics of this approach insist that the market does not solve the problem, but state intervention does. Methods: This article presents Frey's (1997) homo economicus maturus model, whose main merit is to show that both critics are incorrectly because individuals respond not only to monetary incentives in a positive way, as traditional models demonstrate, but they can also present adverse psychological reactions. This model is applied to the dilemma of organ donation with the inclusion of the technological factor, through the replacement of human organs by non-human organs (xenotransplantation). Results: The main result of the article is to show how a technological advance can improve well-being without changing the moral dilemma of individuals. Conclusions: The extension of the traditional economic model allows an analysis with new possibilities about technological changes and the moral dilemmas that they can bring.
Subject(s)
Humans , Transplantation, Heterologous , Organ TransplantationABSTRACT
BACKGROUND@#Small cell lung cancer (SCLC) is characterized by poor differentiation, high malignancy and rapid growth fast, short double time, early and extensive metastatic malignancy. In clinical, chemotherapy is the main treatment method, while resistance to multiple chemotherapy drugs in six to nine months has been a major clinical challenge in SCLC treatment. Therefore, It has important clinical value to building SCLC aninimal model which is similar to patients with SCLC. Animal model of xenotransplantation (PDX) from the patients with small cell lung cancer can well retain the characteristics of primary tumor and is an ideal preclinical animal model. The study is aimed to establish SCLC PDX animal model and induce the chemoresistance model to help to study the mechanism of chemoresistance and individual treatment.@*METHODS@#Fresh surgical excision or puncture specimens from SCLC patients were transplanted into B-NSGTM mice subcutaneous tissues with severe immunodeficiency in one hour after operation the B-NSGTM mice subcutaneous in 1 hour, and inject chemotherapy drugs intraperitoneally after its tumor growed to 400 mm³ with EP which is cisplatin 8 mg/kg eight days and etoposide 5 mg/kg every two days until 8 cycles. Measure the tumor volum and mice weights regularly, then re-engrafted the largest tumor and continue chemotherapy.@*RESULTS@#Nine cases were conducted for B-NSG mice modeling. Three of nine cases could be engrafted to new B-NSG mice at least two generation. The SCLC PDX animal models have been established successfully. After adopting chemotherapy drugs, the chemoresistance PDX models have been established. High homogeneity was found between xenograft tumor and patient's tumor in histopathology, immunohistochemical phenotype (Syn, CD56, Ki67).@*CONCLUSIONS@#The SCLC PDX animal model and the chemoresistance PDX animal model have been successfully constructed, the success rate is 33%, which provides a platform for the clinical research, seeking for biological markers and choosing individual treatment methods of SCLC.
Subject(s)
Animals , Female , Humans , Antineoplastic Combined Chemotherapy Protocols , Pharmacology , Cisplatin , Disease Models, Animal , Drug Resistance, Neoplasm , Etoposide , Interleukin Receptor Common gamma Subunit , Genetics , Lung Neoplasms , Drug Therapy , Metabolism , Pathology , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Small Cell Lung Carcinoma , Drug Therapy , Metabolism , Pathology , Transplantation, Heterologous , Methods , Xenograft Model Antitumor AssaysABSTRACT
Abstract Tumor xenograft model (PDTX) derived from leukemia patients is an animal model in which the leukemia cells or primary cell lines of patients are transplanted directly into immunodeficient mice.The emergence of nude mice and SCID mice opened early xenotransplantation, then the NSG, NOG mice and the improved model and humanized mice based on there mice significantly improves the success rate of transplantation. The late presented transplantation of leukemia LSC and transplantation of patient-derived and induced pluripotent stem cells obtained based on iPSC technology provide new insight for the anderstanding leukemia genesis and development, and the new type humanized mouse model with normal lymphatic hematopoietic reconstruction provides a new platform of leukemia cell therapy and immunotherapy for leukemia therapy. PDTX is an important platform for the study of the pathogenesis and drug resistance mechanism of leukemia, as well as the development of new drugs and individualized treatment. In this paper, the recent progress in the construction and application of models of immunodeficient mice and their models is reviewed.
