RESUMEN
Monoclonal antibodies have been applied in a wide range of biological and medical studies since the advent of cell fusion technology. Although cell fusion techniques have been improved by using myelomas and reagents, researchers still find it difficult to produce monoclonal antibodies because of the long protocols, high costs, and low efficiency of obtaining hybridomas. To solve these problems, we first developed an iliac lymph node method in 1995 using rats. In this method, an antigen emulsion is injected intramuscularly into the tail base, and then B lymphocytes are isolated from the enlarged iliac lymph nodes. This method is approximately 10 times more productive than the conventional spleen method. Here, we present further improvements to the iliac lymph node method to render it easily applicable in both mice and rats. We found that the frequency of hybridomas secreting specific antibodies was over five times higher using the electro cell fusion method than using the polyethylene glycol (PEG) fusion method. This frequency using the iliac lymph node method with electro cell fusion is at least 50 times higher than that using the traditional spleen method, thereby leading to the reduction in the number of mice or rats to be sacrificed. In addition, only a single injection for immunization is necessary for the iliac lymph node method, opposed to three for the spleen method. Therefore, this method is rapid, inexpensive, and ethical for producing monoclonal antibodies.
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Anticuerpos Monoclonales , Antígenos , Animales , Fusión Celular , Hibridomas , Ganglios Linfáticos , Ratones , RatasRESUMEN
Collagenofibrotic glomerulopathy or LMX1B-associated nephropathy is a rare disease in which type III collagen accumulates in the glomeruli. We herein report a 64-year-old Japanese woman with an elevated serum creatinine level and persistent proteinuria for 7 years. An electron microscopic study using tannic acid showed curved and frayed collagen fibers within mesangial and subendothelial regions compatible with type III collagen depositions. The distribution of type IV collagen α1-6 chains was normal. Since no pathogenic mutations were identified in the LMX1B gene, she was diagnosed with collagenofibrotic glomerulopathy and treated with angiotensin II receptor blocker and calcium antagonist to control her blood pressure.
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Enfermedades Renales , Glomérulos Renales , Colágeno Tipo III , Femenino , Mesangio Glomerular , Humanos , Persona de Mediana Edad , ProteinuriaRESUMEN
We used suncus (Suncus murinus; house musk shrew) to generate partner cells for cell fusion to produce suncus monoclonal antibodies. Suncus are insectivores that are genetically distant to rodents, and recognize antigens and epitopes that are not immunogenic in mice and rats, which are the animals most commonly used in basic life science research and from which monoclonal antibodies are usually produced. To date, monoclonal antibodies from suncus have not been generated due to the lack of a plasmacytoma fusion partner. To obtain suncus plasmacytoma cell lines suitable as a cell fusion partner, we injected suncus at both sides of the tail base with antigen emulsion, collected the lymph nodes and spleens, and cultured the cells to obtain immortalized lymphoid cell lines visually resembling mouse SP2/0-Ag14 myeloma cells. Three suncus immunized with the antigen provided 4 cell lines of suncus plasmacytoma, but they did not secrete immunoglobulins. Antibody-producing hybrid cells were generated from these cell lines using a cell fusion technique. Using one of the cell lines as a fusion partner, we obtained six lines of immunoglobulin-producing hybrid cells which secreted an unidentified monoclonal IgG. When these 6 lines were used as new fusion partners, we obtained several hybrid cell lines which secreted immunogen-specific monoclonal antibodies. These hybrid cells can be cloned and cryopreserved. We also obtained another good fusion partner which initially secreted antibody but later stopped doing so. These suncus-suncus hybrid cell lines will be useful for the production of suncus monoclonal antibodies.
