RESUMO
In our previous study, the hinge fragment (225-232/225'-232') of human immunoglobulin G1 (IgG1) was used as a space peptide linker for synthesizing the GnRH3-hinge-MVP chimeric peptide, whereby three repeated gonadotrophin-releasing hormone (GnRH) units and a T-cell epitope from measles virus fusion protein (MVP) were amide-bond-linked at the N and C terminus, respectively, to the hinge peptide for producing anti-GnRH antibody responses. To investigate whether or not the hinge region fragment can improve the immunogenicity of GnRH, we further synthesized and purified GnRH3-hinge-MVP, GnRH3-hinge and GnRH3-MVP using recombinant DNA technology. Under high pH conditions, GnRH3-hinge-MVP was capable of forming double-chain structures. Immunization of male mice with the immunogens of GnRH3-hinge-MVP resulted in the generation of high-titre antibodies specific for GnRH. The synthetic GnRH3-hinge and GnRH3-MVP induced a lower titre of anti-GnRH antibody than GnRH3-hinge-MVP. This was followed by a decrease in serum testosterone levels, which resulted in a low level of expression of the relaxin-like factor gene in the testis. Our data suggest that peptide and T-cell epitopes oriented at the N-terminus or C-terminus of hinge peptides simplify the antigenic peptide conjugates and may be considered as potential synthetic immunogens.
Assuntos
Epitopos de Linfócito T/imunologia , Hormônio Liberador de Gonadotropina/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos/sangue , Expressão Gênica/imunologia , Hormônio Liberador de Gonadotropina/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Imunoglobulina G/imunologia , Insulina/imunologia , Insulina/metabolismo , Masculino , Camundongos , Oxirredução , Proteínas/imunologia , Proteínas/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Testículo/imunologia , Testículo/metabolismo , Testosterona/sangue , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismoRESUMO
The elevated expression and receptor binding of gastrin-releasing peptide (GRP) in various types of cancer, especially in malignant melanoma of the skin, suggest that GRP might be a putative target for immunotherapy in neoplastic diseases. We have therefore constructed a novel DNA vaccine coding for six tandem repeats of a fragment of GRP from amino acids 18 to 27 (GRP6) flanked by helper T-cell epitopes for increased immunogenicity, including HSP65, a tetanus toxoid fragment from amino acids 830 to 844 (T), pan-HLA-DR-binding epitope (PADRE) (P), and two repeats of a mycobacterial HSP70 fragment from amino acids 407 to 426 (M). The anti-GRP DNA vaccine (pCR3.1-VS-HSP65-TP-GRP6-M2) was constructed on a backbone of a pCR3.1 plasmid vector with eight 5'-GACGTT-3' CpG motifs and the VEGF183 signal peptide (VS). Intramuscular (IM) injections of anti-GRP vaccine in mice stimulated the production of high titers of specific antibodies against GRP and suppressed the growth of subcutaneous tumors of B16-F10 melanoma cells. Parallel results were obtained in vitro, showing inhibition of B16-F10 cell proliferation by GRP antisera. IM injections of the DNA vaccine also significantly attenuated tumor-induced angiogenesis associated with intradermal tumors of B16-F10 cells. In addition, lung invasion of intravenously injected cells was highly diminished, suggesting potent antimetastatic activity of the DNA vaccine. These findings support the highly immunogenic and potent antitumorigenic activity of specific anti-GRP antibodies elicited by the anti-GRP DNA vaccine.
