RESUMO
Mutations in the C-terminal region of nucleophosmin in acute myeloid leukemia (AML) result in aberrant cytoplasmic nucleophosmin (cNPM) in leukemic blast cells which is detectable by immunocytochemistry in bone marrow trephine (BMT) biopsy sections. We tested whether cNPM is detectable by immunocytochemistry in air-dried smears of AML with nucleophosmin1 (NPM1) mutations. An immunoalkaline phosphatase method was developed using the OCI-AML3 cell line, known to have mutated NPM1, and assessed on blood and marrow smears of 60 AML cases. NPM was detectable in all blast cell nucleoli and cNPM in 21 of 31 of NPM1 mutated and 15 of 29 wild-type cases. Paired air-dried smears and BMT biopsies from the same case (mutated and wild-type) gave discrepancies in cNPM expression and there was no correlation in 10 of 22 cases. Due to the high false positive and negative rates for cNPM in cell smears, this method should not be used as a surrogate for NPM1 mutations in AML.
Assuntos
Medula Óssea/patologia , Técnicas Citológicas/métodos , Citoplasma/metabolismo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Humanos , Técnicas Imunoenzimáticas , Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Nucleofosmina , Reação em Cadeia da Polimerase , PrognósticoRESUMO
Pancreatic islets implanted heterotopically (i.e., into the kidney, spleen, or liver) become poorly revascularized following transplantation. We hypothesized that islets implanted into the pancreas would become better revascularized. Islets isolated from transgenic mice expressing enhanced yellow fluorescent protein (EYFP) in all somatic cells were cultured before they were implanted into the pancreas or beneath the renal capsule of athymic mice. Vascular density was evaluated in histological sections 1 month posttransplantation. EYFP was used as reporter for the transgene to identify the transplanted islets. Islet endothelial cells were visualized by staining with the lectin Bandeiraea simplicifolia (BS-1). Capillary numbers in intrapancreatically implanted islets were only slightly lower than those counted in endogenous islets, whereas islets implanted beneath the renal capsule had a markedly lower vascular density. In order to determine if this high graft vascular density at the intrapancreatic site reflected expansion of remnant donor endothelial cells or increased ingrowth of blood vessels from the host, also islets from Tie2-green fluorescent protein (GFP) mice (i.e., islets with fluorescent endothelial cells) were transplanted into the pancreas or beneath the renal capsule of athymic mice. These islet grafts revealed that the new vascular structures formed in the islet grafts contained very few GFP-positive cells, and thus mainly were of recipient origin. The reason(s) for the much better ingrowth of blood vessels at the intrapancreatic site merits further studies, because this may help us form strategies to overcome the barrier for ingrowth of host vessels also into islets in heterotopic implantation sites.
Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/citologia , Animais , Células Endoteliais , Sobrevivência de Enxerto , Humanos , Imuno-Histoquímica , Proteínas Luminescentes/análise , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica/fisiologiaRESUMO
OBJECTIVE: The aim of the study was to increase the number of human islet beta-cells after transplantation with injections of human growth hormone (hGH). INTERVENTIONS: Human islets and fetal rat islets were transplanted under the left kidney capsule and under the right kidney capsule, respectively in nude normoglycemic mice which were then given a daily injection of 200 microg hGH for 1-4 weeks. MAIN OUTCOME MEASURE: Beta-cell proliferation was determined using thymidine incorporation and the beta-cell area was assessed using light microscopy. RESULTS: Mice given hGH increased their body weight one week after transplantation and had a more efficient removal of glucose after 3 and 4 weeks. Treatment with hGH resulted in increased beta-cell proliferation in human and fetal rat beta-cells, and the beta-cell area tended to increase. However, serum insulin concentrations and pancreas insulin content remained unchanged. CONCLUSIONS: hGH increased the proliferation of transplanted human beta-cells as well as improving the glucose tolerance of the transplanted mice.