RESUMEN
Collagen IV is a major component of basement membranes (BMs). The α1(IV) chain, encoded by the COL4A1 gene, is expressed ubiquitously and associates with the α2(IV) chain to form the α1α1α2(IV) heterotrimer. Several COL4A1 mutations affecting a conformational domain containing integrin-binding sites are responsible for the systemic syndrome of hereditary angiopathy, nephropathy, aneurysms, and cramps (HANAC). To analyze the pathophysiology of HANAC, Col4a1 mutant mice bearing the p.Gly498Val mutation were generated. Analysis of the skeletal muscles of Col4a1G498V mutant animals showed morphologic characteristics of a muscular dystrophy phenotype with myofiber atrophy, centronucleation, focal inflammatory infiltrates, and fibrosis. Abnormal ultrastructural aspects of muscle BMs was associated with reduced extracellular secretion of the mutant α1α1α2(IV) trimer. In addition to muscular dystrophic features, endothelial cell defects of the muscle capillaries were observed, with intracytoplasmic accumulation of the mutant α1α1α2(IV) molecules, endoplasmic reticulum cisternae dilation, and up-regulation of endoplasmic reticulum stress markers. Induction of the unfolded protein response in Col4a1 mutant muscle tissue resulted in an excess of apoptosis in endothelial cells. HANAC mutant animals also presented with a muscular functional impairment and increased serum creatine kinase levels reflecting altered muscle fiber sarcolemma. This extensive description of the muscular phenotype of the Col4a1 HANAC murine model suggests a potential contribution of primary endothelial cell defects, together with muscle BM alterations, to the development of COL4A1-related myopathy.
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Vasos Sanguíneos/anomalías , Colágeno Tipo IV/genética , Calambre Muscular/genética , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/patología , Mutación/genética , Enfermedad de Raynaud/genética , Animales , Apoptosis , Vasos Sanguíneos/patología , Peso Corporal , Creatina Quinasa/sangre , Distrofina/metabolismo , Estrés del Retículo Endoplásmico , Células Endoteliales/patología , Células Endoteliales/ultraestructura , Matriz Extracelular/metabolismo , Integrina beta1/metabolismo , Ratones , Ratones Mutantes , Músculo Esquelético/ultraestructura , Tamaño de los Órganos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismoRESUMEN
Alport syndrome is caused by mutations in the genes encoding α3, α4, or α5 (IV) chains. Unlike X-linked Alport mice, α5 and α6 (IV) chains are detected in the glomerular basement membrane of autosomal recessive Alport mice, however, the significance of this finding remains to be investigated. We therefore generated mice lacking both α3 and α6 (IV) chains and compared their renal function and survival with Col4a3 knockout mice of 129 × 1/Sv background. No significant difference was observed in the renal function or survival of the two groups, or when the mice were backcrossed once to C57BL/6 background. However, the survival of backcrossed double knockout mice was significantly longer than that of the mice of 129 × 1/Sv background, which suggests that other modifier genes were involved in this phenomenon. In further studies we identified two Alport patients who had a homozygous mutation in intron 46 of COL4A4. The α5 and α6 (IV) chains were focally detected in the glomerular basement membrane of these patients. These findings indicate that although α5 and α6 (IV) chains are induced in the glomerular basement membrane in autosomal recessive Alport syndrome, their induction does not seem to play a major compensatory role.
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Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Genes Recesivos , Nefritis Hereditaria/genética , Adolescente , Animales , Cruzamientos Genéticos , Femenino , Membrana Basal Glomerular/metabolismo , Homocigoto , Humanos , Intrones , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fragmentos de Péptidos/metabolismoRESUMEN
Collagen IV is a major component of basement membranes, and mutations in COL4A1, which encodes collagen IV alpha chain 1, cause a multisystemic disease encompassing cerebrovascular, eye and kidney defects. However, COL4A1 renal disease remains poorly characterized and its pathomolecular mechanisms are unknown. We show that Col4a1 mutations in mice cause hypotension and renal disease, including proteinuria and defects in Bowman's capsule and the glomerular basement membrane, indicating a role for Col4a1 in glomerular filtration. Impaired sodium reabsorption in the loop of Henle and distal nephron despite elevated aldosterone levels indicates that tubular defects contribute to the hypotension, highlighting a novel role for the basement membrane in vascular homeostasis by modulation of the tubular response to aldosterone. Col4a1 mutations also cause diabetes insipidus, whereby the tubular defects lead to polyuria associated with medullary atrophy and a subsequent reduction in the ability to upregulate aquaporin 2 and concentrate urine. Moreover, haematuria, haemorrhage and vascular basement membrane defects confirm an important vascular component. Interestingly, although structural and compositional basement membrane defects occurred in the glomerulus and Bowman's capsule, no tubular basement membrane defects were detected. By contrast, medullary atrophy was associated with chronic ER stress, providing evidence for cell-type-dependent molecular mechanisms of Col4a1 mutations. These data show that both basement membrane defects and ER stress contribute to Col4a1 renal disease, which has important implications for the development of treatment strategies for collagenopathies.
