Assuntos
Derme/fisiologia , Epiderme/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Subpopulações de Linfócitos T/imunologia , Animais , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Derme/imunologia , Epiderme/imunologia , Humanos , Interleucina-7/genética , Interleucina-7/fisiologia , Camundongos , Camundongos TransgênicosRESUMO
Matrix metalloproteinases (MMPs) are thought to play a key role in tumor invasion and metastasis. The role of MMP-9 (gelatinase B) in tumor metastasis was examined in MMP-9-deficient mice produced by gene targeting using embryonic stem cells. MMP-9-deficient mice develop normally and are fertile. In these mice, the number of metastatic colonies of B16-BL6 melanoma cells or Lewis lung carcinoma cells that were implanted intravenously fell by 45% for B16-BL6 melanoma and 59% for Lewis lung carcinoma (p = 0.03 and p = 0.0043, respectively). Gelatin zymography showed that both tumor cell lines did not secrete MMP-9 by themselves but the host cells surrounding the tumor cells secrete MMP-9 in vivo. These results indicated that host-derived MMP-9 plays an important role in the process of tumor metastasis.
Assuntos
Colagenases/fisiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/patologia , Animais , Colagenases/deficiência , Gelatina/análise , Gelatinases/metabolismo , Infusões Intravenosas , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/fisiologiaRESUMO
The liver and activation-regulated chemokine (LARC), also termed MIP-3alpha and Exodus, is a novel human CC chemokine with a selective chemotactic activity for lymphocytes and dendritic cells. Here we describe genomic and cDNA clones encoding the murine orthologue of LARC (mLARC). The gene consists of four exons and three introns. The 5'-noncoding region of about 400 bp contains typical TATA and CAAT boxes but no other potential regulatory elements so far described. The cDNA encodes a CC chemokine of 97 amino acid residues with the highest homology to human LARC (64% amino acid identity). The 3'-noncoding region contains as many as five potential mRNA destabilization signals. mLARC was strongly and transiently induced in the murine monocytoid cell line J774 by lipopolysaccharide (LPS) but not by cytokines such as TNF-alpha, IFN-gamma, IL-1beta or IL-4. In normal mice, mLARC mRNA was expressed selectively in intestinal tissues such as small intestine and colon. Upon treatment with LPS, mLARC expression was enhanced in intestinal tissues and induced in some lymphoid tissues such as lymph nodes. Because of alternative splicing, there are two types of transcripts encoding mLARC and its variant mLARCvar with and without an N-terminal alanine in the mature protein, respectively. Both types of transcripts appeared to be expressed in various mouse tissues. In situ hybridization revealed that epithelial cells of intestinal tissues, especially those lining lymphoid follicles, expressed mLARC. Localization of LARC mRNA in epithelial cells was also demonstrated in a human appendix. Furthermore, mLARC was efficiently chemotactic for cells such as gammadelta type T cells in intestinal epithelium and naive B cells in Peyer's patches. Thus, in both humans and mice, LARC may be physiologically involved in formation and function of the mucosal lymphoid tissues by attracting lymphocytes and dendritic cells toward epithelial cells.
Assuntos
Quimiocinas CC/biossíntese , Quimiocinas CC/imunologia , Mucosa Intestinal/imunologia , Proteínas Inflamatórias de Macrófagos , Receptores de Quimiocinas , Sequência de Aminoácidos , Animais , Sequência de Bases , Quimiocina CCL20 , DNA Complementar/análise , Humanos , Imunidade nas Mucosas , Hibridização In Situ , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/análise , Receptores CCR6RESUMO
Interleukin (IL)-7 transgenic mice, which we established previously, developed severe dermatitis characterized by massive infiltration of gammadelta T cells in the dermal lesion. To fully understand the pathology of this intriguing skin disease, we examined several immunologic features of dermis infiltrating lymphocytes from the lesional skin of IL-7 transgenic mice. We observed a moderate response to mitogens, a poor response to alloantigens, and the absence of cytotoxic activities to several tumor cell lines and skin derived cells regardless of the presence of IL-2 or IL-7. On the other hand, dermis infiltrating lymphocytes could proliferate with exogenous IL-2 and IL-7. Moreover, reverse transcriptase polymerase chain reaction and fluorescence activated cell sorter analysis revealed that dermis infiltrating lymphocytes expressed various cytokines including IL-4 and IL-7, and several activation markers for T cells (CD44, CD69, IL-2R alpha), in addition to IL-7R alpha. In the sera of the affected mice, hyper epsilon-globulinemia was observed. These findings suggested that dermis infiltrating lymphocytes proliferated in an activated state in the skin lesion in an autocrine and/or paracrine manner and produced Th2 type cytokines that might evoke immunologic abnormalities. This study and previous findings suggest that IL-7 transgenic mouse with dermatitis offer the potential of serving as a useful tool for investigating the immunologic role of cutaneous gammadelta T cells, especially their participation in IgE production in vivo.
