RESUMO
Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by deposition of autoantibodies at the basement membrane zone. In an experimental BP model in mice, the subepidermal blistering is mediated by antibodies directed against the hemidesmosomal protein BP180 (collagen XVII, BPAG2), and depends on complement activation and neutrophil infiltration. Gelatinase B is present in BP blister fluid and can cleave BP180. In this study we investigated the role of gelatinase B in the immunopathogenesis of experimental BP using mice containing targeted disruption of the gelatinase B (MMP-9, 92 kD gelatinase) gene. Gelatinase B-deficient mice were resistant to the blistering effect of intracutaneous anti-mBP180 antibodies, although these mice showed deposition of autoantibodies at the basement membrane zone and neutrophil recruitment to the skin comparable to that observed in the control mice. Interleukin 8 given intradermally concomitantly with pathogenic anti-mBP180 elicited a significant neutrophil recruitment into the skin in gelatinase B-deficient mice, but blistering did not occur. However, gelatinase B-deficient mice reconstituted with neutrophils from normal mice developed blistering in response to anti-mBP180 antibodies. These results implicate neutrophil-derived gelatinase B in the pathogenesis of experimental BP and might lead to novel therapeutic strategies for BP.
Assuntos
Colagenases/fisiologia , Penfigoide Bolhoso/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Autoantígenos/imunologia , Colagenases/genética , Modelos Animais de Doenças , Deleção de Genes , Humanos , Imunidade Inata , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Colágenos não Fibrilares , Coelhos , Colágeno Tipo XVIIRESUMO
Interleukin (IL)-13 is a key mediator of tissue fibrosis caused by T helper cell type 2 inflammation. We hypothesized that the fibrogenic effects of IL-13 are mediated by transforming growth factor (TGF)-beta. To test this hypothesis we compared the regulation of TGF-beta in lungs from wild-type mice and CC10-IL-13 mice in which IL-13 overexpression causes pulmonary fibrosis. IL-13 selectively stimulated TGF-beta(1) production in transgenic animals and macrophages were the major site of TGF-beta(1) production and deposition in these tissues. IL-13 also activated TGF-beta(1) in vivo. This activation was associated with decreased levels of mRNA encoding latent TGF-beta-binding protein-1 and increased mRNA encoding urinary plasminogen activator, matrix metalloproteinase (MMP)-9, and CD44. TGF-beta(1) activation was abrogated by the plasmin/serine protease antagonist aprotinin. It was also decreased in progeny of crosses of CC10-IL-13 mice and MMP-9 null mice but was not altered in crosses with CD44 null animals. IL-13-induced fibrosis was also significantly ameliorated by treatment with the TGF-beta antagonist soluble TGFbetaR-Fc (sTGFbetaR-Fc). These studies demonstrate that IL-13 is a potent stimulator and activator of TGF-beta(1) in vivo. They also demonstrate that this activation is mediated by a plasmin/serine protease- and MMP-9-dependent and CD44-independent mechanism(s) and that the fibrogenic effects of IL-13 are mediated, in great extent, by this TGF-beta pathway.
Assuntos
Interleucina-13/imunologia , Fibrose Pulmonar/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Receptores de Hialuronatos/fisiologia , Interleucina-13/genética , Metaloproteinase 9 da Matriz/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
The insulin-like growth factor (IGF) axis plays key roles in normal tissue growth and development as well as in the progression of several tumour types and their subsequent growth and progression to a metastatic phenotype. This review explores the role of IGF system in normal germ cell development and function in addition to examining the evidence for deregulation of IGF signalling in cancer, with particular relevance to evidence supporting a role in testicular germ cell tumours (TGCTs). Despite the clear preclinical rationale for targeting the IGF axis in cancer, there has been a lack of progress in identifying which patients may benefit from such therapy. Future employment of agents targeting the IGF pathway is expected to concentrate on their use in combination with other treatments to prevent resistance and exploit their potential as chemo- and radiosensitizers.