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Membrana Basal/metabolismo , Colágeno Tipo IV/genética , Estrés del Retículo Endoplásmico , Glomérulos Renales/patología , Túbulos Renales/patología , Mutación , Animales , Humanos , Glomérulos Renales/metabolismo , Túbulos Renales/metabolismo , RatonesRESUMEN
Hereditary angiopathy, nephropathy, aneurysms, and muscle cramps (HANAC) syndrome is an autosomal dominant syndrome caused by mutations in COL4A1 that encodes the α1 chain of collagen IV, a major component of basement membranes. Patients present with cerebral small vessel disease, retinal tortuosity, muscle cramps, and kidney disease consisting of multiple renal cysts, chronic kidney failure, and sometimes hematuria. Mutations producing HANAC syndrome localize within the integrin binding site containing CB3[IV] fragment of the COL4A1 protein. To investigate the pathophysiology of HANAC syndrome, we generated mice harboring the Col4a1 p.Gly498Val mutation identified in a family with the syndrome. Col4a1 G498V mutation resulted in delayed glomerulogenesis and podocyte differentiation without reduction of nephron number, causing albuminuria and hematuria in newborns. The glomerular defects resolved within the first month, but glomerular cysts developed in 3-month-old mutant mice. Abnormal structure of Bowman's capsule was associated with metalloproteinase induction and activation of the glomerular parietal epithelial cells that abnormally expressed CD44,α-SMA, ILK, and DDR1. Inflammatory infiltrates were observed around glomeruli and arterioles. Homozygous Col4a1 G498V mutant mice additionally showed dysmorphic papillae and urinary concentration defects. These results reveal a developmental role for the α1α1α2 collagen IV molecule in the embryonic glomerular basement membrane, affecting podocyte differentiation. The observed association between molecular alteration of the collagenous network in Bowman's capsule of the mature kidney and activation of parietal epithelial cells, matrix remodeling, and inflammation may account for glomerular cyst development and CKD in patients with COL4A1-related disorders.
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Colágeno Tipo IV/genética , Enfermedades Renales Quísticas/etiología , Calambre Muscular/complicaciones , Calambre Muscular/genética , Mutación , Enfermedad de Raynaud/complicaciones , Enfermedad de Raynaud/genética , Factores de Edad , Animales , Animales Recién Nacidos , Enfermedades Renales Quísticas/metabolismo , Glomérulos Renales/metabolismo , Ratones , Calambre Muscular/metabolismo , Calambre Muscular/fisiopatología , Permeabilidad , Enfermedad de Raynaud/metabolismo , Enfermedad de Raynaud/fisiopatologíaRESUMEN
The ocular surface consists of the cornea, conjunctiva, and the limbus that is located in the transitional zone between the cornea and conjunctiva. The corneal epithelial cells are generated through the mitosis of corneal epithelial stem cells in the limbus. This study investigated a rabbit corneal deficiency model prepared by the surgical removal of the corneal and limbal epithelia, which express cytokeratin 12 (K12). After the surgery, K13-expressing conjunctival epithelium migrated onto the corneal surface and completely covered the surface, leading to neovascularization and corneal opacification. However, at 24 and 48 weeks after the surgery, K12-expressing cornea-like cells reappeared on the model ocular surface. These cells formed an island surrounded by invaded conjunctiva and were isolated from the limbus. Interestingly, in the 24-week model surface, α1(IV) and α2(IV) collagen chains, which are normally found in the basement membrane of the native limbus and conjunctiva, and not in the cornea, were continuously deposited throughout the entire basement membrane, including the basement membrane under cornea-like cells. By contrast, in the 48-week model surface, α1(IV) and α2(IV) collagen chains were absent from the basement membrane beneath the central part of cornea-like cells and were localized below the invaded conjunctiva and the transitional zone between cornea-like cells and the invaded conjunctiva, which had similar distribution to the native ocular basement membrane. Moreover, K12, K14, p63, vimentin, and α1(IV) and α2(IV) collagen chains, which are colocalized in the native limbus, were all present at the transitional zone of the 48-week model surface. Therefore, a limbus-like structure appeared to be reconstructed on the surface of the 48-week model as a stem cell niche. This study should aid in the understanding of human corneal deficiency, the correlation between the epithelial cell phenotype and the composition of the basement membrane, and the epithelial stem cell niche.