Subject(s)
Animals , Humans , Mice , Disease Models, Animal , Leukemia , Transplantation, HeterologousABSTRACT
OBJECTIVE: Poly (ADP-ribose) polymerase (PARP) is an important molecule in the early stress response of DNA damage, which is involved in DNA damage repair and cellular senescence. Olaparib, as PARP inhibitor, has an anti-tumor effect on high grade serous ovarian cancer, but its effects on cellular senescence have not been reported. This study intends to explore the role of olaparib in the regulation of senescence in ovarian cancer cells. METHODS: The effects of olaparib on the senescence of ovarian cancer cells were detected by using the senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated heterochromatin aggregation (SAHF). Quantitative real-time polymerase chain reaction was used to analyze the senescence-associated secretory phenotype (SASP). Cell cycle and apoptosis were detected by flow cytometry. The effect of olaparib on tumor growth was analyzed in a nude mouse xenograft transplantation model. RESULTS: Long-term (6 days) treatment with olaparib (5 μM) significantly inhibited the growth of ovarian cancer cells, leading to arrest the cell cycle at G0/G1 phase, significant increase the number of positive SA-β-Gal stained cells and positive SAHF cells. The expression of P16 and retinoblastoma protein (p-RB) were significantly enhanced in SKOV3 cells under olaparib treated, meanwhile, the expression of P53 and p-RB were upregulated in A2780 cells. In OVCAR-3 cells, the expression of P53 was downregulated and p-RB was upregulated. Mice with SKOV3 xenograft transplantation was given olaparib (10 mg/kg/day) via abdominal cavity administration, the tumor volume was reduced (p < 0.01). CONCLUSION: Continuous low dosage administration of olaparib induced senescence under P16 or P53 dependent manner in ovarian cancer.
Subject(s)
Animals , Mice , Abdominal Cavity , Aging , Apoptosis , Cellular Senescence , Cell Cycle , DNA Damage , Flow Cytometry , Heterochromatin , Mice, Nude , Ovarian Neoplasms , Phenotype , Real-Time Polymerase Chain Reaction , Retinoblastoma Protein , Transplantation, Heterologous , Tumor BurdenABSTRACT
A doença periodontal causa uma perda na estrutura de suporte dos elementos dentários. Como consequência, ocorre o aparecimento de sequelas provenientes dessa doença. Por conta disso foram desenvolvidos materiais com a fi nalidade de regeneração tecidual na estrutura de suporte, para que houvesse maior sobrevida desses elementos, aumento na quantidade de gengiva inserida e melhor otimização estética para os pacientes. Dentre os inúmeros materiais, temos a Matriz Colágena Porcina, que simplesmente é uma membrana obtida a partir de suínos, sendo que esta passa por uma cadeia de procedimentos, com a fi nalidade de minimizar e/ou eliminar qualquer tipo de interação alergênica no ser humano. A sua utilização é dada principalmente no aumento de tecidos moles ao redor de dentes afetados com a doença periodontal e em implantes osseointegrados, trazendo como principal vantagem a redução de morbidade do paciente, evitando, assim, a necessidade de um segundo sítio cirúrgico.
Periodontal disease results in the loss of structural support on dental elements. As such, sequels can be seen in the mouth. For this, materials were develop for tissue regeneration in order to increase the amount of attached gingiva and to optimize patient esthetics. The porcine collagen matrix is a product were the collagen undergoes a series of several treatments to minimize any type of allergic reaction to the human body. For example, it can be used for soft tissue augmentation around affected teeth and dental implants. Also, the porcine collagen matrix can reduce patient morbidity because it avoids autologous soft tissue harvesting.
Subject(s)
Humans , Biocompatible Materials , Gingival Recession/therapy , Guided Tissue Regeneration, Periodontal , Heterografts , Tissue Transplantation/methods , Transplantation, HeterologousABSTRACT
Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of α 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.