Assuntos
Células Germinativas/metabolismo , Neoplasias Embrionárias de Células Germinativas/metabolismo , Receptor IGF Tipo 1/metabolismo , Somatomedinas/metabolismo , Neoplasias Testiculares/metabolismo , Animais , Carcinogênese , Resistencia a Medicamentos Antineoplásicos , Humanos , Terapia de Alvo Molecular , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Neoplasias Embrionárias de Células Germinativas/etiologia , Neoplasias Testiculares/tratamento farmacológico , Neoplasias Testiculares/etiologiaRESUMO
Abdominal aortic aneurysms represent a life-threatening condition characterized by chronic inflammation, destructive remodeling of the extracellular matrix, and increased local expression of matrix metalloproteinases (MMPs). Both 92-kD gelatinase (MMP-9) and macrophage elastase (MMP-12) have been implicated in this disease, but it is not known if either is necessary in aneurysmal degeneration. We show here that transient elastase perfusion of the mouse aorta results in delayed aneurysm development that is temporally associated with transmural mononuclear inflammation, increased local production of several elastolytic MMPs, and progressive destruction of the elastic lamellae. Elastase-induced aneurysmal degeneration was suppressed by treatment with a nonselective MMP inhibitor (doxycycline) and by targeted gene disruption of MMP-9, but not by isolated deficiency of MMP-12. Bone marrow transplantation from wild-type mice prevented the aneurysm-resistant phenotype in MMP-9-deficient animals, and wild-type mice acquired aneurysm resistance after transplantation from MMP-9-deficient donors. These results demonstrate that inflammatory cell expression of MMP-9 plays a critical role in an experimental model of aortic aneurysm disease, suggesting that therapeutic strategies targeting MMP-9 may limit the growth of small abdominal aortic aneurysms.
Assuntos
Aneurisma da Aorta Abdominal/prevenção & controle , Metaloproteinase 9 da Matriz/fisiologia , Animais , Aneurisma da Aorta Abdominal/etiologia , Transplante de Medula Óssea , Doxiciclina/farmacologia , Marcação de Genes , Metaloproteinase 9 da Matriz/genética , Camundongos , Elastase Pancreática/fisiologiaRESUMO
Latent transforming growth factor beta (TGF-beta) binding protein 2 (LTBP-2) is an integral component of elastin-containing microfibrils. We studied the expression of LTBP-2 in the developing mouse and rat by in situ hybridization, using tropoelastin expression as a marker of tissues participating in elastic fiber formation. LTBP-2 colocalized with tropoelastin within the perichondrium, lung, dermis, large arterial vessels, epicardium, pericardium, and heart valves at various stages of rodent embryonic development. Both LTBP-2 and tropoelastin expression were seen throughout the lung parenchyma and within the cortex of the spleen in the young adult mouse. In the testes, LTBP-2 expression was seen within lumenal cells of the epididymis in the absence of tropoelastin. Collectively, these results imply that LTBP-2 plays a structural role within elastic fibers in most cases. To investigate its importance in development, mice with a targeted disruption of the Ltbp2 gene were generated. Ltbp2(-/-) mice die between embryonic day 3.5 (E3.5) and E6.5. LTBP-2 expression was not detected by in situ hybridization in E6.5 embryos but was detected in E3.5 blastocysts by reverse transcription-PCR. These results are not consistent with the phenotypes of TGF-beta knockout mice or mice with knockouts of other elastic fiber proteins, implying that LTBP-2 performs a yet undiscovered function in early development, perhaps in implantation.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Animais , Biomarcadores , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Proteínas de Ligação a TGF-beta Latente , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , Gravidez , Ratos , Língua/embriologia , Língua/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Tropoelastina/genética , Tropoelastina/metabolismoRESUMO
Lobular carcinoma in situ (LCIS) and atypical lobular hyperplasia (ALH) of the breast are cytologically similar breast lesions that reportedly carry different relative risks of subsequent development of invasive carcinoma. They are frequently multifocal and bilateral. We have identified the chromosomal copy number changes in 31 LCIS and 14 ALH lesions from 28 cases and also the 7 invasive carcinomas that subsequently developed in 6 of these cases. This was achieved by comparative genomic hybridization analysis of microdissected formalin-fixed, paraffin-embedded material. There was no significant difference between the aberrations found in the unilateral versus the bilateral cases of LCIS. Loss of material from 16p, 16q, 17p, and 22q and also gain of material from 6q were found at a similar high frequency in LCIS and ALH. Loss of these genomic regions may indicate the locations of genes that predispose to the development of the lesions, and the results are consistent with LCIS and ALH representing the same genetic stage of development. Comparison of the comparative genomic hybridization results from LCIS/ALH with those from ductal carcinoma in situ and invasive cancer showed some similarities at the chromosomal level, but it also showed significant differences, including gain of 1q and 8q and evidence for genomic amplification, which were not found in LCIS/ALH. A genetic model is postulated for the possible relationships between noninvasive lobular lesions and invasive breast carcinoma, delineating potential roles for specific chromosome copy number changes.
