RESUMO
The key sites within the gastrointestinal (GI) tract where T cells mediate effector responses and the impact of these responses on intestinal stem cells (ISCs) remain unclear. Using experimental bone marrow transplantation to model immune-mediated GI damage and 3D imaging to analyze T cell localization, we found that the ISC compartment is the primary intestinal site targeted by T cells after transplantation. Recruitment to the crypt base region resulted in direct T cell engagement with the stem cell compartment and loss of crypt base columnar ISCs, which expressed both MHC classes I and II. Vasculature expressing the adhesion molecule MAdCAM-1 clustered near the crypt base, preferentially regulating crypt compartment invasion and ISC reduction without affecting T cell migration to villi. These findings indicate that allogeneic T cells rapidly access the stem cell niche after transplantation, and this targeted recruitment to the stem cell compartment results in ISC loss during immune-mediated GI damage.
Assuntos
Células-Tronco Adultas/imunologia , Transplante de Medula Óssea , Mucosa Intestinal/imunologia , Nicho de Células-Tronco/imunologia , Linfócitos T/imunologia , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Citotoxicidade Imunológica , Feminino , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Modelos Animais , Mucoproteínas , Transplante HomólogoRESUMO
Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.
Assuntos
Células Epiteliais/citologia , Interleucinas/imunologia , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Regeneração , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Doença Enxerto-Hospedeiro/patologia , Humanos , Imunidade nas Mucosas , Interleucinas/deficiência , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Camundongos , Organoides/citologia , Organoides/crescimento & desenvolvimento , Organoides/imunologia , Celulas de Paneth/citologia , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Nicho de Células-Tronco , Interleucina 22RESUMO
Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a ubiquitously expressed transcription factor that is well known for its role in regulating the cellular redox pathway. Although there is mounting evidence suggesting a critical role for Nrf2 in hematopoietic stem cells and innate leukocytes, little is known about its involvement in T-cell biology. In this study, we identified a novel role for Nrf2 in regulating alloreactive T-cell function during allogeneic hematopoietic cell transplantation (allo-HCT). We observed increased expression and nuclear translocation of Nrf2 upon T-cell activation in vitro, especially in CD4+ donor T cells after allo-HCT. Allo-HCT recipients of Nrf2 -/- donor T cells had significantly less acute graft-versus-host disease (GVHD)-induced mortality, morbidity, and pathology. This reduction in GVHD was associated with the persistence of Helios+ donor regulatory T cells in the allograft, as well as defective upregulation of the gut-homing receptor LPAM-1 on alloreactive CD8+ T cells. Additionally, Nrf2 -/- donor CD8+ T cells demonstrated intact cytotoxicity against allogeneic target cells. Tumor-bearing allo-HCT recipients of Nrf2 -/- donor T cells had overall improved survival as a result of preserved graft-versus-tumor activity and reduced GVHD activity. Our findings characterized a previously unrecognized role for Nrf2 in T-cell function, as well as revealed a novel therapeutic target to improve the outcomes of allo-HCT.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Ativação Linfocitária , Fator 2 Relacionado a NF-E2/imunologia , Neoplasias Experimentais/imunologia , Doença Aguda , Aloenxertos , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapiaRESUMO
According to current dogma, there is little or no ongoing neurogenesis in the fully developed adult enteric nervous system. This lack of neurogenesis leaves unanswered the question of how enteric neuronal populations are maintained in adult guts, given previous reports of ongoing neuronal death. Here, we confirm that despite ongoing neuronal cell loss because of apoptosis in the myenteric ganglia of the adult small intestine, total myenteric neuronal numbers remain constant. This observed neuronal homeostasis is maintained by new neurons formed in vivo from dividing precursor cells that are located within myenteric ganglia and express both Nestin and p75NTR, but not the pan-glial marker Sox10. Mutation of the phosphatase and tensin homolog gene in this pool of adult precursors leads to an increase in enteric neuronal number, resulting in ganglioneuromatosis, modeling the corresponding disorder in humans. Taken together, our results show significant turnover and neurogenesis of adult enteric neurons and provide a paradigm for understanding the enteric nervous system in health and disease.