Assuntos
Anticorpos/imunologia , Vacinas Anticâncer/imunologia , Peptídeo Liberador de Gastrina/antagonistas & inibidores , Peptídeo Liberador de Gastrina/imunologia , Melanoma/imunologia , Vacinas de DNA/imunologia , Animais , Vacinas Anticâncer/genética , Linhagem Celular , Ilhas de CpG , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Peptídeo Liberador de Gastrina/genética , Injeções Intramusculares , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/prevenção & controle , Vacinas de DNA/genéticaRESUMO
The regenerative inadequacy of the injured myocardium leads to adverse remodeling, cardiac dysfunction, and heart disease. Stem cell-replacement of damaged myocardium faces major challenges such as inappropriate differentiation, cellular uncoupling, scar formation, and accelerated apoptosis of transplanted cells. These challenges can be met by engineering an in vitro system for delivering stem cells capable of cardiac differentiation, tissue integration, and resistance to oxidative stress. In this study, we describe the formation of three-dimensional (3D) cell aggregates ("cardiospheres") by putative stem cells isolated from adult dog myocardium using poly-L-ornithine. De novo formation of cardiospheres in growth factor-containing medium occurred over a period of 2-3 weeks, but accelerated to 2-3 days when seeded on poly-L-ornithine. Older cardiospheres developed foci of "beating" cells upon co-culture with rat neonatal cardiomyocytes. Cardiospheres contained cells that exhibited characteristics of undifferentiated cells; differentiating cardiomyocytes with organized contractile machinery; and vascular cells capable of forming "vessel-like" networks. They exhibited strong resistance to elevated concentrations of hydrogen peroxide in culture and survived subcutaneous injections without undergoing neoplastic transformation up to 3 weeks post-transplantation. These findings suggest that cardiospheres are potentially useful for delivering functional stem cells to the damaged heart.
Assuntos
Diferenciação Celular , Miocárdio/citologia , Estresse Oxidativo , Células-Tronco/citologia , Animais , Técnicas de Cultura de Células , Técnicas de Cocultura , Cães , Miócitos Cardíacos/citologia , Peptídeos , Ratos , Regeneração , Transplante de Células-Tronco/métodosRESUMO
We have previously shown that anti-GnRH antibodies responses can be induced by synthetic GnRH3-hinge-MVP peptide. In this study, GnRH3-hinge-MVP of conjugation to heat shock protein 65 was used as an adjuvant-free vaccine to assess the therapeutic effects of GnRH immunoneutralisation on tumor development in the mice model. Compared with mice treated with Hsp65 and PBS, mice of the o.t. model receiving in situ treatment GnRH3-hinge-MVP-Hsp65 had significant prolongation of survival and suppression of local tumor growth. Serum levels of both testosterone and luteinizing hormone were reduced by treatment with GnRH3-hinge-MVP-Hsp65 (p<0.05). Further analyses of cell mediated immune responses showed that GnRH3-hinge-MVP-Hsp65 induced stronger lymphocyte proliferative responses and higher levels of IFN-gamma (p<0.001). The conjugation of the recombinant GnRH peptide to Hsp65 could be considered a promising approach for the development of an efficacious vaccine against the prostate cancer.
Assuntos
Formação de Anticorpos , Proteínas de Bactérias/imunologia , Vacinas Anticâncer/imunologia , Chaperoninas/imunologia , Hormônio Liberador de Gonadotropina/imunologia , Imunidade Celular , Neoplasias da Próstata/prevenção & controle , Proteínas Virais/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Chaperonina 60 , Imunoglobulina G/sangue , Interferon gama/metabolismo , Hormônio Luteinizante/sangue , Ativação Linfocitária , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/prevenção & controle , Neoplasias da Próstata/sangue , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Proteínas Recombinantes/imunologia , Testosterona/sangue , Fatores de Tempo , Vacinas Conjugadas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor receptor superfamily that paradoxically mediates neuronal survival and differentiation or apoptotic cell death. Cleavage of p75NTR by a constitutively active metalloprotease could result in shedding of its extracellular domain (p75ECD) and generation of a pro-apoptotic intracellular domain (p75ICD). In this study, we established that exposure of a transgenic mouse photoreceptor cell line to intense light upregulated the expression of p75NTR and of the disintegrin metalloprotease tumor necrosis factor-converting enzyme (TACE) and resulted in apoptotic cell death. Light damage promoted TACE cleavage of p75NTR resulting in shedding of the soluble p75ECD and nuclear translocation of the p75ICD. Overexpression of TACE and p75NTR-induced p75NTR cleavage and secretion of p75ECD, but not nuclear transport of p75ICD. Light-induced cleavage of p75NTR, nuclear localization of p75ICD, and apoptosis were inhibited by IC-3, a metalloprotease inhibitor. Increased levels of p75NTR and TACE were observed in photoreceptor cells of animals with photic injury. Our findings support a role for TACE in the proteolytic cleavage of p75NTR and light-induced apoptosis.