Assuntos
Sobrevivência de Enxerto/efeitos dos fármacos , Hormônio do Crescimento Humano/farmacologia , Células Secretoras de Insulina , Transplante das Ilhotas Pancreáticas , Adulto , Idoso , Animais , Glicemia/metabolismo , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Transplante de Tecido Fetal , Hormônio do Crescimento Humano/sangue , Humanos , Insulina/sangue , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/transplante , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Gravidez , Ratos , Ratos Sprague-Dawley , Transplante Heterólogo , Adulto JovemRESUMO
The detection of genetic abnormalities (eg, translocations, amplifications) in paraffin-embedded samples by the fluorescence in situ hybridization (FISH) technique is usually performed on tissue sections. FISH analysis of nuclei extracted from paraffin-embedded samples is also possible, but the technique is not widely used, principally because of the extra labor involved and the loss of information on tissue architecture. In this article, we report that nuclei extracted from paraffin-embedded tissue often retain at least part of the surrounding cytoplasm. Consequently, immunocytochemical labeling for a range of cellular markers (eg, of lineage or proliferation) can be performed in combination with FISH labeling, allowing specific cell populations to be analyzed for genetic abnormalities. These cell preparations are largely free of the problems associated with tissue sections (eg, truncation artifact, signals in different focal planes) so that interpretation is easy and numerical chromosomal abnormalities are readily assessed. Cells isolated from paraffin sections can be stored in suspension so that arrays can be created as and when needed from a range of neoplasms for investigation by the immunoFISH technique (for example, for studying a new genetic abnormality). This procedure represents a novel methodology, which in some settings offers clear advantages over analysis of tissue sections.
Assuntos
Extratos Celulares , Aberrações Cromossômicas , Hibridização in Situ Fluorescente/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Biópsia , Separação Celular , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Neoplasias/patologia , Tonsila Palatina/patologia , Tonsila Palatina/ultraestrutura , Inclusão em Parafina , Fixação de TecidosRESUMO
The aim of the study was to evaluate the distribution of intraportally transplanted islets in mice. We initially administered 2000 polystyrene microspheres with a diameter of 50 microm intraportally into normoglycemic C57BL/6 mice. In separate experiments other mice were injected similarly with 300 microspheres each with a diameter of 100 or 200 microm. One week later the animals were killed, and the lungs and livers were removed and divided into lobes. The number of microspheres in each individual liver lobe and in the lungs was counted using a stereomicroscope. In other experiments, athymic C57BL/6 mice were similarly implanted with 250 islets isolated from transgenic mice expressing the enhanced yellow fluorescent protein in the islet cells. The distribution of microspheres and islets was independent of size, and fairly homogenous within the liver, with the exception of the caudate lobe, which contained fewer microspheres and islets, respectively. Approximately one third of all microspheres and islets were present as aggregates. Eighty-five to 90% of the implanted microspheres were identified in the liver sections, whereas 60-65% of the implanted islets were recovered. Aggregates or single fluorescent cells were observed in the liver of islet-implanted mice. We conclude that islets and microspheres implanted into the liver distribute fairly homogenously and quite a few of them exist as aggregates or, with respect to islets, as fragments.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Fígado/fisiologia , Microesferas , Transplante Homólogo/métodos , Animais , Estudos de Viabilidade , Corantes Fluorescentes , Sobrevivência de Enxerto , Artéria Hepática , Ilhotas Pancreáticas/ultraestrutura , Fígado/química , Pulmão/química , Pulmão/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Veia Porta/citologia , Veia Porta/fisiologia , Distribuição Tecidual , Transplante Homólogo/fisiologiaRESUMO
The growth of both tumors and nonneoplastic tissues may be influenced by signals from the vascular endothelium. In the present investigation we show that purified proliferating endothelial cells from pancreatic islets can stimulate beta-cell proliferation through secretion of hepatocyte growth factor (HGF). This secretion could be induced by soluble signals from the islets, such as vascular endothelial growth factor-A (VEGF-A) and insulin. During pregnancy, the pancreatic beta-cells display a highly reproducible physiological proliferation. We show that islet endothelial cell proliferation precedes beta-cell proliferation in pregnant animals. Vascular growth was closely associated with endocrine cell proliferation, and prominent expression of HGF was observed in islet endothelium on d 15 of pregnancy, i.e. coinciding with the peak of beta-cell proliferation. In summary, our results suggest the existence of an endothelial-endocrine axis within adult pancreatic islets, which is of importance for adult beta-cell proliferation.
