RESUMO
BACKGROUND: In Henoch-Schönlein nephritis (HSN), a risk factor for unfavorable outcome is prolonged proteinuria, but the value of renal biopsies in prognosis assessment is debatable. METHODS: We evaluated serial renal biopsies from 26 HSN patients. Follow-up biopsy occurred at median 2.1 years after diagnostic biopsy. Patients formed two groups at the follow-up biopsy: patients without proteinuria (group I; n = 11) and with proteinuria (group II; n = 15). Biopsies underwent evaluation according to three classifications: International Study of Kidney Disease in Children (ISKDC), Oxford (MEST-C), and semiquantitative classification (SQC) including an activity and chronicity score. Analysis also included expression of pro-fibrotic (alpha-smooth muscle actin and vimentin) and inflammatory (P-selectin glycoprotein ligand-1) molecules in the diagnostic biopsy specimens. Definition of unfavorable outcome was active renal disease or reduced renal function at last follow-up. RESULTS: Between the biopsies, SQC chronicity score increased in 22 (85%) patients, whereas activity score and ISKDC grade decreased in 21 (81%) and 17 (65%), respectively. Of the MEST-C parameters, endocapillary proliferation (from 83 to 13%; p < 0.001) and crescents (from 63 to 25%; p = 0.022) showed significant reduction, and segmental glomerulosclerosis (from 38 to 79%; p = 0.006) significant increment. These changes occurred similarly in groups I and II. Expression of the pro-fibrotic and inflammatory molecules showed no clinically significant differences between groups I and II. None in group I and five (33%) patients in group II had unfavorable outcome (p = 0.053). CONCLUSIONS: Our results suggest that follow-up biopsies provide limited additional information to clinical symptoms in HSN outcome prediction.
Assuntos
Vasculite por IgA/patologia , Nefrite/patologia , Adolescente , Biópsia , Estudos de Casos e Controles , Criança , Feminino , Taxa de Filtração Glomerular , Humanos , Vasculite por IgA/complicações , Masculino , Nefrite/etiologia , Proteinúria/etiologia , Estudos RetrospectivosRESUMO
Mitochondrial fatty acid synthesis (mtFAS) is an underappreciated but highly conserved metabolic process, indispensable for mitochondrial respiration. It was recently reported that dysfunction of mtFAS causes childhood onset of dystonia and optic atrophy in humans (MEPAN). To study the role of mtFAS in mammals, we generated three different mouse lines with modifications of the Mecr gene, encoding mitochondrial enoyl-CoA/ACP reductase (Mecr). A knock-out-first type mutation, relying on insertion of a strong transcriptional terminator between the first two exons of Mecr, displayed embryonic lethality over a broad window of time and due to a variety of causes. Complete removal of exon 2 or replacing endogenous Mecr by its functional prokaryotic analogue fabI (Mecrtm(fabI)) led to more consistent lethality phenotypes and revealed a hypoplastic placenta. Analyses of several mitochondrial parameters indicate that mitochondrial capacity for aerobic metabolism is reduced upon disrupting mtFAS function. Further analysis of the synthetic Mecrtm(fabI) models disclosed defects in development of placental trophoblasts consistent with disturbed peroxisome proliferator-activated receptor signalling. We conclude that disrupted mtFAS leads to deficiency in mitochondrial respiration, which lies at the root of the observed pantropic effects on embryonic and placental development in these mouse models.
Assuntos
Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Animais , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Feminino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Oxirredutases/metabolismo , Placenta , Placentação/genética , Placentação/fisiologia , GravidezRESUMO
BACKGROUND: Tubulointerstitial nephritis (TIN) is an inflammatory disease of unknown pathogenesis. To evaluate a possible role of regulatory T cells (Tregs) in the pathophysiology of TIN with (TINU) and without uveitis, we investigated the presence and quantity of FOXP3+ T regulatory lymphocytes in diagnostic kidney biopsies from pediatric patients. METHODS: A total of 33 patients (14 TIN and 19 TINU) were enrolled. The quantity of CD4+, FOXP3+ and double-positive T cells in formalin-fixed kidney biopsies was determined using double label immunohistochemistry with anti-human CD4 and FOXP3 antibodies. RESULTS: FOXP3 staining was successful in all 33 patients. In patients with chronic uveitis, the density of FOXP3+ cells was significantly lower (p = 0.046) than in TIN patients without uveitis or with uveitis lasting <3 months. CD4+ staining was successful in 23 patients. The density of all lymphocytes (CD4+, CD4+FOXP3+ and FOXP3+ cells) was significantly lower (p = 0.023) in patients with chronic uveitis than in other patients. CONCLUSIONS: FOXP3+ T cells are present in kidney biopsy samples from TIN and TINU patients. In patients with chronic uveitis, the density of FOXP3+ T cells is significantly lower than in other patients, suggesting a different pathomechanism for these clinical conditions.