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Membrana Basal/metabolismo , Enfermedades de la Córnea/fisiopatología , Epitelio Corneal/fisiología , Regeneración/fisiología , Animales , Movimiento Celular/fisiología , Colágeno Tipo IV/metabolismo , Enfermedades de la Córnea/cirugía , Opacidad de la Córnea/etiología , Epitelio Corneal/cirugía , Inmunohistoquímica , Queratina-12/metabolismo , Queratina-13/metabolismo , Conejos , Factores de TiempoRESUMEN
BACKGROUND: Many mutations in the NPHS1 gene were detected among patients with congenital nephrotic syndrome. Functional analysis of those mutations was done with a stable-expression cell line. Nevertheless, establishing such a cell line is time-consuming. METHODS AND RESULTS: We established an easier method using automatic counting software for functional analysis with transient-transfection cells rather than a stable-expression cell line. We demonstrated maltrafficking to the plasma membrane of abnormal nephrin for immunostaining on transient-expression cells by comparison without Triton X (detecting proteins on the cell membrane only) and with Triton X (detecting proteins both on the cell membrane and inside the cell cytoplasm). We obtained relevant results with data obtained previously using a stable-expression cell line. Furthermore, we conducted functional analysis of NPHS1 mutations in Japanese patients with congenital nephrotic syndrome using this simple method, which revealed that all pathogenic mutations impaired trafficking to the protein plasma membrane. CONCLUSIONS: Functional analysis using transient-expression cells with automatic counting software was useful to demonstrate maltrafficking to the plasma membrane of a protein. All pathogenic mutations detected in Japanese patients impaired trafficking to the protein plasma membrane.
Asunto(s)
Proteínas de la Membrana/genética , Mutación , Síndrome Nefrótico/genética , Pueblo Asiatico/genética , Genotipo , Células HEK293 , Humanos , JapónRESUMEN
Alport post-transplant nephritis (APTN) is an aggressive form of anti-glomerular basement membrane disease that targets the allograft in transplanted patients with X-linked Alport syndrome. Alloantibodies develop against the NC1 domain of α5(IV) collagen, which occurs in normal kidneys, including renal allografts, forming distinct α345(IV) and α1256(IV) networks. Here, we studied the roles of these networks as antigens inciting alloimmunity and as targets of nephritogenic alloantibodies in APTN. We found that patients with APTN, but not those without nephritis, produce two kinds of alloantibodies against allogeneic collagen IV. Some alloantibodies targeted alloepitopes within α5NC1 monomers, shared by α345NC1 and α1256NC1 hexamers. Other alloantibodies specifically targeted alloepitopes that depended on the quaternary structure of α345NC1 hexamers. In Col4a5-null mice, immunization with native forms of allogeneic collagen IV exclusively elicited antibodies to quaternary α345NC1 alloepitopes, whereas alloimmunogens lacking native quaternary structure elicited antibodies to shared α5NC1 alloepitopes. These results imply that quaternary epitopes within α345NC1 hexamers may initiate alloimmune responses after transplant in X-linked Alport patients. Thus, α345NC1 hexamers are the culprit alloantigen and primary target of all alloantibodies mediating APTN, whereas α1256NC1 hexamers become secondary targets of anti-α5NC1 alloantibodies. Reliable detection of alloantibodies by immunoassays using α345NC1 hexamers may improve outcomes by facilitating early, accurate diagnosis.
Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Autoantígenos/inmunología , Colágeno Tipo IV/inmunología , Mapeo Epitopo , Trasplante de Riñón/inmunología , Nefritis Hereditaria/inmunología , Nefritis Hereditaria/cirugía , Animales , Autoantígenos/química , Membrana Basal/inmunología , Bovinos , Colágeno Tipo IV/química , Haplorrinos , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Isoanticuerpos/sangre , Isoanticuerpos/inmunología , Glomérulos Renales/inmunología , Ratones , Ratones Transgénicos , Complicaciones Posoperatorias/inmunología , Dominios y Motivos de Interacción de Proteínas/inmunología , Estructura Cuaternaria de Proteína , Trasplante HomólogoRESUMEN
Mesenchymal stromal cells (MSCs) derived from adipose tissue have immunomodulatory effects, suggesting that they may have therapeutic potential for crescentic GN. Here, we systemically administered adipose-derived stromal cells (ASCs) in a rat model of anti-glomerular basement membrane (anti-GBM) disease and found that this treatment protected against renal injury and decreased proteinuria, crescent formation, and infiltration by glomerular leukocytes, including neutrophils, CD8(+) T cells, and CD68(+) macrophages. Interestingly, ASCs cultured under low-serum conditions (LASCs), but not bone marrow-derived MSCs (BM-MSCs), increased the number of immunoregulatory CD163(+) macrophages in diseased glomeruli. Macrophages cocultured with ASCs, but not with BM-MSCs, adopted an immunoregulatory phenotype. Notably, LASCs polarized macrophages into CD163(+) immunoregulatory cells associated with IL-10 production more efficiently than ASCs cultured under high-serum conditions. Pharmaceutical ablation of PGE2 production, blocking the EP4 receptor, or neutralizing IL-6 in the coculture medium all significantly reversed this LASC-induced conversion of macrophages. Furthermore, pretreating LASCs with aspirin or cyclooxygenase-2 inhibitors impaired the ability of LASCs to ameliorate nephritogenic IgG-mediated renal injury. Taken together, these results suggest that LASCs exert renoprotective effects in anti-GBM GN by promoting the phenotypic conversion of macrophages to immunoregulatory cells, suggesting that LASC transfer may represent a therapeutic strategy for crescentic GN.
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Tejido Adiposo/citología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/terapia , Macrófagos/inmunología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Animales , Técnicas de Cultivo de Célula , Medio de Cultivo Libre de Suero , Citocinas/inmunología , Modelos Animales de Enfermedad , Inmunomodulación , Células Madre Mesenquimatosas/inmunología , Ratas , Ratas Endogámicas WKY , Células del Estroma/citología , Células del Estroma/inmunologíaRESUMEN
Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (nâ=â80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.
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Modelos Biológicos , Folículo Ovárico/embriología , Ovario/embriología , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN , Femenino , Inmunohistoquímica , Masculino , EmbarazoRESUMEN
Vitamin A is essential for lung development and pulmonary cell differentiation. Its deficiency leads to altered lung structure and function and to basement membrane architecture and composition disturbances. Previously, we showed that lack of retinoids thickens the alveolar basement membrane and increases collagen IV, which are reversed by retinoic acid, the main biologically active vitamin A form. This study analyzed how vitamin A deficiency affects the subunit composition of collagen IV and laminin of lung basement membranes and pulmonary matrix metalloproteinase content, plus the recovering effect of all-trans-retinoic acid. Male weanling pups were fed a retinol-adequate/-deficient diet until 60 days old. A subgroup of vitamin-A-deficient pups received daily intraperitoneal all-trans-retinoic acid injections for 10 days. Collagen IV and laminin chain composition were modified in vitamin-A-deficient rats. The protein and mRNA contents of chains α1(IV), α3(IV) and α4(IV) increased; those of chains α2(IV) and α5(IV) remained unchanged; and the protein and mRNA contents of laminin chains α5, ß1 and γ1 decreased. The mRNA of laminin chains α2 and α4 also decreased. Matrix metalloproteinases 2 and 9 decreased, but the tissue inhibitors of metalloproteinases 1 and 2 did not change. Treating vitamin-A-deficient rats with retinoic acid reversed all alterations, but laminin chains α2, α4 and α5 and matrix metalloproteinase 2 remained low. In conclusion, vitamin A deficiency alters the subunit composition of collagen IV and laminin and the lung's proteolytic potential, which are partly reverted by retinoic acid. These alterations could contribute to impaired lung function and predispose to pulmonary disease.