Assuntos
Neoplasias da Mama/genética , Carcinoma in Situ/genética , Carcinoma Lobular/genética , Aberrações Cromossômicas , Hibridização de Ácido Nucleico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Dosagem de Genes , Humanos , Pessoa de Meia-Idade , Modelos GenéticosRESUMO
A high proportion of synovial sarcomas contain the reciprocal translocation t(X;18)(p11.2;q11.2). We have previously localized the breakpoint on the X chromosome between the X chromosome marker DXS255 and an ornithine aminotransferase (OAT) pseudogene region designated OATL2. Subsequently by fluorescence in situ hybridization (FISH) we provided evidence that YACs corresponding to the OATL2 locus spanned the break-point. In order to confirm the position of this breakpoint cosmids corresponding to the OATL2 region were isolated. Most of these cosmids mapped to four cosmid contigs designated C1-C4. Analysis of two contigs, C1- and C4, using FISH established that in four of six synovial sarcomas examined the breakpoint occurs between these two contigs: C1 lies distal to the break-point while C4 is proximal. In contrast we provide evidence that the breakpoint in the remaining two tumours mapped to a second pseudogene region called OATL1 that is telomeric to the OATL2 locus. This heterogeneity of the breakpoint position on the X chromosome explains why in previous mapping studies there have been discrepancies between the results obtained by different laboratories.
Assuntos
Cromossomos Humanos Par 18 , Sarcoma Sinovial/genética , Translocação Genética , Cromossomo X , Mapeamento Cromossômico , Cosmídeos/genética , Humanos , Hibridização in Situ FluorescenteRESUMO
Gain of 12p material is invariably associated with testicular germ cell tumors (TGCTs) of adolescents and adults, most usually as an isochromosome 12p. We analyzed TGCTs with i(12p) using a global approach to expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2-p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype showed higher levels of expression than seminomas. Notably, 12p11.2-p12.1 is amplified in about 10% of TGCTs and CESH analysis of such amplicon cases showed high levels of overexpression from this region. Microarray analysis, including cDNA clones representing most UniGene clusters from 12p11.2-p12.1, was applied to DNA and RNA from 5 TGCTs with amplification of 12p11.2-p12.1 and seven TGCTs with gain of the entire short arm of chromosome 12. Expression profiles were consistent with the CESH data and overexpression of EST595078, MRPS35 and LDHB at 12p11.2-p12.1 was detected in most TGCTs. High-level overexpression of BCAT1 was specific to nonseminomas and overexpression of genes such as CMAS, EKI1, KRAS2, SURB7 and various ESTs correlated with their amplification. Genes such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were also overexpressed. The overexpressed sequences identified, particularly those in the region amplified, represent candidate genes for involvement in TGCT development.
Assuntos
Cromossomos Humanos Par 12 , Amplificação de Genes , Perfilação da Expressão Gênica , Germinoma/genética , Neoplasias Testiculares/genética , Adolescente , Adulto , Humanos , Masculino , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Staphylococcus aureus infections developed in 16 of 721 infants born at a general hospital during a six-month period (October 1972 to March 1973). Twelve of the 16 affected infants had cervical adenitis, which usually became manifest two to four months after they were discharged from the hospital. Although most infants with adenitis underwent incision and drainage procedures, physicians noted few constitutional symptoms. Staphylococcal phage types from infants with adenitis were identical to phage types commonly recovered from colonized newborns and nursery personnel at the hospital. Despite the long period between hospital discharge and onset of clinical symptoms, cases of cervical adenitis were probably nosocomial in origin.