Assuntos
Nefrite Intersticial/imunologia , Linfócitos T Reguladores/imunologia , Uveíte/imunologia , Adolescente , Biópsia , Criança , Feminino , Fatores de Transcrição Forkhead/imunologia , Humanos , MasculinoRESUMO
BACKGROUND: Histological findings from primary kidney biopsies were correlated with patient outcomes in a national cohort of paediatric Henoch-Schönlein nephritis (HSN) patients. METHODS: Primary kidney biopsies from 53 HSN patients were re-evaluated using the ISKDC (International Study of Kidney Disease in Children) classification and a modified semiquantitative classification (SQC) that scores renal findings and also takes into account activity, chronicity and tubulointerstitial indices. The ISKDC and SQC classifications were evaluated comparatively in four outcome groups: no signs of renal disease (outcome A, n = 27), minor urinary abnormalities (outcome B, n = 18), active renal disease (outcome C, n = 3) and renal insufficiency, end-stage renal disease or succumbed due to HSN (outcome D, n = 5). For the receiver operating characteristic and logistic regression analyses, outcomes A and B were considered to be favourable and outcomes C and D to be unfavourable. The median follow-up time was 7.3 years. RESULTS: The patients with an unfavourable outcome (C and D), considered together due to low patient numbers, had significantly higher total biopsy SQC scores and activity indices than those who had a favourable one (groups A and B). The chronicity and tubulointerstitial indices differed significantly only between group C + D and group A. The difference in areas under the curve between the total biopsy SQC scores and ISKDC findings was 0.15 [p = 0.04, normal-based 95% confidence interval (CI) 0.007-0.29, bias-controlled 95% CI -0.004 to 0.28]. CONCLUSIONS: Our results suggest that the modified SQC is more sensitive than ISKDC classification for predicting the outcome in HSN cases.
Assuntos
Vasculite por IgA/patologia , Falência Renal Crônica/patologia , Nefrite/patologia , Proteinúria/patologia , Adolescente , Biópsia , Criança , Estudos de Viabilidade , Feminino , Seguimentos , Taxa de Filtração Glomerular , Humanos , Vasculite por IgA/classificação , Vasculite por IgA/complicações , Vasculite por IgA/urina , Rim/patologia , Falência Renal Crônica/classificação , Falência Renal Crônica/etiologia , Falência Renal Crônica/urina , Masculino , Nefrite/classificação , Nefrite/etiologia , Nefrite/urina , Prognóstico , Proteinúria/etiologia , Proteinúria/urina , Curva ROC , Estudos RetrospectivosRESUMO
As the second most common skin malignancy, cutaneous squamous cell carcinoma (cSCC) is an increasing health concern, while its pathogenesis at molecular level remains largely unknown. We studied the expression and localisation of two homologous basement membrane (BM) collagens, types XV and XVIII, at different stages of cSCC. These collagens are involved in angiogenesis and tumorigenesis, but their role in cancer development is incompletely understood. Quantitative RT-PCR analysis revealed upregulation of collagen XVIII, but not collagen XV, in primary cSCC cells in comparison with normal human epidermal keratinocytes. In addition, the Ha-ras-transformed invasive cell line II-4 expressed high levels of collagen XVIII mRNA, indicating upregulation in the course of malignant transformation. Immunohistochemical analyses of a large human tissue microarray material showed that collagen XVIII is expressed by tumor cells from grade 1 onwards, while keratinocytes in normal skin and in premalignant lesions showed negative staining for it. Collagen XV appeared instead as deposits in the tumor stroma. Our findings in human cSCCs and in mouse cSCCs from the DMBA-TPA skin carcinogenesis model showed that collagen XVIII, but not collagen XV or the BM markers collagen IV or laminin, was selectively reduced in the tumor vasculature, and this decrease associated significantly with cancer progression. Our results demonstrate that collagens XV and XVIII are expressed in different sites of cSCC and may contribute in a distinct manner to processes related to cSCC tumorigenesis, identifying these collagens as potential biomarkers in the disease.