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Colágeno Tipo IV/metabolismo , Laminina/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Tretinoina/farmacología , Deficiencia de Vitamina A/metabolismo , Animales , Membrana Basal/metabolismo , Colágeno Tipo IV/genética , Femenino , Expresión Génica , Laminina/genética , Masculino , Ratas , Ratas Wistar , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Vitamina A/sangre , Deficiencia de Vitamina A/tratamiento farmacológicoRESUMEN
The aim of this study was to compare the expressions of basal lamina (BL) collagen IV α chains and matrix metalloproteinases (MMP)-2 and MMP-9 in oral dysplasia (OED) and invasive carcinoma. Ten cases each of OEDs, carcinomas-in situ and oral squamous cell carcinomas (OSCCs) were examined by immunohistochemistry. Another 5 cases, each of normal and hyperplastic oral mucosa, served as controls. Results showed that α1(IV)/α2(IV) and α5(IV)/α6(IV) chains were intact in BLs of control and OEDs. In BLs of carcinoma-in situ, α1(IV)/α2(IV) chains preceded α5(IV)/α6(IV) chains in showing incipient signs of disruption. OSCCs exhibited varying degrees of collagen α(IV) chain degradation. MMP-2 and MMP-9 were absent in controls and OED, but weakly detectable in carcinoma-in situ. In OSCC, these proteolytic enzymes were expressed in areas corresponding to collagen α(IV) chain loss. Enzymatic activity was enhanced in higher grade OSCC, and along the tumor advancing front. Overall the present findings suggest that loss of BL collagen α(IV) chains coincided with gain of expression for MMP-2 and MMP-9, and that these protein alterations are crucial events during progression from OED to OSCC.
Asunto(s)
Carcinoma/enzimología , Colágeno Tipo IV/química , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Boca/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Membrana Basal/enzimología , Carcinoma in Situ , Estudios de Casos y Controles , Colágeno Tipo IV/metabolismo , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Mucosa Bucal/patología , Invasividad NeoplásicaRESUMEN
BACKGROUND: The destruction of the basement membrane (BM) is the first step in cancer invasion and metastasis. Type IV collagen is a major component of the BM, and is composed of six genetically distinct α(IV) chains; α1(IV) to α6(IV). The loss of α5(IV) and α6(IV) chains from the epithelial BM at the early stage of cancer invasion has been reported in several types of cancers. However, the expression of α5(IV) and α6(IV) chains in extrahepatic bile duct carcinoma (EBDC) remains unclear. METHODS: We examined the expression of α(IV) chains by immunohistochemistry using 71 resected EBDC specimens. Prognostic significance of α(IV) chains was examined by Cox regression and Kaplan-Meier analyses. RESULTS: In the invasive cancer, the expression of α6(IV) chain in the BM was lost partially or completely preceded by the loss of α2(IV) chain. The loss of α6(IV) chain in the BM of the invasive cancer was related to the tumor classification, TNM stages, and the expression of α2(IV) chain. The patients with α2(IV)-negative and α6(IV)-negative chains had significantly poorer prognosis than those with α2(IV)-positive and α6(IV)-positive/negative chains (P = 0.04). CONCLUSIONS: The loss of α2(IV) and α6(IV) chains might be a useful prognostic factor in patients with EBDC.