Subject(s)
Adult , Female , Humans , Pregnancy , Cell Line , Cytidine , DNA , Ear , Embryonic Development , Fibroblasts , Flow Cytometry , Fluorescent Antibody Technique , Genetics , Heterografts , Kidney , Korea , Physiology , Skin , Swine , Swine, Miniature , Transplantation, HeterologousABSTRACT
Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.
Subject(s)
Animals , Alleles , Endogenous Retroviruses , Genes, MHC Class I , Genes, MHC Class II , Genome , Heterografts , Leukocytes , Mass Screening , Recombination, Genetic , Retroviridae , RNA , Swine , Transgenes , Transplantation, Heterologous , ViremiaABSTRACT
O adenocarcinoma ductal pancreático (PDAC, pancreatic ductal adenocarcinoma), o tipo mais prevalente de câncer do pâncreas, é uma neoplasia extremamente agressiva e com elevado índice de letalidade. Há uma necessidade premente de identificação de vulnerabilidades no PDAC que possam ser exploradas como alvos terapêuticos, e a utilização de modelos pré-clínicos que recapitulem a complexidade biológica e heterogeneidade clínica da doença é um aspecto central para a realização dessa tarefa. Os xenotransplantes de tecido tumoral derivado de pacientes (PDX, patient-derived tumor tissue xenografts), realizados em camundongos imunodeficientes, replicam com grande similaridade as principais características do tumor original e, assim, constituem uma ferramenta valiosa para o teste de drogas e estudos funcionais. Neste trabalho, 17 amostras cirúrgicas de PDAC humano foram implantadas subcutaneamente em camundongos nude atímicos. Sete tumores (41%) foram enxertados com sucesso e têm sido mantidos em sucessivas gerações de animais receptores. O exame histológico de seis desses xenoenxertos identificou características morfológicas compatíveis com os padrões reconhecidos no PDAC humano, assim como uma consistente similaridade de seu status de diferenciação histológica em relação aos perfis verificados nos tumoresoriginais. O cultivo in vitro de células derivadas de um dos xenotumores resultou em uma nova linhagem de câncer de pâncreas, com morfologia e cinética de crescimento comparáveis às de outras linhagens celulares de câncer pancreático. O potencial tumorigênico dessa nova linhagem foi validado in vivo, com uma consistente formação de tumores após inoculação em camundongos nude. A fim de aproveitar esse recurso para a investigação de potenciais alvos terapêuticos no PDAC, um rastreamento de vulnerabilidades moleculares foi realizado por meio de silenciamento gênico em larga-escala com RNA de interferência (RNAi). Uma biblioteca lentiviral de 4492 shRNAs (short hairpin RNAs), alvejando cerca de 350 genes envolvidos na regulação epigenética, foi empregada para a triagem de genes de suscetibilidade nas células derivadas de PDX, e em outras cinco linhagens tumorais pancreáticas (AsPC-1, BxPC-3, Capan-1, MIA PaCa-2 e PANC-1). Inicialmente, foi realizada uma série de experimentos preliminares, visando à amplificação e controle de qualidade da biblioteca de silenciamento, à produção de vetores lentivirais e à padronização das condições experimentais para a transdução e seleção das células-alvo. Apenas três das linhagens avaliadas (AsPC-1, MIA PaCa-2 e PANC-1) mostraram-se permissíveis à transdução pelos vetores lentivirais, e foram assim utilizadas no screening de alvos epigenéticos. A análise dos dados obtidos nesse ensaio está em curso e os resultados serão utilizados para a definição de potenciais alvos candidatos. Em conclusão, recursos valiosos para apoiar a pesquisa sobre o câncer de pâncreas foram desenvolvidos. A coleção de PDXs estabelecida, bem como a linhagem celular recém-derivada, constituem uma fonte permanente e estável de células de PDAC para análises moleculares e estudos funcionais que busquem elucidar aspectos da doença ainda pouco compreendidos. Adicionalmente, os reagentes gerados e a expertise adquirida com os ensaiosrealizados com a biblioteca de shRNAs contra alvos epigenéticos serão de grande utilidade em futuras investigações para identificar genes com funções importantes na manutenção do fenótipo tumoral, e consequentemente com potencial para serem explorados terapeuticamente
Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is a highly aggressive and lethal neoplasm. There is a pressing need to identify vulnerabilities in PDAC suited to be exploited as therapeutic targets, and the use of preclinical models recapitulating the biological complexity and clinical heterogeneity of the disease is central to this task. Patient-derived tumor tissue xenografts (PDX), established in immunodeficient mice, replicate with great similarity the main characteristics of the original tumor and thus constitute a valuable tool for drug testing and functional studies. In this work, 17 surgical samples of human PDAC were implanted subcutaneously in athymic nude mice. Seven tumors (41%) were successfully grafted and have been maintained through successive generations of recipient animals. Histological examination of six of these xenografts identified morphological characteristics compatible with the recognized patterns of human PDAC, as well as a consistent similarity of their histological differentiation status in relation to the profiles verified in the original tumors. In vitro culture of cells derived from one of these xenografts resulted in a new pancreatic cancer cell line, with morphology and growth kinetics comparable to those of other pancreatic tumor cells. The tumorigenic potential of this freshly derived cell line was validated in vivo, with a consistent tumor formation following inoculation into nude mice. To take advantage ofthis resource to investigate potential therapeutic targets in PDAC, a screening of molecular vulnerabilities was performed through large-scale gene silencing with RNA interference (RNAi). A lentiviral library containing 4492 short hairpin RNAs (shRNAs), targeting about 350 genes involved in epigenetic regulation, was employed for the search of susceptibility genes in the PDX-derived cells and in other five pancreatic tumor cell lines (AsPC-1, BxPC -3, Capan-1, MIA PaCa-2 and PANC-1). Initially, a series of preliminary experiments were carried out aiming at the amplification and quality control of the silencing library, production of lentiviral vectors and adjustment of the experimental conditions for transduction and selection of the target cells. Only three of the cell lines evaluated (AsPC-1, MIA PaCa-2 and PANC-1) were permissible for transduction by the lentiviral vectors, and were accordingly used in the screening of epigenetic targets. The analysis of data obtained in this trial is ongoing and the results will be used for definition of potential candidate targets. In conclusion, valuable resources to support research on pancreatic cancer have been developed. The established collection of PDXs as well as the newly derived cell line constitutes a permanent and stable source of PDAC cells for molecular analyzes and functional studies seeking to elucidate aspects of this disease that are still poorly understood. Additionally, both the reagents generated and the expertise gained from the RNAi assay against epigenetic targets will have inordinate usefulness in future investigations to identify genes with major functions in maintaining the malignant phenotype, and consequently with the potential to be exploited therapeutically
Subject(s)
Animals , Female , Mice , Pancreatic Neoplasms/physiopathology , Cell Line, Tumor/classification , Heterografts/metabolism , Transplantation, Heterologous/instrumentation , Gene Library , RNA, Small Interfering , RNA Interference , Epigenomics/standardsABSTRACT
Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Methods Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F +T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. Results The experiments indicated that sensory function improved when transgenic hESCs were used. The regeneration of sensory fibers indeed led to increased reflexes, upon stimulation of the paw ipsilateral to the lesion, as seen by von-Frey evaluation, which was supported by immunohistochemistry. Conclusions Overall, the present data demonstrated that transgenic embryonic stem cells, engineered to overexpress FGF-2 in an inducible fashion, could be employed to support regeneration aiming at the recovery of both motor and sensory functions.(AU)
Subject(s)
Animals , Male , Rats , Sciatic Nerve/transplantation , Transplantation, Heterologous/rehabilitation , Fibrin Tissue Adhesive , Embryonic Stem Cells , Nerve Regeneration , Mice, Inbred C57BLABSTRACT