Assuntos
Membrana Basal/metabolismo , Carcinoma de Células Escamosas/metabolismo , Colágeno Tipo XVIII/metabolismo , Colágeno/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Humanos , CamundongosRESUMO
The kidney filtration barrier consists of the capillary endothelium, the glomerular basement membrane and the slit diaphragm localized between foot processes of neighbouring podocytes. We report that collagen XVII, a transmembrane molecule known to be required for epithelial adhesion, is expressed in podocytes of normal human and mouse kidneys and in endothelial cells of the glomerular filtration barrier. Immunoelectron microscopy has revealed that collagen XVII is localized in foot processes of podocytes and in the glomerular basement membrane. Its role in kidney has been analysed in knockout mice, which survive to birth but have high neonatal mortality and skin blistering and structural abnormalities in their glomeruli. Morphometric analysis has shown increases in glomerular volume fraction and surface densities of knockout kidneys, indicating an increased glomerular amount in the cortex. Collagen XVII deficiency causes effacement of podocyte foot processes; however, major slit diaphragm disruptions have not been detected. The glomerular basement membrane is split in areas in which glomerular and endothelial basement membranes meet. Differences in the expression of collagen IV, integrins α3 or ß1, laminin α5 and nephrin have not been observed in mutant mice compared with controls. We propose that collagen XVII has a function in the attachment of podocyte foot processes to the glomerular basement membrane. It probably contributes to podocyte maturation and might have a role in glomerular filtration.
Assuntos
Autoantígenos/metabolismo , Membrana Basal Glomerular/metabolismo , Barreira de Filtração Glomerular/metabolismo , Colágenos não Fibrilares/metabolismo , Animais , Pré-Escolar , Feminino , Membrana Basal Glomerular/ultraestrutura , Barreira de Filtração Glomerular/ultraestrutura , Humanos , Camundongos , Camundongos Knockout , Colágenos não Fibrilares/deficiência , Fenótipo , Colágeno Tipo XVIIRESUMO
Knowledge about how to treat severe Henoch-Schönlein nephritis (HSN) is scarce. The aim of our study is to compare cyclosporine A (CyA) and methylprednisolone pulses (MP) in the treatment of severe HSN. Out of 24 pediatric HSN patients with nephrotic-range proteinuria or crescentic HSN in kidney biopsy, seven were randomized to receive CyA for 12 months at an initial dose of 5 mg/kg and eight to receive 3 MP pulses of 30 mg/kg followed by prednisone for 4 months. The other nine patients received identical treatment without randomization. Kidney biopsies were performed at inclusion and after 2 years. The primary outcomes were the duration of proteinuria and hematuria, estimated glomerular filtration rate, and renal biopsy histology. All the 11 CyA-treated patients achieved resolution of nephrotic-range proteinuria within 3 months, while the MP-group response was slower, and in 6/13 was not achieved with the initial treatment. Additional immunosuppressive treatment was needed in none of the CyA-treated patients but in six patients treated with MP (difference in proportion 46%, p = 0.008). The 2-year control biopsies were similarly improved in both groups. After mean 6.1 years (2.2-10.4 years), 16 patients (eight CyA, eight MP) had no renal symptoms and six (three CyA, three MP) had persistent nephropathy but normal renal function. One MP-treated patient had reduced renal function and another had developed ESRD and received a renal transplant. CyA gave a 100% resolution of nephrotic-range proteinuria and a 100% renal survival rate without additional therapy after a mean follow-up of 6 years. Treatment of HSN with CyA is efficacious, safe and not inferior to MP.
Assuntos
Ciclosporina/uso terapêutico , Vasculite por IgA/tratamento farmacológico , Imunossupressores/uso terapêutico , Metilprednisolona/uso terapêutico , Nefrite/tratamento farmacológico , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Vasculite por IgA/complicações , Masculino , Nefrite/etiologiaRESUMO
In pemphigoid gestationis (PG), autoantibodies target collagen XVII, a hemidesmosomal transmembrane protein, which is an important element in cutaneous epithelial adhesion and signalling. We report that collagen XVII is expressed in the first trimester and term syncytial and cytotrophoblastic cells of normal placenta and in epithelial cells of amniotic membrane. Immunoelectron microscopy confirmed the localization of collagen XVII to the hemidesmosomes of amniotic epithelium. Examination of three PG placentas showed mild villitis, but there were no differences between collagen XVII expression levels or immunostaining signals as compared to normal placenta. Collagen XVII expression was also detected in cultured extravillous trophoblast HTR-8/SVneo cells, where collagen XVII expression was upregulated by PMA and TGF-beta1. Interestingly, the presence of Col15, the cell migration domain of collagen XVII, induced the migration of HTR-8/SVneo cells in transmigration assay. Analysis of amniotic fluid samples at different gestational weeks revealed that a large quantity of collagen XVII ectodomain was shed into amniotic fluid throughout pregnancy. Biochemical and immunoblotting analysis indicated that the ectodomain in amniotic fluid is structurally very similar to the ectodomain produced by cultured keratinocytes. Cultured cells from amniotic fluid samples also expressed collagen XVII. Our results suggest that collagen XVII may contribute to the invasion of extravillous trophoblasts during placental development and is also required for the integrity of amniotic basement membrane. Although the exact pathomechanism of PG is still largely unknown, the clinical symptoms of PG are initiated after the expression of collagen XVII in placenta during the first trimester of pregnancy.