Assuntos
Células Endoteliais/citologia , Endotélio Vascular/citologia , Regulação da Expressão Gênica no Desenvolvimento , Ilhotas Pancreáticas/citologia , Animais , Proliferação de Células , Células Cultivadas , Meios de Cultura/metabolismo , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Microscopia de Fluorescência , Modelos Biológicos , Gravidez , Prenhez , Prolactina/metabolismo , Ratos , Ratos Wistar , Ovinos , Transdução de Sinais , Trombospondina 1/biossíntese , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
For their survival and optimal function, pancreatic islets depend posttransplantation on a rapid and adequate revascularization. Native islets display a marked size-dependent heterogeneity in both angioarchitecture and degree of blood perfusion. This study evaluated whether there also are differences in the degree of revascularization of islets of different size when transplanted. Mouse pancreatic islets were isolated by collagenase digestion, and cultured in vitro for 4-7 days before transplantation. Groups of 200 islets with a diameter either exceeding or being below 100 microm were implanted beneath the left renal capsule of syngeneic C57 BL/6 mice. One month posttransplantation, graft-bearing kidneys were removed. Histological specimens were prepared and stained for endothelium with the lectin Bandeiraea simplicifolia. Pancreata from nontransplanted control animals were prepared similarly. The vascular density in transplanted islets was markedly lower than in native islets. However, islet transplants composed of small islets (<100 microm in diameter) had a vascular density in the endocrine tissue twice that in transplants of larger islets (>100 microm). The connective tissue stroma surrounding smaller islets was also more revascularized than in corresponding grafts with large islets. The vascular density in the connective tissue stroma surrounding the individual islets in the grafts was markedly higher than in the endocrine parts per se. These combined observations indicate that smaller islets have a higher capacity to stimulate regrowth of blood vessels following transplantation. Further studies on islet differences with regard to revascularization capacity may teach us strategies for treatment of transplanted islets to improve their revascularization.
Assuntos
Transplante das Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/patologia , Neovascularização Fisiológica/fisiologia , Animais , Biometria , Sobrevivência Celular/fisiologia , Células Cultivadas , Endotélio Vascular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/fisiologiaRESUMO
An adequate revascularization is crucial for islet survival and function after transplantation. Previous studies have suggested that islet revascularization is concluded within 14 days after transplantation. We investigated if the vascular density of transplanted islets and endogenous pancreatic islets differs. Cultured islets were syngeneically transplanted into the kidney, liver, or spleen of C57BL/6 mice. One month later, the graft-bearing organ was removed, and histological specimens were prepared and stained for endothelium with the lectin Bandeiraea simplicifolia. Pancreata from nontransplanted control animals were prepared similarly. Uniform staining of endothelium within the grafts and endogenous islets was obtained. The vascular density was markedly decreased in transplanted islets at all implantation sites, but preferentially in islets implanted into the spleen. The vascular density in the connective tissue surrounding the transplanted islets was very high compared with that of graft intra-islet capillaries. A much lower vascular density was detected in connective tissue surrounding implanted microspheres of a size similar to the islets, which suggests that the islets per se induced blood vessel formation in their vicinity. We conclude that the vascular density in revascularized transplanted islets is markedly decreased compared with endogenous islets. This has potential implications for islet graft metabolism and function.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/irrigação sanguínea , Neovascularização Fisiológica/fisiologia , Lectinas de Plantas , Animais , Tecido Conjuntivo/irrigação sanguínea , Sobrevivência de Enxerto/fisiologia , Ilhotas Pancreáticas/citologia , Rim/cirurgia , Lectinas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/cirurgiaRESUMO
A drawback in pancreatic islet transplantation is the large number of islets needed to obtain insulin independence in patients with diabetes. This most likely reflects extensive posttransplantation islet cell death and functional impairment of the remaining endocrine cells. We aimed to develop an experimental method to retrieve transplanted islets from the mouse liver, which would enable comparisons of transplanted and endogenous islets and provide valuable information on functional changes induced by intraportal transplantation. Transplanted islets were obtained by retrograde perfusion of the liver with collagenase. The identity of retrieved tissue as transplanted islets was confirmed by intravital staining, immunohistochemistry, and electron microscopy. The retrieved islets, irrespective of whether they had resided in diabetic or nondiabetic recipients, had a markedly lower insulin content and glucose-stimulated insulin release when compared with isolated endogenous islets. The glucose oxidation rate was also markedly lower in the retrieved islets, suggesting mitochondrial dysfunction. These disturbances in insulin content, insulin release, and glucose oxidation rate were not reversed by a few days of culture after retrieval. The results implicate changes in islet function after intraportal transplantation. Such dysfunction may contribute to the high number of islets needed for successful transplantation in diabetic individuals.