Abstract Purpose: To assess the efficacy of allogeneic mesenchymal stem cells and xenogenic platelet rich plasma in the treatment of bone failure of osteoporotic rabbits secondary to estrogenic deprivation and iatrogenic hypercortisolism. Methods: Eight female rabbits underwent ovarian resection and corticoid therapy to induce clinical status of osteoporosis. Four failures were produced in the tibiae, with each failure being treated with hemostatic sponge, allogenic mesenchymal stem cells, xenogenic platelet-rich plasma and the association between both. The animals were divided into two groups, evaluated radiographically and histopathologically at 30 and 60 days post treatment. Results: A radiographically confirmed consolidation of bone failures treated with allogeneic mesenchymal stem cells, associated with the histopathological image of mature and immature bone tissue, without evidence of osteopenia, was compared with the other groups, in which radiolucent failures with osteopenia and fibrosis were still present, denoting the satisfactory effect of the first treatment in detriment to the others. Conclusion: The treatment of bone failures of rabbits with secondary osteoporosis with allogeneic mesenchymal stem cells induced greater bone consolidation with mature and immature bone tissue production (p<0.01), when compared to the other treatments.
Subject(s)
Animals , Female , Rats , Osteoporosis/complications , Tibia/pathology , Bone Regeneration/physiology , Mesenchymal Stem Cell Transplantation , Platelet-Rich Plasma , Tibia/injuries , Time Factors , Transplantation, HeterologousABSTRACT
En el laboratorio de la Dra Pasqualini, por serendipitismo surgió un modelo de cáncer de mama murino que se caracteriza por el hecho de que es uno de los pocos modelos de ratón en los cuales los carcinomas mamarios inducidos modelan al tipo de tumor más frecuente de la mujer: el carcinoma mamario luminal que expresa receptores hormonales y es modulable por terapia endócrina. Luego ya en el IBYME demostramos que en este modelo los receptores de estrógeno interaccionan con los de progesterona en los promotores de genes blanco y que la proporción de isoformas de receptor de progesterona es fundamental para predecir la respuesta a antiprogestágenos. Hemos validado estas observaciones en modelos de xenotransplantes de líneas celulares de cáncer de mama humano creciendo en ratones inmunosuprimidos y posteriormente, utilizando muestras de pacientes de cáncer de mama humano, hemos demostrado que sólo aquellas con mayor expresión de isofoma A que de isofoma B, responden ex vivo al tratamiento con antiprogestágenos. Proponemos que estas pacientes serían candidatas a tratamientos combinados de antiestrógenos y antiprogestágenos. Estudios de tumores de pacientes creciendo en ratones inmunosuprimidos ayudarán a discernir cuáles tumores responden mejor a una u otra terapia endocrina
By serendipity, at Dr. Christiane Dosne Pasqualini´s Laboratory,, we have developed one of the few mouse experimental models of breast cancer in which induced-tumors share many features of human luminal breast cancer: ductal histology, hormone receptor expression and hormone responsiveness. After moving to IBYME, we were able to demonstrate that estrogen receptors interact with progesterone receptors at the promoter of key target genes and that the ratio of progesterone receptor isoforms A and B is essential to predict antiprogestin responsiveness. After validating this data in human breast cancer cell xenografts, modified to express different PR isoform ratios, we designed a study aimed to evaluate the response of human breast cancers to antiprogestins ex-vivo and correlate this data with the PR isoform ratio. Only those samples with higher levels of isoform A than isoform B were inhibited by antiprogestins. We propose that selected breast cancer patients, those with higher levels of isoform A than isoform B, might be candidates for adjuvant treatment with antiprogestins in combination with standard endocrine therapy. Future experiments generating patient derived xenografts will help to understand and better select patients for personalized endocrine treatments.