Assuntos
Autoantígenos/química , Membrana Basal/metabolismo , Membrana Celular/metabolismo , Membranas Extraembrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Colágenos não Fibrilares/química , Penfigoide Gestacional/imunologia , Placenta/metabolismo , Trofoblastos/metabolismo , Adulto , Âmnio/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Penfigoide Gestacional/metabolismo , Gravidez , Colágeno Tipo XVIIRESUMO
Type XVII collagen (collagen XVII) is a component of hemidesmosomes, which connect epithelial cells to the underlying basement membrane. Previously, an association has been suggested between neurological disorders and the skin disease bullous pemphigoid, where autoimmunity is directed against collagen XVII. Furthermore, the lack of alpha6 integrin, a ligand of collagen XVII, has been implicated in defects of cortical organization in the mouse brain. In this study, we demonstrate for the first time the presence of collagen XVII in neurons of the human brain by in situ hybridisation, immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). We propose that collagen XVII may be involved in the pathogenesis of various disorders affecting neuronal migration or synaptic plasticity.
Assuntos
Autoantígenos/análise , Sistema Nervoso Central/citologia , Neurônios/química , Colágenos não Fibrilares/análise , Adulto , Idoso , Animais , Autoantígenos/genética , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neurônios/citologia , Colágenos não Fibrilares/genética , Colágeno Tipo XVIIRESUMO
PURPOSE: This study was designed to clarify the expression of 2 closely related collagen (Col) types XVIII and XV, and the proteolytically derived endostatin fragment of ColXVIII in normal, keratoconus, and scarred human corneas. METHODS: Immunohistochemistry, in situ hybridization, immunoelectron microscopy, and Western immunoblotting were used for human corneal samples obtained from penetrating keratoplasty. RESULTS: In the normal cornea, ColXVIII was immunolocalized to the corneal and conjunctival epithelial basement membrane (EBM), Descemet s membrane, and the limbal and conjunctival capillaries. Immunoreaction for endostatin was otherwise similar, but it also was present in corneal epithelial cells. Western immunoblotting showed that normal cornea contains several endostatin fragments ranging from 20 to 100 kDa. ColXV was present in the EBM of the limbus and conjunctiva, but not in EBM of the clear cornea. In situ hybridization revealed that corneal basal epithelial cells were responsible for the synthesis of ColXVIII mRNA. Keratoconus cases were characterized by an irregular EBM immunoreactivity for ColXVIII and endostatin and patchy immunoreactivity beneath EBM. In scarred corneas, highly increased immunoreactivity for ColXVIII, endostatin, and ColXV was present within stroma. CONCLUSIONS: The results indicate that ColXVIII and ColXV are differentially expressed in normal human corneas. Constant expression of ColXVIII by corneal EBM suggests that it is an important structural molecule. Aberrant expression of ColXVIII, endostatin, and ColXV in keratoconus and scarred corneas emphasizes the active role these molecules in the wound healing process.
Assuntos
Colágeno Tipo XVIII/metabolismo , Colágeno/metabolismo , Córnea/metabolismo , Endostatinas/metabolismo , Ceratite/metabolismo , Ceratocone/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Feminino , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Masculino , Microscopia Imunoeletrônica , Pessoa de Meia-IdadeRESUMO
Immunohistochemistry was used to study the distribution of laminin (Ln) chains, collagen types IV (alpha 1/2), VII, and XVIII and Lutheran antigen (Lu) in 36 frozen ovarian carcinoma samples. Surface epithelial basement membrane (BM) of the normal ovary showed immunoreactivity for Ln alpha1, alpha3-alpha5, beta1-3, gamma1, and gamma2 chains and type IV and XVIII collagens. Chains of Ln-5 (alpha3beta3gamma2) and Ln-10 (alpha5beta1gamma1) as well as type IV and XVIII collagens were found in most tumor BMs, but Ln alpha2 chain and type VII collagen were detected only in few tumors. Contrary to serous tumors, BMs of mucinous carcinomas showed Ln alpha4 chain, but not Ln alpha1 and beta2 chains. Ln alpha1 chain was found in most endometrioid carcinomas, whereas chains of Ln-5 were only moderately detectable in comparison with serous and mucinous carcinomas. In the normal ovary, Lu immunoreactivity was confined to basal aspect in the ovarian epithelial cells, but in tumor specimens Lu immunostainings showed variable polarized and nonpolarized patterns. The results suggest that the three types of ovarian carcinoma have distinct differences in their Ln distribution and can be grouped based on their expression pattern. This suggests that they may have histogenetically different precursors and may help to distinguish these tumors from each other.