Assuntos
Diabetes Mellitus Experimental/cirurgia , Transplante das Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/patologia , Fígado/patologia , Coleta de Tecidos e Órgãos/métodos , Animais , Separação Celular , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/ultraestrutura , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Camundongos Endogâmicos C57BL , Perfusão , Teofilina/farmacologia , Transplante IsogênicoRESUMO
Pancreatic islet function and glucose homeostasis have been characterized in the transgenic YC-3.0 mouse, which expresses the yellow chameleon 3.0 (YC-3.0) protein under the control of the beta-actin and the cytomegalovirus promoters. Fluorescence from the enhanced yellow fluorescent protein (EYFP), one part of the yellow chameleon protein, was used as a reporter of transgene expression. EYFP was expressed in different quantities throughout most cell types, including islet endocrine and stromal cells. No adverse effects of the transgene on animal health, growth or fertility were observed. Likewise, in vivo glucose homeostasis, mean arterial blood pressure and regional blood flow values were normal. Furthermore, the transgenic YC-3.0 mouse had a normal beta-cell volume and mass as well as glucose-stimulated insulin release in vitro, compared with the C57BL/6 control mouse. Isolated islets from YC-3.0 animals continuously expressed the transgene and reversed hyperglycemia when transplanted under the renal capsule of alloxan-diabetic nude mice. We conclude that isolated pancreatic islets from YC-3.0 animals implanted into recipients without any EYFP expression, constitute a novel and versatile model for studies of islet engraftment.
Assuntos
Proteínas de Bactérias/genética , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Proteínas Luminescentes/genética , Animais , Glicemia/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/cirurgia , Corantes Fluorescentes/metabolismo , Expressão Gênica , Genes Reporter , Camundongos , Camundongos Nus , Camundongos Transgênicos , Modelos Animais , TransgenesRESUMO
The blood vessels of the pancreatic islets are of crucial importance for oxygen and metabolite supply, and dispersal of secreted hormones. In addition to this, endothelial cells have an important role in the revascularization process after islet transplantation. Studies have reported signs of poor engraftment of transplanted islets, presumably due to impaired revascularization. The aims of this study were to investigate islet endothelial cells and the revascularization process of transplanted islets. The lectin Bandeiraea simplicifolia was found to consistently stain endothelium of both endogenous and transplanted pancreatic islets. By using this marker, we investigated the vascular density of both endogenous and transplanted islets of C57BL/6 mice. One month post-transplantation, a time point when the implants are assumed to be completely revascularized, the graft vascular density was decreased at all investigated implantation sites when compared to endogenous islets. Furthermore, most of the blood vessels were located in the graft connective tissue stroma. Similar results were obtained six months post-transplantation and in cured diabetic animals after one month. In order to evaluate the function of intraportally transplanted islets, we developed a method to retrieve such islets. Enzymatic and mechanic treatment of the liver enabled us to re-isolate the transplanted islets for further in vitro studies. These islets had decreased insulin release, insulin content and glucose oxidation rate when compared to non-transplanted control islets. To understand the role of islet endothelium in the revascularization of transplanted islets we performed angiogenesis microarray studies on islet endothelial cells, from non-cultured, cultured and transplanted islets. We found that the islet endothelium expressed mRNA for both inhibitors and inducers of angiogenesis, and that this expression differed with time. In conclusion, these results provide a useful platform for further studies on the islet endothelium.