Asunto(s)
Membrana Basal , Neoplasias de los Conductos Biliares/química , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Extrahepáticos , Biomarcadores de Tumor/análisis , Carcinoma/química , Carcinoma/patología , Colágeno Tipo IV/análisis , Anciano , Análisis de Varianza , Membrana Basal/química , Membrana Basal/patología , Conductos Biliares Extrahepáticos/química , Conductos Biliares Extrahepáticos/patología , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Antígeno Ki-67/análisis , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Proteína p53 Supresora de Tumor/análisisRESUMEN
Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults and a significant cause of end-stage renal disease, yet current therapies are nonspecific, toxic, and often ineffective. The development of novel targeted therapies requires a detailed understanding of the pathogenic mechanisms, but progress is hampered by the lack of a robust mouse model of disease. We report that DBA/1 mice as well as congenic FcγRIII(-/-) and FcRγ(-/-) mice immunized with a fragment of α3(IV) collagen developed massive albuminuria and nephrotic syndrome, because of subepithelial deposits of mouse IgG and C3 with corresponding basement membrane reaction and podocyte foot process effacement. The clinical presentation and histopathologic findings were characteristic of MN. Although immunized mice produced genuine anti-α3NC1 autoantibodies that bound to kidney and lung basement membranes, neither crescentic glomerulonephritis nor alveolitis ensued, likely because of the predominance of mouse IgG1 over IgG2a and IgG2b autoantibodies. The ablation of activating IgG Fc receptors did not ameliorate injury, implicating subepithelial deposition of immune complexes and consequent complement activation as a major effector pathway. We have thus established an active model of murine MN. This model, leveraged by the availability of genetically engineered mice and mouse-specific reagents, will be instrumental in studying the pathogenesis of MN and evaluating the efficacy of novel experimental therapies.
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Autoantígenos/toxicidad , Colágeno Tipo IV/toxicidad , Modelos Animales de Enfermedad , Glomerulonefritis Membranosa/inmunología , Síndrome Nefrótico/etiología , Albuminuria/etiología , Albuminuria/inmunología , Animales , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Membrana Basal/inmunología , Colágeno Tipo IV/inmunología , Complemento C3/inmunología , Glomerulonefritis Membranosa/complicaciones , Inmunización , Inmunoglobulina G/inmunología , Riñón/inmunología , Riñón/patología , Riñón/fisiopatología , Ratones , Ratones Congénicos , Ratones Endogámicos DBA , Síndrome Nefrótico/inmunología , Alveolos Pulmonares/inmunología , Receptores de IgG , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/toxicidadRESUMEN
Several types of collagen contain cryptic antiangiogenic noncollagenous domains that are released upon proteolysis of extracellular matrix (ECM). Among those is Arresten, a collagen-derived antiangiogenic factor (CDAF) that is processed from α1 collagen IV. However, the conditions under which Arresten is released from collagen IV in vivo or whether the protein functions in tumor suppressor pathways remain unknown. Here, we show that p53 induces the expression of α1 collagen IV and release of Arresten-containing fragments from the ECM. Comparison of the transcriptional activation of COL4A1 with other CDAF-containing genes revealed that COL4A1 is a major antiangiogenic gene induced by p53 in human adenocarinoma cells. p53 directly activated transcription of the COL4A1 gene by binding to an enhancer region 26 kbp downstream of its 3' end. p53 also stabilized the expression of full-length α1 collagen IV by upregulation of α(II) prolyl-hydroxylase and increased the release of Arresten in the ECM through a matrix metalloproteinase (MMP)-dependent mechanism. The resulting upregulation of α1 collagen IV and production of Arresten by the tumor cells significantly inhibited angiogenesis and limited tumor growth in vivo. Furthermore, we show that immunostaining of Arresten correlated with p53 status in human prostate cancer specimens. Our findings, therefore, link the production of Arresten to the p53 tumor suppressor pathway and show a novel mechanism through which p53 can inhibit angiogenesis.