Assuntos
Adenocarcinoma/metabolismo , Laminina/biossíntese , Neoplasias Ovarianas/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/ultraestrutura , Membrana Basal/metabolismo , Carcinoma Endometrioide/diagnóstico , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/ultraestrutura , Cistadenocarcinoma Mucinoso/diagnóstico , Cistadenocarcinoma Mucinoso/metabolismo , Cistadenocarcinoma Mucinoso/ultraestrutura , Cistadenocarcinoma Seroso/diagnóstico , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/ultraestrutura , Diagnóstico Diferencial , Epitélio/metabolismo , Epitélio/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/ultraestrutura , Ovário/metabolismo , Ovário/ultraestrutura , Isoformas de Proteínas/biossínteseRESUMO
PURPOSE: To study extracellular matrix (ECM) metabolism by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in aqueous humor (AH) samples collected from primary open-angle glaucoma (POAG), exfoliation syndrome (EXS), and exfoliation glaucoma (EXG) in relation to samples derived from cataract control patients. MATERIALS AND METHODS: Seventy-one AH samples were collected during cataract extraction and trabeculectomy. The expression and molecular forms of MMP-2, -8, -9, -13, and -14 and tissue inhibitor of metalloproteinases-1 and -2 (TIMPs) were analyzed by Western immunoblotting. Gelatinase and collagenase activities were studied by zymography and type I collagen degradation assays, respectively. MMP-2 and TIMP-2 concentrations were measured by ELISA assays. RESULTS: By Western immunoblotting all the studied MMPs were mainly in their latent form in all diagnostic groups. Zymography demonstrated that MMP-2 represents the major gelatinase in AH. Similarly, type I collagenolytic activity was low and similar in cataract and glaucoma samples. In ELISA measurements the TIMP-2 levels were significantly elevated in glaucoma and EXS samples in comparison to cataract controls (P < 0.05). CONCLUSION: TIMP-2 is elevated in glaucomatous process over MMP-2, which support and further extend the conjuncture that the ECM accumulation rather than degradation predominates in the pathogenesis of POAG and EXG.
Assuntos
Humor Aquoso/enzimologia , Síndrome de Exfoliação/enzimologia , Glaucoma de Ângulo Aberto/enzimologia , Metaloproteinases da Matriz/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Feminino , Humanos , Pressão Intraocular , Masculino , Pessoa de Meia-IdadeRESUMO
Collagen XVII has a well-established role as an adhesion molecule and a cell surface receptor located in the type I hemidesmosome of stratified epithelia. Its ectodomain is constitutively shed from the cell surface and suggested to regulate the adhesion, migration, and signaling of cutaneous epithelial cells. Collagen XVII was not previously thought to be expressed by colon epithelial cells. Immunohistochemical analysis of tissue microarray samples of 141 cases of colorectal carcinoma showed that collagen XVII is expressed in normal human colonic mucosa and colorectal carcinoma. In colorectal carcinoma, increased collagen XVII expression was significantly associated with higher TNM stage. It also correlated with infiltrative growth pattern and tumor budding as well as lymph node and distant metastasis. Increased collagen XVII expression was associated with decreased disease-free and cancer-specific survival. Immunofluorescence staining of collagen XVII and its well-known binding partner laminin γ2 chain demonstrated a partial colocalization in normal and tumor tissue. In vitro, the overexpression of murine collagen XVII promoted the invasion of CaCo-2 colon carcinoma cells through Matrigel (BD Biosciences; Bedford, MA). To conclude, this study reports for the first time the expression of collagen XVII in colon epithelium and the association of increased collagen XVII immunoexpression with poor outcome in colorectal carcinoma.