Assuntos
Dermatite/genética , Dermatite/imunologia , Interleucina-7/genética , Camundongos Transgênicos/genética , Animais , Biomarcadores , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Citocinas/genética , Citocinas/farmacologia , Dermatite/sangue , Imunoglobulina E/análise , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Linfócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mitógenos/farmacologia , RNA Mensageiro/metabolismo , Pele/imunologia , Baço/citologia , Baço/fisiologia , Células Th1/metabolismo , Células Th2/metabolismo , Células Tumorais CultivadasRESUMO
Longitudinal bone growth is determined by the process of endochondral ossification in the cartilaginous growth plate, which is located at both ends of vertebrae and long bones and involves many systemic hormones and local regulators. Natriuretic peptides organize a family of three structurally related peptides: atrial natriuretic peptide, brain natriuretic peptide (BNP), and C-type natriuretic peptide. Atrial natriuretic peptide and BNP are cardiac hormones that are produced predominantly by the atrium and ventricle, respectively. C-type natriuretic peptide occurs in a wide variety of tissues, where it acts as a local regulator. These peptides can influence body fluid homeostasis and blood pressure control through the activation of two guanylyl cyclase (GC)-coupled natriuretic peptide receptor subtypes-GC-A and GC-B. We report here marked skeletal overgrowth in transgenic mice that overexpress BNP. Transgenic mice with elevated plasma BNP concentrations exhibited deformed bony skeletons characterized by kyphosis, elongated limbs and paws, and crooked tails. Bone abnormalities resulted from a high turnover of endochondral ossification accompanied by overgrowth of the growth plate. Studies using an in vitro organ culture of embryonic mouse tibias revealed that BNP increases the height of cartilaginous primordium directly, thereby stimulating the total longitudinal bone growth. The present study demonstrates that natriuretic peptides can affect the process of endochondral ossification.
Assuntos
Desenvolvimento Ósseo/genética , Osso e Ossos/anormalidades , Proteínas do Tecido Nervoso/fisiologia , Osteogênese/genética , Envelhecimento , Animais , Osso e Ossos/patologia , Desenvolvimento Embrionário e Fetal , Humanos , Camundongos , Camundongos Transgênicos , Peptídeo Natriurético Encefálico , Proteínas do Tecido Nervoso/sangue , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Componente Amiloide P Sérico/biossíntese , Componente Amiloide P Sérico/genética , Tíbia/embriologia , Tíbia/patologiaRESUMO
We have demonstrated that intestinal epithelial cells produce interleukin 7 (IL-7), and IL-7 serves as a potent regulatory factor for proliferation of intestinal mucosal lymphocytes expressing functional IL-7 receptor. To clarify the mechanism by which locally produced IL-7 regulates the mucosal lymphocytes, we investigated IL-7 transgenic mice. Here we report that transgenic mice expressing murine IL-7 cDNA driver by the SRalpha promoter developed chronic colitis in concert with the expression of SRalpha/IL-7 transgene in the colonic mucosa. IL-7 transgenic but not littermate mice developed chronic colitis at 4-12 wk of age, with histopathological similarity to ulcerative colitis in humans. Southern blot hybridization and competitive PCR demonstrated that the expression of IL-7 messenger RNA was increased in the colonic mucosal lymphocytes but not in the colonic epithelial cells. IL-7 protein accumulation was decreased in the goblet cell-depleted colonic epithelium in the transgenic mice. Immunohistochemical and cytokine production analysis showed that lymphoid infiltrates in the lamina propria were dominated by T helper cell type 1 CD4+ T cells. Flow cytometric analysis demonstrated that CD4+ intraepithelial T cells were increased, but T cell receptor gamma/delta T cells and CD8alpha/alpha cells were not increased in the area of chronic inflammation. Increased IL-7 receptor expression in mucosal lymphocytes was demonstrated in the transgenic mice. These findings suggest that chronic inflammation in the colonic mucosa may be mediated by dysregulation of colonic epithelial cell-derived IL-7, and this murine model of chronic colitis may contribute to the understanding of the pathogenesis of human inflammatory bowel disease.