Assuntos
Endotélio Vascular/citologia , Ilhotas Pancreáticas/irrigação sanguínea , Animais , Ilhotas Pancreáticas/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Pancreatic islets are avascular immediately after transplantation. Although the islets are rapidly revascularized, it is uncertain whether the revascularization produces an adequate oxygenation of the transplanted islet tissue. We measured pO(2), blood flow and vascular density in mouse or human islets 1 month after transplantation to nude mice. Tissue pO(2) was measured with Clark microelectrodes. Blood perfusion was measured with laser-Doppler flow cytometry, whereas vascular density was determined in histological specimens stained for the lectin Bandeiraea simplicifolia (BS-1). Both the transplanted mouse and human islets had a pO(2) 15-20% of that in endogenous mouse islets. Moreover, the vascular density of the transplanted islets was decreased compared with that of endogenous mouse and human islets. Graft blood perfusion was approximately 50% of renal cortex blood flow. A negative correlation was found between donor age and blood perfusion of the human islet grafts. A similar correlation was seen between donor age and the total vascular density of these grafts. In conclusion, transplanted human islets had a markedly decreased vascular density and pO(2) compared with endogenous islets. This has potential implications for clinical islet transplantations, because poor vascular engraftment may significantly increase the number of islets needed to obtain insulin independence.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/irrigação sanguínea , Neovascularização Fisiológica , Adulto , Animais , Glicemia/análise , Pressão Sanguínea , Vasos Sanguíneos/patologia , Peso Corporal , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Oxigênio/metabolismo , Pressão Parcial , Fluxo Sanguíneo RegionalRESUMO
BACKGROUND: Pancreatic islets are avascular immediately after transplantation and depend on revascularization. Recently, the authors found decreased vascular density in mouse islets 1 month after implantation into nondiabetic recipients. This study investigated possible differences in revascularization between islets implanted into nondiabetic and diabetic recipients, and also evaluated changes in vascular density up to 6 months posttransplantation. METHODS: Islets were syngenically transplanted beneath the renal capsule of normoglycemic or alloxan-diabetic C57BL/6 mice. One to 6 months later, the animals were killed and the grafts removed. Histologic slides were prepared and stained with Bandeiraea simplicifolia. RESULTS: The vascular density in all transplanted islets was decreased compared with native islets. There were no differences in the islet graft vascular density between nondiabetic and diabetic animals. No improvement over time occurred. CONCLUSIONS: The vascular density is decreased in islets implanted to cure diabetic recipients. No improvement occurs in transplanted islets after 1 month posttransplantation.
Assuntos
Diabetes Mellitus Experimental/cirurgia , Glucose/metabolismo , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/irrigação sanguínea , Neovascularização Fisiológica , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Período Pós-Operatório , Valores de Referência , Fatores de TempoRESUMO
We have previously recorded a decreased oxygen tension and blood flow in syngeneically transplanted rat pancreatic islets. The present study related measurements of oxygen tension and blood flow to the vascular density in such grafts implanted beneath the renal capsule. We also evaluated whether transplanted fetal islets are better revascularized than adult islets, and if the degree of revascularization is directly related to the islet vascular endothelial growth factor (VEGF) production. Tissue pO2 was measured using Clark microelectrodes, whereas islet graft blood flow was measured with laser-Doppler flowmetry. The vascular density of endogenous and transplanted islets was quantified in histological specimens stained with the lectin Bandeiraea simplicifolia (BS-1). Tissue pO2 in the transplanted adult and fetal islet grafts was similar and markedly lower than in the endogenous islets. The blood perfusion of both the adult and fetal islet grafts was 60-65% of that in the renal cortex. Administration of D-glucose did not affect tissue pO2 in either the endogenous or transplanted islets, nor graft blood perfusion. The number of capillaries found in the transplanted adult and fetal islets was similar and markedly lower than in endogenous islets. However, in the connective tissue stroma, which constituted approximately 20% of all islet grafts, the vascular density was higher than in the corresponding endocrine parts of these grafts. Incubated adult islets released higher amounts of VEGF than fetal islets. In conclusion, the previously described low oxygen tension of syngeneically transplanted adult rat islets is related to a low vascular density. Similar low oxygen tension and vascular density are seen in grafted fetal islets. The amount of VEGF production does not correlate to the degree of revascularization of the grafts.