Assuntos
Autoantígenos/análise , Biomarcadores Tumorais/análise , Neoplasias Colorretais/química , Mucosa Intestinal/química , Invasividade Neoplásica/patologia , Neoplasias de Células Escamosas/química , Neoplasias de Células Escamosas/secundário , Colágenos não Fibrilares/análise , Idoso , Membrana Basal/química , Membrana Basal/patologia , Células CACO-2/química , Células CACO-2/patologia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Intervalo Livre de Doença , Feminino , Imunofluorescência , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias de Células Escamosas/mortalidade , Neoplasias de Células Escamosas/patologia , Neoplasias de Células Escamosas/terapia , Curva ROC , Análise de Sobrevida , Resultado do Tratamento , Colágeno Tipo XVIIRESUMO
Human type XVIII collagen was found to be expressed as three variants, termed NC1-303, NC1-493 and NC1-728, differing in their N-terminal non-collagenous domains (NC1). The corresponding gene was found to be approximately 105 kb in size and contain 43 exons. The short variant is derived from utilization of an upstream promoter associated with the first two exons of the gene. The two other variants are derived from a downstream promoter and alternative splicing of exon 3, resulting in 192 residues of shared sequences characterized by a putative approximately 30 residue conserved coiled-coil motif and 235 residues of sequences specific to NC1-728. The NC1-728 variant has a conserved cysteine-rich domain homologous with the ligand-binding part of the frizzled proteins. A polyclonal antibody specific to the NC1-728 variant was generated, and immunostaining of fetal tissues revealed staining in lung and skeletal muscle. Human serum contained 173- and 144-kDa alpha1(XVIII) chains corresponding to the NC1-728 and NC1-493 variants, respectively. A 200-kDa polypeptide was detected in cells transfected with a cDNA construct corresponding to the full-length NC1-728 variant, and EBNA-293 cells endogenously synthesizing low amounts of type XVIII collagen had a 45-kDa fragment in their culture medium that corresponded to most of the NC1 domain of the NC1-728 variant, suggesting processing of the N-terminal frizzled-containing domain.
Assuntos
Colágeno Tipo XVIII/química , Colágeno Tipo XVIII/genética , Processamento Alternativo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Cisteína/química , DNA Complementar/metabolismo , Éxons , Variação Genética , Humanos , Imuno-Histoquímica , Íntrons , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Peptídeos/química , Testes de Precipitina , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Peripheral lymphoid tissues act as important organs of immunological defense. Characteristic of their architecture is the rich reticular fiber meshwork composed of various extracellular matrix (ECM) molecules with which the stationary non-lymphatic cells stay in intimate contact and form channels through which the lymphatic cells travel. Here we studied the distribution of various laminin (Ln) chains and different types of collagens in human spleen, lymph node, and tonsil to clarify their chain-specific distribution. The most widely distributed proteins in all these organs were Ln chains alpha5, beta1, gamma1 and collagen types IV and XVIII, which were present in practically all compartments. Conversely, Ln alpha1, alpha2, alpha4, and type VII collagen showed a more restricted expression pattern. A unique feature was that Ln alpha3-, beta3-, and gamma2-chains, which normally are not localized to the vascular wall in non-lymphatic tissues, were present also in capillary basement membranes (BMs) of the follicular structures of lymph node and tonsil and in Ln alpha1-chain and type VII collagen also in the splenic white pulp. We also found that collagen XVII was exclusively present in the ring fibers of the spleen. The results indicate that BMs of lymphatic tissues contain a variety of macromolecules that probably contribute strongly to immunological events. In addition, capillaries of the lymphoid tissue exhibit a specified BM composition resembling that in epithelial BMs of non-lymphoid tissues.
Assuntos
Colágeno/biossíntese , Laminina/biossíntese , Tecido Linfoide/metabolismo , Membrana Basal/metabolismo , Humanos , Imuno-Histoquímica , Linfonodos/metabolismo , Tonsila Palatina/metabolismo , Baço/metabolismoRESUMO
Basement membranes (BMs) play an important role in anchoring epithelial cells and separating them from the adjacent stroma. Altered composition and assembly of BMs may influence carcinoma cell growth and invasion. Using immunohistochemistry and in situ hybridization, we investigated the expressions of the BMs components laminin-5 (Ln-5) subunits and collagen types IV, VII and XVII in normal endometrium and compared them to the expression pattern in hyperplastic and neoplastic endometrium. Chains of Ln-5 (alpha3beta3gamma2) and types IV, VII and XVII collagens were observed in normal endometrium. In hyperplastic endometrium, laminin gamma2 chain and type XVII collagen showed intensified expression in foci of dispersed epithelial cells. Individual carcinoma cells in adenocarcinomas of low differentiation grade displayed increased laminin gamma2 chain and type XVII collagen immunoreactivity and mRNA synthesis, whereas type VII collagen usually showed decreased expression. Laminin and type IV collagen showed BM disruptions, especially in tumors with low differentiation. Our results indicate that all the BM anchoring molecules investigated are expressed in normal endometrium, but the expression of laminin gamma2 chain and collagen type XVII is altered in endometrial adenocarcinomas, which support their role in malignant growth.