Asunto(s)
Adenocarcinoma/metabolismo , Inhibidores de la Angiogénesis/genética , Colágeno Tipo IV/genética , Neoplasias de la Próstata/genética , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular Tumoral , Colágeno Tipo IV/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Microvascular rarefaction following an episode of acute kidney injury (AKI) is associated with renal hypoxia and progression toward chronic kidney disease. The mechanisms contributing to microvascular rarefaction are not well-understood, although disruption in local angioregulatory substances is thought to contribute. Matrix metalloproteinase (MMP)-9 is an endopeptidase important in modifying the extracellular matrix (ECM) and remodeling the vasculature. We examined the role of MMP-9 gene deletion on microvascular rarefaction in a rodent model of ischemic AKI. MMP-9-null mice and background control (FVB/NJ) mice were subjected to bilateral renal artery clamping for 20 min followed by reperfusion for 14, 28, or 56 days. Serum creatinine level in MMP-9-null mice 24 h after injury [1.4 (SD 0.8) mg/dl] was not significantly different from FVB/NJ mice [1.5 (SD 0.6) mg/dl]. Four weeks after ischemic injury, FVB/NJ mice demonstrated a 30-40% loss of microvascular density compared with sham-operated (SO) mice. In contrast, microvascular density was not significantly different in the MMP-9-null mice at this time following injury compared with SO mice. FVB/NJ mice had a 50% decrease in tissue vascular endothelial growth factor (VEGF) 2 wk after ischemic insult compared with SO mice. A significant difference in VEGF was not observed in MMP-9-null mice compared with SO mice. There was no significant difference in the liberation of angioinhibitory fragments from the ECM between MMP-9-null mice and FVB/NJ mice following ischemic injury. In conclusion, MMP-9 deletion stabilizes microvascular density following ischemic AKI in part by preserving tissue VEGF levels.
Asunto(s)
Lesión Renal Aguda/patología , Capilares/patología , Isquemia/patología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/fisiología , Animales , Western Blotting , Colágeno/metabolismo , Fibrosis , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Riñón/patología , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteinuria/genética , Circulación Renal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The distribution of the collagen chains from α1(IV) to α6(IV) could serve as a basis for the characterization of type IV collagen. In this study, immunohistochemistry of the ocular anterior segment of adult mice was performed using specific monoclonal antibodies against each chain in the series from α1(IV) to α6(IV). The results show that the components of type IV collagen in vascular basement membranes are α1(IV) and α2(IV) with or without α5(IV) and α6(IV) chains and those in epithelium and muscle basement membranes are α1(IV), α2(IV), α5(IV), and α6(IV) chains. In corneal endothelium, pigmented epithelium of iris and ciliary body, and trabecular meshwork, α3(IV) and α4(IV) chains are also expressed in addition to α1(IV), α2(IV), α5(IV), and α6(IV) chains. Moreover, we investigated the change in molecular composition in ciliary body during postnatal development. α3(IV) and α4(IV) chains were also expressed in addition to α1(IV), α2(IV), α5(IV), and α6(IV) chains in ciliary pigmented epithelium basement membrane from 7 days after birth. This result suggests that the basement membranes gradually change their biochemical features owing to temporal regulation. Taken together, these findings suggest that the different distribution and the developmental expression of α1(IV) to α6(IV) chains are associated with the tissue-specific function of type IV collagen in basement membranes.
Asunto(s)
Membrana Basal/metabolismo , Colágeno Tipo IV/metabolismo , Ojo/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Cuerpo Ciliar/metabolismo , Endotelio/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epitelio/metabolismo , Ojo/crecimiento & desarrollo , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/genéticaRESUMEN
Here we report two brothers with electron-microscopically diagnosed Alport's syndrome (AS) who showed normal staining patterns for the alpha1(IV)-alpha4(IV) chains of collagen type IV, but abnormal expression of the alpha5(IV) and alpha6(IV) chains. Both patients had microscopic hematuria and mild proteinuria from around 10 years old, and had renal biopsies at 23 (older) and 26 (younger) years old due to increased proteinuria (0.5-0.8 g/day) with normal renal function. A skin biopsy of the patients' mother showed similar abnormal staining patterns for the alpha5(IV) and alpha6(IV) chains in the skin basement membranes. Both of them showed slow progression of renal dysfunction and no extrarenal manifestations. The existences of incomplete alpha3,alpha4,alpha5(IV) molecules in the glomerular basement membrane (GBM) and inadequately formed alpha5,alpha5,alpha6(IV) molecules are suggested for these patients. A missense mutation of the COL4A5 gene may present in this family as possible X-linked inheritance and a mild form of AS.