Assuntos
Apoptose , Sistema Nervoso Entérico/metabolismo , Nestina/metabolismo , Neurogênese , Receptores de Fator de Crescimento Neural/metabolismo , Fatores de Transcrição SOXE/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Nestina/genética , Receptores de Fator de Crescimento Neural/genética , Fatores de Transcrição SOXE/genéticaRESUMO
BACKGROUND & AIMS: As in other tumor types, progression of pancreatic cancer may require a functionally unique population of cancer stem cells. Although such cells have been identified in many invasive cancers, it is not clear whether they emerge during early or late stages of tumorigenesis. Using mouse models and human pancreatic cancer cell lines, we investigated whether preinvasive pancreatic neoplasia contains a subpopulation of cells with distinct morphologies and cancer stem cell-like properties. METHODS: Pancreatic tissue samples were collected from the KC(Pdx1), KPC(Pdx1), and KC(iMist1) mouse models of pancreatic intraepithelial neoplasia (PanIN) and analyzed by confocal and electron microscopy, lineage tracing, and fluorescence-activated cell sorting. Subpopulations of human pancreatic ductal adenocarcinoma (PDAC) cells were similarly analyzed and also used in complementary DNA microarray analyses. RESULTS: The microtubule regulator DCLK1 marked a morphologically distinct and functionally unique population of pancreatic cancer-initiating cells. These cells displayed morphological and molecular features of gastrointestinal tuft cells. Cells that expressed DCLK1 also expressed high levels of ATAT1, HES1, HEY1, IGF1R, and ABL1, and manipulation of these pathways in PDAC cell lines inhibited their clonogenic potential. Pharmacological inhibition of γ-secretase activity reduced the abundance of these cells in murine PanIN in a manner that correlated with inhibition of PanIN progression. CONCLUSIONS: Human PDAC cells and pancreatic neoplasms in mice contain morphologically and functionally distinct subpopulations that have cancer stem cell-like properties. These populations can be identified at the earliest stages of pancreatic tumorigenesis and provide new cellular and molecular targets for pancreatic cancer treatment and/or chemoprevention.
Assuntos
Carcinoma in Situ/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/metabolismo , Animais , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica , Modelos Animais de Doenças , Quinases Semelhantes a Duplacortina , Citometria de Fluxo , Humanos , Camundongos , Microscopia Eletrônica , Invasividade Neoplásica , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Células-Tronco/patologiaRESUMO
Synchronization of preimplantation embryo development to blastocysts is one of the prerequisites for normal embryo implantation. While previous studies have ascribed an adverse effect to aberrant opioid signaling on embryo and fetal development, it has remained unclear whether the opioid system is operative in early pregnancy events. In the present study, employing multiple pharmacological and genetic approaches, we demonstrated that preimplantation embryos spanning the zygote to blastocyst express the opioid receptor subtypes and the oviduct expresses endogenous opioid precursors dynamically, which suggest that opioid signaling is functionally operative during preimplantation embryo development. Subsequent analysis further revealed that an aberrantly activated opioid signaling by morphine can remarkably derail normal preimplantation embryo development via inhibiting intracellular calcium mobilization, while a cotreatment of naloxone with morphine can remarkably reverse the adverse effects of morphine on preimplantation embryo development. Besides shedding light on the pathophysiological significance of the opioid system during early embryo development in mice, our findings have potential clinical relevance because an abused use of illicit opiate drugs is frequently associated with retarded fetal development and pregnancy failure in women.
Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Mórula/metabolismo , Receptores Opioides/metabolismo , Transdução de Sinais/fisiologia , Animais , Blastocisto/efeitos dos fármacos , Cálcio/metabolismo , Implantação do Embrião/efeitos dos fármacos , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Camundongos , Morfina/farmacologia , Mórula/efeitos dos fármacos , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Entorpecentes/farmacologia , Gravidez , Transdução de Sinais/efeitos dos fármacosRESUMO
Precise measurement of the binding activity changes of therapeutic antibodies is important to determine the potential critical quality attributes (CQAs) in developability assessment at the early stage of antibody development. Here, we report a surface plasmon resonance (SPR)-based relative binding activity method, which incorporates both binding affinity and binding response and allows us to determine relative binding activity of antibodies with high accuracy and precision. We applied the SPR-based relative binding activity method in multiple forced degradation studies of antibody developability assessment. The current developability assessment strategy provided comprehensive, precise characterization of antibody binding activity in the stability studies, enabling us to perform correlation analysis and establish the structure-function relationship between relative binding activity and quality attributes. The impact of a given quality attribute on binding activity could be confidently determined without isolating antibody variants. We identified several potential CQAs, including Asp isomerization, Asn deamidation, and fragmentation. Some potential CQAs affected binding affinity of antibody and resulted in a reduction of binding activity. Certain potential CQAs impaired antibody binding to antigen and led to a loss of binding activity. A few potential CQAs could influence both binding affinity and binding response and cause a substantial decrease in antibody binding activity. Specifically, we identified low abundance Asn33 deamidation in the light chain complementarity-determining region as a potential CQA, in which all the stressed antibody samples showed Asn33 deamidation abundances ranging from 4.2% to 27.5% and a mild binding affinity change from 1.76 nM to 2.16 nM.
Assuntos
Anticorpos Monoclonais , Ressonância de Plasmônio de Superfície , Ressonância de Plasmônio de Superfície/métodos , Humanos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Ligação Proteica , AnimaisRESUMO
Corticosteroid treatment (CST) failure is associated with poor outcomes for patients with gastrointestinal (GI) graft-versus-host disease (GVHD). CST is intended to target the immune system, but the glucocorticoid receptor (GR) is widely expressed, including within the intestines, where its effects are poorly understood. Here, we report that corticosteroids (CS) directly targeted intestinal epithelium, potentially worsening immune-mediated GI damage. CS administered to mice in vivo and intestinal organoid cultures ex vivo reduced epithelial proliferation. Following irradiation, immediate CST mitigated GI damage but delayed treatment attenuated regeneration and exacerbated damage. In a murine steroid-refractory (SR) GVHD model, CST impaired epithelial regeneration, worsened crypt loss, and reduced intestinal stem cell (ISC) frequencies. CST also exacerbated immune-mediated damage in organoid cultures with SR, GR-deficient T cells or IFN-γ. These findings correlated with CS-dependent changes in apoptosis-related gene expression and STAT3-related epithelial proliferation. Conversely, IL-22 administration enhanced STAT3 activity and overcame CS-mediated attenuation of regeneration, reducing crypt loss and promoting ISC expansion in steroid-treated mice with GVHD. Therefore, CST has the potential to exacerbate GI damage if it fails to control the damage-inducing immune response, but this risk may be countered by strategies augmenting epithelial regeneration, thus providing a rationale for clinical approaches combining such tissue-targeted therapies with immunosuppression.
Assuntos
Doença Enxerto-Hospedeiro , Intestinos , Humanos , Camundongos , Animais , Mucosa Intestinal/metabolismo , Corticosteroides , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/metabolismo , Esteroides/metabolismo , Regeneração/efeitos da radiaçãoRESUMO