Assuntos
Adenocarcinoma/metabolismo , Autoantígenos/metabolismo , Membrana Basal/metabolismo , Neoplasias do Endométrio/metabolismo , Endométrio/metabolismo , Laminina/metabolismo , Colágenos não Fibrilares/metabolismo , Adenocarcinoma/patologia , Autoantígenos/genética , Membrana Basal/química , Membrana Basal/citologia , Neoplasias do Endométrio/patologia , Endométrio/química , Endométrio/citologia , Endométrio/patologia , Feminino , Humanos , Hibridização In Situ , Laminina/genética , Colágenos não Fibrilares/genética , Pele/citologia , Pele/metabolismo , Colágeno Tipo XVIIRESUMO
Prolyl 4-hydroxylase is the key enzyme of synthesis of collagens. Hydroxylation of a sufficient number of proline residues to hydroxyproline is necessary for the stability of triple helices in collagenous proteins, because non-hydroxylated non-triple-helical collagen polypeptide chains are degraded intracellularly. We studied 15 primary chondrosarcomas and osteosarcomas, 17 benign bone tumours and one case of fibrous dysplasia and chordoma using immunofluorescence staining with antibodies against the alpha(I) and alpha(II) subunits of type I and II prolyl 4-hydroxylases, and with antibodies against collagen types I and II. Type I prolyl 4-hydroxylase was found to be the predominant isoenzyme in both types of bone sarcoma, whereas the type II enzyme was more readily expressed by benign tumours. A feature of collagen staining, that was common to both sarcoma types, was that collagen types I and II were mainly found within cancer cells and were rarely present extracellularly. Extracellular collagen staining was more obvious in benign tumours. The results show that expression of prolyl 4-hydroxylase isoenzymes is altered in bone sarcomas as compared with normal bone tissue. Chondrous cells, which normally express mainly the type II isoenzyme, switch their expression pattern to that of type I. The findings provide evidence that type I is the major isoenzyme in malignant bone tumours, and probably in malignant neoplasms in general. The pattern of enzyme expression is considered to be associated with dedifferentiation of cancer cells.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Ósseas/enzimologia , Condrossarcoma/enzimologia , Cordoma/enzimologia , Displasia Fibrosa Óssea/enzimologia , Osteossarcoma/enzimologia , Pró-Colágeno-Prolina Dioxigenase/biossíntese , Animais , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Condrossarcoma/diagnóstico , Condrossarcoma/patologia , Cordoma/diagnóstico , Cordoma/patologia , Colágeno Tipo I/biossíntese , Colágeno Tipo II/biossíntese , Displasia Fibrosa Óssea/diagnóstico , Displasia Fibrosa Óssea/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/metabolismo , Microscopia de Fluorescência , Osteossarcoma/diagnóstico , Osteossarcoma/patologia , Valor Preditivo dos Testes , RatosRESUMO
Long-term fructose consumption has been shown to evoke leptin resistance, to elevate triglyceride levels and to induce insulin resistance and hepatic steatosis. Autophagy has been suggested to function in processes such as lipid storage in adipose tissue and inflammation in liver. Autophagy and the leptin system have also been suggested to regulate each other. This study aimed to identify the changes caused by fetal undernourishment and postnatal fructose diet in the gene expression of leptin, its receptors (LEPR-a, LEPR-b, LEPR-c, LEPR-e and LEPR-f) and autophagy genes in the white adipose tissue (WAT) and liver of adult male rats in order to clarify the mechanism behind the metabolic alterations. The data clearly revealed that the long-term postnatal fructose diet decreased leptin levels (p < 0.001), LEPR (p < 0.001), especially LEPR-b (p = 0.011) and LEPR-f (p = 0.005), as well as SOCS3 (p < 0.001), ACC (p = 0.006), ATG7 (p < 0.001), MAP1LC3ß (p < 0.001) and LAMP2 (p = 0.004) mRNA expression in WAT. Furthermore, LEPR (p < 0.001), especially LEPR-b (p = 0.001) and LEPR-f (p < 0.001), ACC (p = 0.010), ATG7 (p = 0.024), MAP1LC3ß (p = 0.003) and LAMP2 (p < 0.001) mRNA expression in the liver was increased in fructose-fed rats. In addition, the LEPR expression in liver and MAP1LC3ß expression in WAT together explained 55.7 % of the variation in the plasma triglyceride levels of the rats (R adj. (2) = 0.557, p < 0.001). These results, together with increased p62 levels in WAT (p < 0.001), could indicate decreased adipose tissue lipid storing capacity as well as alterations in liver metabolism which may represent a plausible mechanism through which fructose consumption could disturb lipid metabolism and result in elevated triglyceride levels.