Assuntos
Infecção Hospitalar/microbiologia , Surtos de Doenças , Linfadenite/microbiologia , Infecções Estafilocócicas , Infecção Hospitalar/prevenção & controle , Surtos de Doenças/prevenção & controle , Feminino , Humanos , Lactente , Linfadenite/prevenção & controle , Estudos Retrospectivos , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureusRESUMO
Karyotypes of three sublines of the human cell line U937 showed considerable variation, but all contained four consistent marker chromosomes (i.e., 3q-, 11q-, 16p+, and 17p- chromosomes). The 11q- chromosome appeared to be derived from either an interstitial deletion in bands 11q21-23 or from a translocation with an unidentified chromosome. The presence of this chromosome was of particular interest because rearrangements of chromosome #11 at band 11q23 are often associated with malignancies of the monocytic lineage. The possible significance of these findings is discussed.
Assuntos
Aberrações Cromossômicas , Marcadores Genéticos , Monócitos/ultraestrutura , Células Tumorais Cultivadas , Humanos , Cariotipagem , Células-Tronco/ultraestrutura , Células Tumorais Cultivadas/patologiaRESUMO
Cytogenetic and fluorescent in situ hybridization (FISH) analysis has been performed on consecutive samples, taken 4 weeks apart, from a phyllodes breast tumor. This revealed the presence of two different chromosome 1 derivatives, namely a dic(1;10)(q10;q24) in the first sample and an i(1) (q10) in the second. In one cell out of 25 from the second sample both derivative chromosomes were seen. A chromosome 21 was lost in both samples. These results are consistent with phyllodes tumors having a clonal origin.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 1 , Tumor Filoide/genética , Translocação Genética , Idoso , Feminino , Humanos , Hibridização in Situ Fluorescente , CariotipagemRESUMO
Alveolar rhabdomyosarcoma is an aggressive childhood tumor that exhibits muscle cell differentiation. Cytogenetically, it is characterized by t(2;13)(q35;q14); no consistent secondary abnormalities have been reported. Cytogenetic analysis of bone marrow in a case of alveolar rhabdomyosarcoma revealed t(2;13)(q35;q14) and der(16)t(1;16)(q21;q13). The present case and a review of the literature suggest that up to 11% of these tumors possess der(16)t(1;16)(q21;q13). This is similar to the incidence observed in the Ewing family of tumors, where unbalanced der(16)t(1;16) translocations, resulting in partial trisomy of 1q, are regarded as a consistent secondary cytogenetic change.
Assuntos
Cromossomos Humanos Par 16 , Cromossomos Humanos Par 1 , Rabdomiossarcoma Alveolar/genética , Neoplasias de Tecidos Moles/genética , Translocação Genética , Criança , Humanos , Cariotipagem , MasculinoRESUMO
We report the cytogenetic findings in a case of nodular fasciitis of the breast. The abnormalities found in all 11 metaphases available for analysis were -2, -2, -13, der(15)t(2;15)(q31;q26), + der(?) t(?;2), + mar1, + mar2. Other consistent abnormalities were also identified. Fluorescence in situ hybridization (FISH) was used to confirm the origin of some of the chromosomes. A large acrocentric chromosome was confirmed to be derived from chromosome 15 with chromosome 2 material translocated onto the q arm. The metacentric der(?)t(?;2) was demonstrated to have part of chromosome 2 on the q arm. No other chromosome 2 material was found. Eight of 11 cells were tetraploid and had two copies of a del(6)(q16q24).
Assuntos
Doenças Mamárias/genética , Fasciite/genética , Idoso , Idoso de 80 Anos ou mais , Aberrações Cromossômicas , Humanos , CariotipagemRESUMO
McBride and Ozer (1) were the first to show that purified metaphase chromosomes could act as vectors in transferring genetic information into mammalian cells. This technique, termed chromosome-mediated gene transfer (CMGT), involves the transfer of subchromosomal fragments from the cells of one species (the donor, usually human) to those of another (the recipient, usually a rodent) that have the ability to maintain them. CMGT thus fills a gap between the isolation of whole chromosomes, produced by cell fusion and microcell-mediated gene transfer, and the isolation of relatively short stretches of DNA by cloning or transfection methods.