AIMS/HYPOTHESIS: Islet Schwann (glial) cells and pericytes are the microorgan's accessory cells positioned at the external and internal boundaries facing the exocrine pancreas and endothelium, respectively, adjacent to the endocrine cells. Plasticity of glial cells and pericytes is shown in the glial scar formation after injury to the central nervous system. It remains unclear whether similar reactive cellular responses occur in insulitis. We applied three-dimensional (3D) histology to perform qualitative and quantitative analyses of the islet Schwann cell network and pericytes in normal, streptozotocin-injected (positive control of gliosis) and NOD mouse models. METHODS: Vessel painting paired with immunostaining of mouse pancreatic tissue was used to reveal the islet Schwann cells and pericytes and their association with vasculature. Transparent islet specimens were prepared by optical clearing to facilitate 3D confocal microscopy for panoramic visualisation of the tissue networks. RESULTS: In-depth microscopy showed that the islet Schwann cell network extends from the peri-islet domain into the core. One week after streptozotocin injection, we observed intra-islet perivascular gliosis and an increase in pericyte density. In early/moderate insulitis in the NOD mice, perilesional gliosis occurred at the front of the lymphocytic infiltration with atypical islet Schwann cell morphologies, including excessive branching and perivascular gliosis. Meanwhile, pericytes aggregated on the walls of the feeding arteriole at the peri- and intralesional domains with a marked increase in surface marker density. CONCLUSIONS/INTERPRETATION: The reactive cellular responses demonstrate plasticity and suggest a stop-gap mechanism consisting of the Schwann cells and pericytes in association with the islet lesion and vasculature when injury occurs.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Pericitos/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pericitos/patologia , Células de Schwann/patologia , Estreptozocina/toxicidadeRESUMO
Because of the dispersed nature of nerves and blood vessels, standard histology cannot provide a global and associated observation of the enteric nervous system (ENS) and vascular network. We prepared transparent mouse intestine and combined vessel painting and three-dimensional (3-D) neurohistology for joint visualization of the ENS and vasculature. Cardiac perfusion of the fluorescent wheat germ agglutinin (vessel painting) was used to label the ileal blood vessels. The pan-neuronal marker PGP9.5, sympathetic neuronal marker tyrosine hydroxylase (TH), serotonin, and glial markers S100B and GFAP were used as the immunostaining targets of neural tissues. The fluorescently labeled specimens were immersed in the optical clearing solution to improve photon penetration for 3-D confocal microscopy. Notably, we simultaneously revealed the ileal microstructure, vasculature, and innervation with micrometer-level resolution. Four examples are given: 1) the morphology of the TH-labeled sympathetic nerves: sparse in epithelium, perivascular at the submucosa, and intraganglionic at myenteric plexus; 2) distinct patterns of the extrinsic perivascular and intrinsic pericryptic innervation at the submucosal-mucosal interface; 3) different associations of serotonin cells with the mucosal neurovascular elements in the villi and crypts; and 4) the periganglionic capillary network at the myenteric plexus and its contact with glial fibers. Our 3-D imaging approach provides a useful tool to simultaneously reveal the nerves and blood vessels in a space continuum for panoramic illustration and analysis of the neurovascular complex to better understand the intestinal physiology and diseases.
Assuntos
Sistema Nervoso Entérico/anatomia & histologia , Intestinos/irrigação sanguínea , Intestinos/inervação , Animais , Vasos Sanguíneos/anatomia & histologia , Vasos Sanguíneos/inervação , Capilares/anatomia & histologia , Feminino , Corantes Fluorescentes , Proteína Glial Fibrilar Ácida/metabolismo , Imageamento Tridimensional , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Plexo Mientérico/anatomia & histologia , Fibras Nervosas/fisiologia , Fatores de Crescimento Neural/metabolismo , Rede Nervosa/anatomia & histologia , Neuroglia/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Serotonina/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Intestinal stem cells (ISCs) maintain the epithelial lining of the intestines, but mechanisms regulating ISCs and their niche after damage remain poorly understood. Utilizing radiation injury to model intestinal pathology, we report here that the Interleukin-33 (IL-33)/ST2 axis, an immunomodulatory pathway monitored clinically as an intestinal injury biomarker, regulates intrinsic epithelial regeneration by inducing production of epidermal growth factor (EGF). Three-dimensional imaging and lineage-specific RiboTag induction within the stem cell compartment indicated that ISCs expressed IL-33 in response to radiation injury. Neighboring Paneth cells responded to IL-33 by augmenting production of EGF, which promoted ISC recovery and epithelial regeneration. These findings reveal an unknown pathway of niche regulation and crypt regeneration whereby the niche responds dynamically upon injury and the stem cells orchestrate regeneration by regulating their niche. This regenerative circuit also highlights the breadth of IL-33 activity beyond immunomodulation and the therapeutic potential of EGF administration for treatment of intestinal injury.