RESUMO
Major cause of prematurity is spontaneous preterm birth (PTB) associated with intrauterine inflammation. Our aim was to establish a model of endotoxin Lipopolysaccharide-induced PTB of live-born pups and to study early immune activation in fetal and maternal compartments. Expression of several proteins that bind microbes (Toll-like receptors TLR4, TLR2; surfactant proteins SP-A, SP-D) was analyzed. At 16 or 17 d of gestation, C57BL/6 dams received a single dose of intraperitoneal LPS, leading to PTB within 17 h. Cytokine levels increased in maternal serum, followed by a modest increase in fetal serum and in amniotic fluid. In uterus, placenta, and fetal membranes, LPS mostly increased the expressions of TLR, SPs, and cytokines. The number of TLR2-positive macrophages increased in labyrinthine placenta. In fetal lung, intestine, liver, and brain there were modest changes in cytokine expressions. In fetal lung, SP and TLR mRNAs decreased and TLR2-positive macrophages redistributed around vessels. LPS-induced fetal deaths associated with early age (16 d gestation) rather than with proinflammatory activation. Here we propose that maternal LPS response leads to PTB and acute decrease of immune proteins in epithelial lining of fetal lung. Instead, acceleration of lung maturity has been previously observed in intraamniotic inflammation.
Assuntos
Colectinas/metabolismo , Citocinas/metabolismo , Feto/imunologia , Inflamação/imunologia , Troca Materno-Fetal , Nascimento Prematuro/etiologia , Receptores Toll-Like/metabolismo , Doenças Uterinas/imunologia , Líquido Amniótico/imunologia , Animais , Quimiocina CCL2/metabolismo , Colectinas/sangue , Citocinas/sangue , Membranas Extraembrionárias/imunologia , Feminino , Sangue Fetal/imunologia , Morte Fetal , Idade Gestacional , Imuno-Histoquímica , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Pulmão/embriologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Placenta/imunologia , Gravidez , Nascimento Prematuro/imunologia , Nascimento Prematuro/fisiopatologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Fatores de Tempo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/sangue , Fator de Necrose Tumoral alfa/metabolismo , Doenças Uterinas/induzido quimicamente , Doenças Uterinas/fisiopatologia , Útero/imunologiaRESUMO
Overexpression of type XIII collagen molecules with an 83-amino-acid residue in-frame deletion of part of the ectodomain leads to fetal lethality in Col13a1COL2del transgenic mice. We characterize here the functional disturbances in the cardiovascular system of mouse fetuses overexpressing mutant type XIII collagen. Doppler ultrasonography was performed at 12.5 days of gestation on 33 fetuses resulting from heterozygous matings of seven female mice and on 16 fetuses from two matings between heterozygous and wild-type mice. Nine fetuses had atrioventricular valve regurgitation (AVVR), and all of them were transgene-positive. The fetuses with AVVR had a lower outflow mean velocity (Vmean; P<0.005) and a greater proportion of isovolumetric relaxation time (IRT%) in the cardiac cycle (P<0.0001) than those without AVVR, and their ductus venosus pulsatility indices for veins (DV PIV) and the umbilical artery pulsatility indices were increased. A positive correlation was found between IRT% and DV PIV, and a negative correlation was seen between outflow V(mean) and DV PIV. Morphological analysis of the heart revealed no differences between the two groups of fetuses, but histological analysis showed the trabeculation of the ventricles to be reduced and the myocardium to be thinner in the fetuses with AVVR. Based on in situ hybridization, type XIII collagen mRNAs were normal constituents of these structures. Moreover, a positive correlation was found between outflow Vmean and myocardial thickness. IRT% and DV PIV correlated negatively with myocardial thickness. Thus, overexpression of mutant type XIII collagen results in mid-gestation cardiac dysfunction in mouse fetuses, and these disturbances in cardiac function may lead to death in utero.