Assuntos
Colite/genética , Interleucina-7/metabolismo , Mucosa Intestinal/metabolismo , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Southern Blotting , Colite/etiologia , Colite/imunologia , Colite/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Inflamação/imunologia , Interleucina-7/genética , Interleucina-7/farmacologia , Mucosa Intestinal/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-7 , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Brain natriuretic peptide (BNP) is a cardiac hormone that occurs predominantly in the ventricle. To study the roles of BNP in chronic cardiovascular regulation, we isolated mouse BNP cDNA and genomic clones, and generated transgenic mice with elevated plasma BNP concentration. The mouse BNP gene was organized into three exons and two introns. Two BNP mRNA species were identified, which were generated by the alternative mRNA splicing. The ventricle was a major site of BNP production in mice. Mouse preproBNP was a 121- (or 120-) residue peptide, and its COOH-terminal 45-residue peptide was the major storage form in the heart. Transgenic mice carrying the human serum amyloid P component/mouse BNP fusion gene were generated so that the hormone expression is targeted to the liver. In the liver of these mice, considerable levels of BNP mRNA and peptide were detected, reaching up to 10-fold greater than in the ventricle. These animals showed 10- to 100-fold increase in plasma BNP concentration accompanied by elevated plasma cyclic GMP concentration, and had significantly lower blood pressure than their nontransgenic littermates. The present study demonstrates that these mice provide a useful model system with which to assess the roles of BNP in cardiovascular regulation and suggests the potential usefulness of BNP as a long-term therapeutic agent.
Assuntos
Química Encefálica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Reações Cruzadas , GMP Cíclico/sangue , DNA Complementar/genética , Expressão Gênica , Ventrículos do Coração/metabolismo , Fígado/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeo Natriurético Encefálico , Proteínas do Tecido Nervoso/sangue , Radioimunoensaio , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Transgenic mice constitutively expressing IL-7 developed severe dermatitis with erythroderma and alopecia. The skin lesions were characterized by massive infiltration of mononuclear cells. Immunofluorescence staining showed that most of the infiltrating cells were T cells with the majority bearing the gamma delta TCR other than the V gamma 5 moiety. Furthermore, the number of gamma delta T cells had increased in the lymphoid organs of the dermatitis animals. These findings indicate the strong relationship between the expression of IL-7 and the development of gamma delta T cells in vivo and the pathological involvement of proliferated and/or activated gamma delta T cells in skin disease.
Assuntos
Dermatite/imunologia , Interleucina-7/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T/fisiologia , Animais , Anticorpos Monoclonais , Sequência de Bases , Movimento Celular , Dermatite/patologia , Citometria de Fluxo , Imuno-Histoquímica , Interleucina-7/biossíntese , Interleucina-7/sangue , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Dados de Sequência MolecularRESUMO
Previously, we showed that transgenic expression of the MHC (major histocompatibility complex) class II I-E molecules prevented insulitis in non-obese diabetic (NOD) mice at the age of 19 weeks. To rule out the possibility that the I-E expression merely delays the onset of insulitis, we have further characterized the expression and function of the I-E molecule expressed in transgenic NOD mice and confirmed our previous observations. Northern blot analysis showed that the transgenic E alpha d gene was expressed in a pattern similar to the endogenous E alpha d gene in BALB/c mice. The newly expressed I-E molecules were recognized as an alloantigen by the T lymphocytes of normal NOD mice as shown by mixed lymphocyte reaction (MLR). Transgenic NOD mice were resistant to the treatment by cyclophosphamide, which effectively induces diabetes in normal NOD mice, and did not develop diabetes up to 40 weeks of age. On the basis of these findings, we discuss the role of I-E molecules in the prevention of diabetes in NOD mice.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Expressão Gênica , Terapia Genética , Antígenos de Histocompatibilidade Classe II/genética , Animais , Animais Geneticamente Modificados , Ilhotas Pancreáticas/patologia , Linfócitos/fisiologia , Camundongos , Camundongos Mutantes , Monócitos/fisiologiaRESUMO
Insulin-dependent diabetes mellitus is characterized by the infiltration of lymphocytes into the islets of Langerhans of the pancreas (insulitis) followed by destruction of insulin-secreting beta-cells leading to overt diabetes. The best model for the disease is the non-obese diabetic (NOD) mouse. Two unusual features of the class II major histocompatibility complex (MHC) of the NOD mouse are the absence of I-E and the presence of unique I-A molecules (I-ANOD), in which aspartic acid at position 57 of the beta-chain is replaced by serine. This feature is also found in the HLA-DQ chain of many Caucasians with insulin-dependent diabetes mellitus. We have previously reported that the expression of I-E prevents the development of insulitis in NOD mouse. Here we report that the expression of I-Ak (A alpha kA beta k) in transgenic NOD mice can also prevent insulitis, and that this protection is seen not only when the I-A beta-chain has aspartic acid as residue 57, but also when this residue is serine. These results show that the single amino-acid substitution at position 57 of the I-A beta-chain from aspartic acid to serine is not sufficient for the development of the disease.