Assuntos
Interleucina-33 , Lesões por Radiação , Humanos , Fator de Crescimento Epidérmico , Imageamento Tridimensional , ImunomodulaçãoRESUMO
BACKGROUND & AIMS: The intrinsic opacity of mouse intestinal tissue prevents its evaluation by high-resolution, in-depth optical microscopy. Instead, intestinal tissue is usually sectioned to expose the interior domains of the mucosa and submucosa for microscopic examination. However, microtome sectioning can cause distortions and artifacts that prevent acquisition of an accurate view of the sample. We therefore attempted to develop a microtome-free 3-dimensional (3D) confocal imaging method for characterization of mouse intestine. METHODS: We applied an optical-clearing solution, FocusClear, to permeate and reduce the opacity of mouse colon and ileum. Tissues were labeled with fluorescent probes and examined by confocal microscopy with efficient fluorescence excitation and emission in the FocusClear solution. The voxel-based confocal micrographs were processed with Amira software for 3D visualization and analysis. RESULTS: Treatment of tissues with the optical-clearing solution improved photon penetration, resulting in the acquisition of images with subcellular-level resolution across the mucosa, submucosa, and muscle layers. Collectively, the acquired image stacks were processed by projection algorithms for 3D analysis of the spatial relations in villi, crypts, and connective tissues. These imaging technologies allowed for identification of spatiotemporal changes in crypt morphology of colon tissues from mice with dextran sulfate sodium-induced colitis as well as detection of transgenic fluorescent proteins expressed in the colon and ileum. CONCLUSIONS: This new optical method for penetrative imaging of mouse intestine does not require tissue sectioning and provides a useful tool for 3D presentation and analysis of diseased and transgenic intestine in an integrated fashion.
Assuntos
Colite Ulcerativa/patologia , Colo/ultraestrutura , Íleo/ultraestrutura , Interpretação de Imagem Assistida por Computador , Imageamento Tridimensional/métodos , Microscopia Confocal/instrumentação , Animais , Colo/patologia , Modelos Animais de Doenças , Feminino , Tecnologia de Fibra Óptica , Íleo/patologia , Mucosa Intestinal/patologia , Mucosa Intestinal/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Microscopia Confocal/métodos , Probabilidade , Distribuição Aleatória , Valores de Referência , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Prostasin is a glycosylphosphatidylinositol-anchored extracellular serine protease with a role in epidermal growth factor receptor (EGFR) signal modulation. EGFR signaling has been shown to be important for regulating cytotrophoblast (CT) cell proliferation in human placenta. We investigated the impact of prostasin expression regulation on this cellular function as well as the molecular mechanisms involved in human cytotrophoblastic cells. METHODS: An immortalized normal human CT cell line (B6Tert-1) was used as an in vitro cell model. Prostasin expression in B6Tert-1 cells was knocked down by transfection of a short interfering RNA. Lentivirus-mediated expression of recombinant human prostasin under tetracycline regulation was performed to obtain stable B6Tert-1 cell sublines that over-expressed prostasin. Changes in cell proliferation and EGFR signaling were evaluated by immunocytochemistry for Ki67 and western blot analysis, respectively, in B6Tert-1 cells with knocked-down or increased prostasin expression. RESULTS: Prostasin knock-down in B6Tert-1 cells resulted in inhibition of cell proliferation, in association with down-regulated EGFR protein expression (both P < 0.05 versus control) as well as reduced phosphorylation of c-raf, mitogen-activated protein kinase (MAPK) kinases (MEK1/2) and extracellular signal-regulated kinases (Erk1/2) (all P < 0.05 versus control). Over-expression of prostasin led to up-regulation of the EGFR protein, but had no effect on cell proliferation or phosphorylation of MAPK signaling molecules in the B6Tert-1 cells. CONCLUSIONS: Prostasin may regulate trophoblast cell proliferation via modulating the EGFR-MAPK signaling pathway.
Assuntos
Proliferação de Células/efeitos dos fármacos , Receptores ErbB/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Serina Endopeptidases/fisiologia , Trofoblastos/fisiologia , Linhagem Celular , Feminino , Inativação Gênica , Humanos , GravidezRESUMO
Intestinal microvasculature plays a central role in nutrient absorption and immune response against infections. Microscopic visualization of intestinal microvasculature under normal and pathological conditions such as inflammatory bowel disease is essential for understanding the pathophysiology of the disease. Despite the intensive need to characterize the intestinal microstructure and vasculature in an integrated fashion, 3-dimensional (3D) visualization of the gastrointestinal tissue is often limited by the spatial resolution of the imaging tools. In this research, we aimed to apply optical clearing to minimize the random light scattering in the mouse ileum, thereby facilitating photon penetration for high-resolution, 3D optical microscopy of the tissue network without microtome sectioning. We applied cardiac perfusion of lipophilic dialkylcarbocyanine dye DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) to label the mouse blood vessels, including the intestinal microvasculature. The labeled and paraformaldehyde-fixed ileum was immersed in the aqueous optical-clearing solution to improve photon penetration. Optical clearing revealed the interior domain of the mouse ileal mucosa and submucosa, where random light scattering was suppressed and the size of the microstructure in the fixed specimen remained the same. Using fluorescent labeling, the intestinal microstructure and vasculature were simultaneously imaged by 3D confocal microscopy to allow for an integrated visualization of the tissue network with high definition. This new optical approach provides a useful tool for 3D presentation and analysis of the microvasculature for better understanding the intestinal physiology.