Assuntos
Transplante de Tecido Fetal , Sobrevivência de Enxerto/fisiologia , Transplante das Ilhotas Pancreáticas , Rim/irrigação sanguínea , Oxigênio/sangue , Fatores Etários , Animais , Glicemia , Pressão Sanguínea , Fatores de Crescimento Endotelial/biossíntese , Feminino , Hematócrito , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Rim/cirurgia , Linfocinas/biossíntese , Masculino , Neovascularização Fisiológica , Gravidez , Ratos , Ratos Endogâmicos WF , Circulação Renal , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Despite recent advances in clinical islet transplantation, a surprisingly large number of islets (approximately 1 million) are still required to obtain insulin independence in type 1 diabetes. The reasons for this are obscure and likely multifactorial. One explanation may be disturbances in engraftment of the transplanted islets, i.e. the adaptation of the islet transplant to its new surroundings with regard to e.g. revascularization and blood perfusion. Endogenous islets have a dense glomerular-like angioarchitecture. Transplantation of isolated islets causes a disruption of their vascular connections, making the islets dependent on the formation of new blood vessels for optimal function. Evidence from experimental islet transplantation indicates an insufficient revascularization of transplanted islets with subsequent chronically decreased blood perfusion and oxygen tension, which has metabolic consequences within the tissue.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/irrigação sanguínea , Neovascularização Patológica/etiologia , Neovascularização Fisiológica/fisiologia , Animais , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 1/cirurgia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante das Ilhotas Pancreáticas/fisiologia , Camundongos , Microcirculação/metabolismo , Microcirculação/patologia , Microcirculação/fisiopatologiaRESUMO
The detection of genetic abnormalities in paraffin sections by fluorescence in situ hybridization (FISH) is widely used in clinical practice to detect amplification of the ERB2 gene in breast carcinoma and various chromosomal translocations in lymphomas and soft tissue tumors. However, interpretation of FISH signals in tissue sections may be difficult due to overlapping nuclei and nuclear truncation artifacts. Some of these shortcomings may be avoided by the use of isolated nuclear preparations. However, identification of cell populations may be difficult in detached cells removed from their histological context. We have described an optimized immunoFISH technique on isolated nuclear suspension, which combines the benefits of studying isolated cells derived from paraffin embedded tissues by FISH analysis with the ability to detect cell lineage and other markers by immunofluorescence.
Assuntos
Núcleo Celular/metabolismo , Hibridização in Situ Fluorescente/métodos , Inclusão em Parafina/métodos , Animais , Biópsia , Separação Celular , ImunofluorescênciaRESUMO
c-Myc (Myc) is a mediator of glucotoxicity but could also independently compromise ß-cell survival and function. We have shown that after Myc activation in adult ß-cells in vivo, apoptosis is preceded by hyperglycemia, suggesting glucotoxicity might contribute to Myc-induced apoptosis. To address this question conditional Myc was activated in ß-cells of adult pIns-c-MycER(TAM) mice in vivo in the presence or absence of various glucose-lowering treatments, including exogenous insulin and prior to transplantation with wild-type islets. Changes in blood glucose levels were subsequently correlated with changes in ß-cell mass and markers of function/differentiation. Activation of c-Myc resulted in reduced insulin secretion, hyperglycemia and loss of ß-cell differentiation, followed by reduction in mass. Glucose-lowering interventions did not prevent loss of ß-cells. Therefore, Myc can cause diabetes by direct effects on ß-cell apoptosis even in the absence of potentially confounding secondary hyperglycemia. Moreover, as loss of ß-cell differentiation/function and hyperglycemia are not prevented by preventing ß-cell apoptosis, we conclude that Myc might contribute to the pathogenesis of diabetes by directly coupling cell cycle entry and ß-cell failure through two distinct pathways.