Assuntos
Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Ilhotas Pancreáticas/patologia , Linfócitos/patologia , Animais , Ácido Aspártico , Sequência de Bases , Diabetes Mellitus Experimental/patologia , Feminino , Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Conformação Proteica , SerinaRESUMO
The transgenic mice were produced by injecting eggs of B6 and C3H/HeJ mice with the human E mu-myc gene. Preferential development of B lymphomas was observed in the B6 transgenic mice, whereas the C3H/HeJ transgenic mice developed mostly T lymphomas. The phenotypic activation of B lineage cells but not of T lineage cells was detected in the prelymphomatous transgenic mice of both strains. The transgene was similarly expressed in B and T cells of the transgenic mice of both strains. These results suggest that a high incidence of T lymphomas in the C3H/HeJ transgenic mice may not be due to the preferential activation of or the preferential E mu-myc expression in T lymphocytes. When the bone marrow or fetal liver cells from the prelymphomatous transgenic mice of both strains were transferred into irradiated normal C3H/HeJ mice, most of the recipients developed T lymphomas. Moreover, even when irradiated B6 mice received the hematopoietic stem cells from the prelymphomatous B6 transgenic mice, the incidence of T lymphoma increased up to 50%. These findings suggest that B6 and C3H/HeJ mice might provide the environment that supports the development or growth of B and T lymphomas, respectively, and that such an environment could be modified by irradiation of the mice.
Assuntos
Elementos Facilitadores Genéticos , Genes de Imunoglobulinas , Linfoma/etiologia , Proto-Oncogenes , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Transplante de Medula Óssea , Antígenos CD8 , Ativação Linfocitária , Linfoma/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/análise , Especificidade da EspécieRESUMO
To analyze the regulation of human serum amyloid P component (SAP) gene expression, we have produced seven transgenic mice. The 3.3 kb human SAP genes containing about 0.8 kb of 5' and 1.5 kb of 3' flanking region were injected into fertilized eggs of C57BL/6 mice. In five of the seven transgenic mice, human SAP was detected in the sera and serum concentrations were higher than that of human serum in three lines. The human SAP gene was expressed only in the liver. Amounts of human mRNA in the liver and serum concentrations of human SAP were roughly proportional to the copy number of the integrated gene. Human SAP production lowered the serum levels of mouse endogenous SAP. With the intraperitoneal administration of lipopolysaccharide, the mRNA levels in the liver and serum levels of mouse SAP increased several-fold in both the control and transgenic mice. On the other hand, neither the mRNA nor the serum levels of human SAP increased significantly.
Assuntos
Regulação da Expressão Gênica , Camundongos Transgênicos/metabolismo , Componente Amiloide P Sérico/genética , Animais , Southern Blotting , Western Blotting , Humanos , Inflamação/metabolismo , Injeções Intraperitoneais , Lipopolissacarídeos/farmacologia , Camundongos , RNA Mensageiro/fisiologia , Mapeamento por Restrição , Componente Amiloide P Sérico/análiseRESUMO
Two lines of E alpha d-expressing NOD mice were established by continuously backcrossing [E alpha d B6 transgenic mice x NOD] F1 to parental NOD or directly microinjecting the E alpha d gene into fertilized NOD eggs. Similarly, A beta k-expressing transgenic NOD mice were produced. Subsequent histological examination of pancreatic tissues revealed that autoimmune insulitis was prevented in E alpha d backcross and transgenic mice but not in A beta k transgenic mice.