Assuntos
Íleo/irrigação sanguínea , Íleo/ultraestrutura , Interpretação de Imagem Assistida por Computador , Imageamento Tridimensional , Microscopia Confocal , Microvasos/ultraestrutura , Animais , Feminino , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Fosfoglicerato Quinase/genéticaRESUMO
Apolipoprotein M (APOM), a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in plasma, is involved in lipid and lipoprotein metabolism. Through comparative mapping, we have mapped this gene to SSC7 p1.1 in which many QTLs affecting fat deposition traits have been reported. As a candidate gene for fat deposition traits, in this study, we obtained the 742-bp mRNA sequence of porcine APOM including the full coding region and encoding a protein of 188 amino acids. The sequence was deposited into the GenBank under the accession no. DQ329240. Semi-quantitative RT-PCR results showed that the porcine APOM gene is expressed predominantly in liver and kidney tissue. The genomic sequence of this gene which contains six exons and five introns, is 3,621 bp in length (DQ272488). Bioinformatic analysis of the 5' regulatory region has revealed that classical TATA-box element and species conserved Hepatocyte nuclear factor-1a (HNF-1alpha) biding site were represented in this region. A G2289C single nucleotide polymorphism (SNP) in the intron 2 of porcine APOM gene detected as an Eco130I PCR-restriction fragment length polymorphism (PCR-RFLP) showed allele frequency differences among three purebreds. Association of the genotypes with fat deposition traits showed that different genotypes of porcine APOM gene were significantly associated with leaf fat weight (P < 0.05), backfat thickness at shoulder (P < 0.05), backfat thickness at thorax-waist (P < 0.05), backfat thickness at buttock (P < 0.01) and average backfat thickness over shoulder, thorax-waist and buttock (P < 0.01).
Assuntos
Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Sus scrofa/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Composição Corporal/genética , Mapeamento Cromossômico , Biologia Computacional , Primers do DNA/genética , Componentes do Gene , Perfilação da Expressão Gênica , Frequência do Gene , Estudo de Associação Genômica Ampla , Genótipo , Rim/metabolismo , Fígado/metabolismo , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Prostasin is down-regulated during inflammation and in invasive cancers, and plays a role in regulation of inflammatory gene expression and invasion. METHODS: We used the human benign prostatic hyperplasia cell line BPH-1 to investigate gene expression changes associated with siRNA-mediated loss of prostasin expression. Quantitative PCR and/or western blotting were used to evaluate the expression changes of iNOS, ICAM-1, cyclin D1, IL-6, and IL-8. Gene expression changes were also evaluated in the presence of a PAR-2 antagonist. The PC-3 human prostate cancer cell line was used for evaluation of gene expression in response to prostasin re-expression. RESULTS: Prostasin silencing in BPH-1 was associated with up-regulation of iNOS, ICAM-1, IL-6, and IL-8, and down-regulation of cyclin D1; as well as reduced proliferation and invasion. The iNOS up-regulation and cyclin D1 down-regulation associated with prostasin silencing were inhibited by a PAR-2 antagonist. Re-expression of prostasin, a serine active-site mutant, and a GPI-anchor-free mutant, in the PC-3 cells resulted in PAR-2 and cyclin D1 transcription up-regulation. Transcription up-regulation of IL-6 and IL-8 was associated with re-expression of the serine active-site mutant prostasin in the PC-3 cells. Transcription up-regulation of IL-8, but to a lesser extent, was also observed in PC-3 cells expressing the wild-type prostasin. Expression of a serine protease active prostasin, GPI-anchored or anchor-free, prevented the IL-6 induction in response to PAR-2. The GPI-anchor-free prostasin also prevented the IL-8 induction. CONCLUSIONS: Prostasin plays a negative regulatory role on PAR-2-mediated signaling in prostate epithelial cells.