Assuntos
Genes myc/fisiologia , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Células Secretoras de Insulina/citologia , Insulina/metabolismo , Animais , Contagem de Células , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Feminino , Hiperglicemia/genética , Hiperglicemia/fisiopatologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Tamanho do Órgão/genética , Pâncreas/metabolismo , Pâncreas/patologia , Transgenes/fisiologia , Regulação para CimaRESUMO
Double immunoenzymatic labeling of 2 different molecules in tissue sections is a widely used technique. However, it is time consuming since the 2 immunoenzymatic procedures are carried out in sequence, and they must also be optimally performed to avoid unwanted background labeling. In this paper, we report that double immunoenzymatic staining performed using automated immunostaining apparatus considerably reduces the requirements in terms of time and is also highly reproducible and free of background. Three tissue markers can also be visualized by performing (after immunoperoxidase labeling) 2 sequential immuno-alkaline phosphatase procedures using different substrates. Furthermore, single or double detection of mRNA by in situ hybridization can be combined with immunoenzymatic labeling. Finally, automated labeling could also be performed on peripheral blood and bone marrow smears, opening the possibility of using this procedure in the analysis of hematologic/cytology samples.
Assuntos
Biomarcadores/metabolismo , Técnicas Imunoenzimáticas/métodos , RNA Mensageiro/metabolismo , Coloração e Rotulagem , Análise Serial de Tecidos , Anticorpos , Humanos , Técnicas Imunoenzimáticas/instrumentação , Imuno-Histoquímica , Hibridização In Situ , Leucócitos/química , Leucócitos/citologia , Leucócitos/imunologia , Linfonodos/química , Linfonodos/citologia , Linfonodos/imunologia , Tonsila Palatina/química , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Inclusão em Parafina , Coloração e Rotulagem/instrumentação , Análise Serial de Tecidos/instrumentação , Análise Serial de Tecidos/métodosRESUMO
OBJECTIVE: Clinical islet transplantations are performed through infusion of islets via the portal vein into the liver. This study aimed at characterizing the influence of the implantation microenvironment on islet graft metabolism and function. RESEARCH DESIGN AND METHODS: Islets were transplanted into their normal environment, i.e., the pancreas, or intraportally into the liver of mice. One month posttransplantation, the transplanted islets were retrieved and investigated for changes in function and gene expression. RESULTS: Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis, and glucose oxidation rate were markedly decreased in islets retrieved from the liver, both when compared with islets transplanted into the pancreas and endogenous islets. Islets transplanted into the pancreas showed normal insulin content, (pro)insulin biosynthesis, and glucose oxidation rate but increased basal insulin secretion and impaired glucose stimulation index. Gene expression data for retrieved islets showed downregulation of pancreatic and duodenal homeobox gene-1, GLUT-2, glucokinase, mitochondrial glycerol-phosphate dehydrogenase, and pyruvate carboxylase, preferentially in intraportally transplanted islets. CONCLUSIONS: Islets transplanted into their normal microenvironment, i.e., the pancreas, display gene expression changes when compared with endogenous islets but only moderate changes in metabolic functions. In contrast, site-specific properties of the liver markedly impaired the metabolic functions of intraportally transplanted islets.
Assuntos
Transplante das Ilhotas Pancreáticas/patologia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Genes Reporter , Glucose/farmacologia , Glucose/fisiologia , Insulina/metabolismo , Insulina/fisiologia , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Transplante das Ilhotas Pancreáticas/métodos , Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Transplante HeterotópicoRESUMO
BACKGROUND/AIMS: An important reason for the large amount of islets required for successful islet transplantation is likely to be inadequate engraftment of the transplanted islets. Thus, the revascularization is of major importance for graft survival. In order to study the expression of angiogenic peptides and receptors on islet endothelial cells (EC), we needed methods giving access to such endothelium. Therefore, we developed methods to isolate EC from islets transplanted intraportally or beneath the kidney capsule. METHODS: Pancreatic islets were isolated, cultured and syngeneically transplanted into the liver or beneath the kidney capsule of C57BL/6 mice. One month post-transplantation, the islets were retrieved and EC from these islets were explanted. EC were also collected from freshly isolated and cultured non-transplanted islets. The EC were purified with Dynabeads and identified with immunocytochemistry. Angiogenesis GEArray technology was used to study angiogenic gene expression. RESULTS: Several angiogenic genes were expressed in EC; e.g. endostatin, pigment-epithelial derived factor, vascular endothelial growth factor and angiopoietin-2, and their expression were affected by culture. CONCLUSION: The expression of angiogenesis-related genes in islet EC from non-transplanted islets is affected by culture. Moreover, we also describe a technique, which makes it possible to obtain EC from transplanted islets.