Assuntos
Neoplasias da Mama/genética , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/genética , Rearranjo Gênico , Tumor Filoide/genética , Ribonucleoproteínas/genética , Fatores de Transcrição/genética , Feminino , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Proteína FUS de Ligação a RNA , Fator de Transcrição CHOPRESUMO
Pulmonary surfactant protein A (SP-A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro-organisms, and mediates microbial lysis and clearance. SP-A also modulates multiple immune-related functions including cytokine production and chemotaxis for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril-associated protein 4 (MFAP4) and SP-A. MFAP4 is a collagen-binding molecule containing a C-terminal fibrinogen-like domain and a N-terminal located integrin-binding motif. We produced recombinant MFAP4 with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium-dependent binding between MFAP4 and human SP-A1 and SP-A2. No binding was seen to recombinant SP-A composed of the neck region and carbohydrate recognition domain of SP-A indicating that the interaction between MFAP4 and SP-A is mediated via the collagen domain of SP-A. Monoclonal antibodies directed against MFAP4 and SP-A were used for immunohistochemical analysis, which demonstrates that the two molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP-A via the collagen region in vitro, and that MFAP4 and SP-A colocates in different lung compartments indicating that the interaction may be operative in vivo.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Pulmão/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Animais , Sítios de Ligação/imunologia , Western Blotting , Líquido da Lavagem Broncoalveolar/imunologia , Células CHO , Cálcio/imunologia , Proteínas de Transporte/imunologia , Cricetinae , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/imunologia , Matriz Extracelular/ultraestrutura , Proteínas da Matriz Extracelular/imunologia , Glicoproteínas/imunologia , Humanos , Imuno-Histoquímica , Pulmão/imunologia , Microscopia Imunoeletrônica , Ligação Proteica , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Phosphorylation of mannose residues on N-linked oligosaccharide side chains of lysosomal enzymes targets them to lysosomes. We used site-directed mutagenesis to observe the effect of eliminating selective glycosylation sites from human beta-glucuronidase on enzyme sorting. Expression studies allowed us to determine which of four potential sites were glycosylated, preferentially phosphorylated, and required for catalytic activity. All four sites of the human enzyme were glycosylated, whereas in the mouse and rat enzymes, only three of four sites are used. Sites 2 and 3 were preferentially phosphorylated. Elimination of sites 2 and 3 in combination markedly decreased sorting to lysosomes and increased enzyme secretion. Each of the four glycosylation sites could be eliminated individually without drastic reduction in catalytic activity. Activity was progressively lost as combinations of two, three, and four sites were eliminated. Wild-type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for realization of enzyme activity. However, active enzyme could be deglycosylated with only minimal loss of activity. Mutant enzyme completely lacking glycosylation did not form tetramers. Partial restoration of tetramerization was achieved by the co-expression of normal rat enzyme, provided that the normal rat enzyme supplied at least two subunits to the tetramer.
Assuntos
Glucuronidase/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Sequência de Bases , Linhagem Celular , Glucuronidase/genética , Glucuronidase/isolamento & purificação , Glicosilação , Humanos , Lisossomos/enzimologia , Substâncias Macromoleculares , Metionina/metabolismo , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Fosforilação , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , TransfecçãoRESUMO
Increased expression of matrix metalloproteinases, particularly gelatinase B (MMP-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B+/+ littermates. Twenty-one days after bleomycin the two groups of mice were indistinguishable in terms of pulmonary histology and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, whereas bronchiolization was prominent in the lungs of gelatinase B+/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 and 14 days after bleomycin in gelatinase B+/+ mice, and whole lung gelatinase B mRNA was increased at the same times. Many bronchiolized cells displayed Clara cell features by electron microscopy. Some bronchiolized cells stained with antibody to helix transcription factor 4, a factor associated with the ciliated cell phenotype. Thus, fibrosing alveolitis develops after intratracheal bleomycin irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into the regions of alveolar injury.