Assuntos
Ciclina D1/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Reação em Cadeia da Polimerase , Próstata/patologia , Hiperplasia Prostática/patologia , RNA Interferente Pequeno/farmacologia , Serina Endopeptidases/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Controlled invasion of the uterine wall by the trophoblast cells is pivotal for the successful pregnancy, and various kinds of protease are involved in this process. Serine protease prostasin has been shown to participate in the proteolytic activation of epithelial sodium channel as well as cleavage of epidermal growth factor receptor extracellular domain in human epithelial cells. Its physiological significance in human placentation has been suggested but not validated. In the present study, we found that prostasin was expressed at a relatively high level in human placenta trophoblasts in early pregnant weeks. In the in vitro cultured human choriocarcinomal JEG-3 cells, treatment with functional antibody against prostasin led to promotion in cell invasion capability, as well as increase in the production of MMP-2, MMP-26, TIMP-1, and TIMP-4. Our data indicated that this serine protease may function as an invasion suppressor in human trophoblast, participating in the invasion-restrictive regulation of trophoblasts to avoid their over-penetration into the uterine wall.
Assuntos
Coriocarcinoma/patologia , Invasividade Neoplásica , Serina Endopeptidases/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinases da Matriz Secretadas/biossíntese , Gravidez , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidores Teciduais de Metaloproteinases/biossíntese , Trofoblastos/citologia , Útero/citologia , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
Expression of prostasin in the PC-3 human prostate carcinoma cells inhibited in vitro invasion, but the molecular mechanisms are unknown. Wild-type human prostasin or a serine active-site mutant prostasin was expressed in the PC-3 cells. Molecular changes were measured at the mRNA and the protein levels. Cell signaling changes were evaluated by measuring phosphorylation of the extracellular signal-regulated kinases (Erk1/2) following epidermal growth factor (EGF) treatment of the cells. Protein expression of the EGF receptor (EGFR) was differentially down-regulated by the wild-type and the active-site mutant prostasin. The mRNA expression of EGFR and the transcription repressor SLUG was reduced in cells expressing wild-type prostasin but not the active-site mutant. Phosphorylation of Erk1/2 in response to EGF was greatly reduced by the wild-type prostasin but not by the active-site mutant. The mRNA expression of the urokinase-type plasminogen activator (uPA), the uPA receptor (uPAR), cyclooxygenase-2 (COX-2), and the inducible nitric oxide synthase (iNOS) was decreased by the wild-type and the active-site mutant prostasin. The mRNA or protein expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), matriptase, and E-cadherin was greatly increased by the active-site mutant prostasin. In conclusion, prostasin expression elicits both protease-dependent and independent molecular changes in the PC-3 cells.
Assuntos
Linhagem Celular Tumoral , Neoplasias da Próstata/metabolismo , Serina Endopeptidases/metabolismo , Sítios de Ligação , Caderinas/genética , Caderinas/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Serina Endopeptidases/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Nerves are a notable feature of the tumor microenvironment in some epithelial tumors, but their role in the malignant progression of pancreatic ductal adenocarcinoma (PDAC) is uncertain. Here, we identify dense innervation in the microenvironment of precancerous pancreatic lesions, known as pancreatic intraepithelial neoplasms (PanIN), and describe a unique subpopulation of neuroendocrine PanIN cells that express the neuropeptide substance P (SP) receptor neurokinin 1-R (NK1-R). Using organoid culture, we demonstrated that sensory neurons promoted the proliferation of PanIN organoids via SP-NK1-R signaling and STAT3 activation. Nerve-responsive neuroendocrine cells exerted trophic influences and potentiated global PanIN organoid growth. Sensory denervation of a genetically engineered mouse model of PDAC led to loss of STAT3 activation, a decrease in the neoplastic neuroendocrine cell population, and impaired PanIN progression to tumor. Overall, our data provide evidence that nerves of the PanIN microenvironment promote oncogenesis, likely via direct signaling to neoplastic neuroendocrine cells capable of trophic influences. These findings identify neuroepithelial cross-talk as a potential novel target in PDAC treatment. Cancer Res; 77(8); 1868-79. ©2017 AACR.