Assuntos
Doenças Autoimunes/genética , Diabetes Mellitus Experimental/genética , Antígenos de Histocompatibilidade Classe II/genética , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Cruzamentos Genéticos , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Feminino , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos TransgênicosRESUMO
We have created a transgenic mouse which showed an autosomal dominant mutation of facial development. This facial malformation was characterized by a short snout and a twisted upper jaw. All offspring showing the dysmorphic phenotype carried the injected gene. In order to analyze the primary cause of this mutation, newborn mice and embryos were examined. The outcome was that the malformation of nasal and premaxillary bone was not the primary defect but was a secondary event. The primary cause of this dysmorphism was a developmental defect in the first branchial arch. Genomic DNA fragments flanking the insertion site of this mutant mouse were cloned. Using these fragments, we have assigned the integration site to chromosome 13. The gene responsible for a previously reported mutant mouse, one which also has a short snout, is also reported to be on chromosome 13. In the fragments flanking the insertion site of the transgenic mouse, at least one fragment was highly conserved in mammals. These results indicate that this malformation is due to the insertional disruption of a host gene. However, the possibility that this mutation is caused by an inappropriate expression of the injected gene still remains to be investigated.
Assuntos
Desenvolvimento Maxilofacial , Camundongos Transgênicos/crescimento & desenvolvimento , Pré-Albumina/genética , Animais , Mapeamento Cromossômico , Clonagem Molecular , Regulação da Expressão Gênica , Genes , Genes Dominantes , Camundongos , Mutação , FenótipoRESUMO
We have isolated a hybrid plasmid, pDB(mei2)2, containing a 7.4-kilobases (kb) DNA fragment from a Schizosaccharomyces pombe genomic library which is able to complement the mei2 mutation of S. pombe. Integration of the cloned DNA sequence at the mei2 site on chromosome I demonstrated that it contained the mei2 gene. This gene was localized on a 4.7-kb HindIII-PvuII fragment in the subclone pFMV402. Transcriptional regulation was studied by Northern blot analysis in which polyadenylated RNA was prepared from a heterozygous (h+N/h-S) diploid strain cultured either in nitrogen-rich growth medium or in nitrogen-free sporulation medium. The size of the major mei2 mRNA, which always gave a broad band, was estimated to be 4.2 +/- 0.2 kb, and a few minor bands (e.g., 3.2 and 1.8 kb) appeared as well. These transcripts appeared more abundantly in sporulating cells than in growing cells. Neither the mating type genes (mat) nor the mei3 gene was essential for transcription of the mei2 gene, since ample mei2 mRNA was detected in sporulation-deficient cells transferred to sporulation medium, such as h+N/h+N and h-S/h-S homozygotes, as well as mei1 and mei3 mutants.
Assuntos
Clonagem Molecular , Desoxirribonucleases de Sítio Específico do Tipo II , Saccharomycetales/genética , Schizosaccharomyces/genética , Transcrição Gênica , Enzimas de Restrição do DNA/metabolismo , DNA Fúngico/análise , Desoxirribonuclease HindIII , Meiose , Plasmídeos , RNA Mensageiro/análiseRESUMO
To analyze the regulation of transthyretin gene expression we have produced transgenic mice by microinjecting cloned human transthyretin genes into fertilized eggs of C57BL/6 mice. The 7.6-kilobase (kb) human transthyretin gene containing about 500 base pairs (bp) in the upstream region was used for microinjection. Seven out of nine transgenic mice had detectable amounts of human transthyretin in serum when analyzed by enzyme-linked immunosorbent assay. Transthyretin mRNA was detected in liver and yolk sac but not in other tissues including brain. The amount of mRNA was variable among transgenic mice and was about one-tenth of mouse endogenous transthyretin mRNA. Human and mouse transthyretin mRNAs were detected in liver of fetus and yolk sac at 13 days of gestation and unlike yolk sac the level of mRNA in liver increased gradually during development and reached the maximum at around 17 days of gestation. Human transthyretin was associated with mouse transthyretin to form tetramers as judged from the dilution curve of enzyme-linked immunosorbent assay and the spur formation in Ouchterlony assay.