Assuntos
Proteínas ADAM/metabolismo , Apoptose/fisiologia , Estimulação Luminosa/efeitos adversos , Células Fotorreceptoras/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Retina/metabolismo , Proteínas ADAM/efeitos da radiação , Proteína ADAM17 , Transporte Ativo do Núcleo Celular/fisiologia , Transporte Ativo do Núcleo Celular/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Linhagem Celular , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/fisiologia , Regulação da Expressão Gênica/efeitos da radiação , Líquido Intracelular/metabolismo , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Camundongos , Camundongos Transgênicos , Células Fotorreceptoras/lesões , Células Fotorreceptoras/efeitos da radiação , Estrutura Terciária de Proteína/fisiologia , Estrutura Terciária de Proteína/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento Neural/efeitos da radiação , Retina/lesões , Retina/efeitos da radiaçãoRESUMO
A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, approximately 63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células Fotorreceptoras/citologia , Epitélio Pigmentado Ocular/metabolismo , Retina/citologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Proteínas do Olho/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Camundongos , Células Fotorreceptoras/efeitos dos fármacos , Epitélio Pigmentado Ocular/efeitos dos fármacos , Retina/efeitos dos fármacos , Células-Tronco/efeitos dos fármacosRESUMO
To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3-hinge-MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3-hinge-MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE-Sephacel chromatography. The purified GnRH3-hinge-MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3-hinge-MVP could be considered as a candidate anti-GnRH vaccine.
Assuntos
Hormônio Liberador de Gonadotropina/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Precipitação Química , Clonagem Molecular , Epitopos de Linfócito T , Escherichia coli/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/imunologia , Humanos , Hidrólise , Imunoglobulina G/genética , Corpos de Inclusão/metabolismo , Vírus do Sarampo/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Virais/genéticaRESUMO
Active immunization against self-peptides have gained widespread acceptance inspite of their low immunogenicity. Recent applications involving multiple copies of self-peptides in linear alignment and conjugation with carrier proteins appear to increase the immune response against self-peptides. As with most vaccines, however, immunogens require supplementation with adjuvants to elicit an optimum immune response. In the present study, we prepared a double-chain mini-protein with each chain containing three linear repeats of the self-peptide gonadotropin-releasing hormone (GnRH3), the hinge region of human IgG1 (hinge), and a T-helper epitope from the measles virus protein (MVP). The GnRH3-hinge-MVP mini-protein was conjugated to purified recombinant heat shock protein 65 (Hsp 65) of Mycobacterium bovis and used to immunize rats primed with subcutaneous injections of Bacillus Calmette-Guerin (BCG) in the absence of adjuvants. The GnRH3-hinge-MVP-Hsp 65 stimulated the production of specific anti-GnRH antibodies in the absence of adjuvants and the antibody titer was comparable to that produced in rats immunized with the dimeric mini-protein in the presence of Freund's adjuvant. Moreover, immunization with the adjuvant-free GnRH3-hinge-MVP-Hsp 65 induced degeneration of the reproductive organs in both male and female rats unlike those immunized in the absence of Hsp 65 or in control animals inoculated with the vehicle only. Histological examination of the affected organs showed atrophy of the seminiferous tubules with diminished spermatogenesis in the testes of male rats. In female rats, the uteri were much smaller in size and the ovaries exhibited reduced follicular development. These findings demonstrated that GnRH3-hinge-MVP-Hsp 65 mounted a strong immune response in the absence of conventional adjuvants, and could prove useful in control of fertility and the treatment of conditions/diseases where GnRH ablation is required.
Assuntos
Hormônio Liberador de Gonadotropina/farmacologia , Vacinas Anticoncepcionais/farmacologia , Animais , Anticorpos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Chaperonina 60 , Chaperoninas/genética , Chaperoninas/imunologia , Doenças do Sistema Endócrino/prevenção & controle , Epitopos de Linfócito T/genética , Feminino , Adjuvante de Freund/imunologia , Genitália Feminina/efeitos dos fármacos , Genitália Masculina/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Masculino , Mycobacterium bovis/imunologia , Folículo Ovariano/patologia , Ratos , Túbulos Seminíferos/patologia , Espermatogênese/imunologia , Útero/patologia , Vacinas Anticoncepcionais/genética , Vacinas Anticoncepcionais/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologiaRESUMO
For many years, graduate students at the University of North Texas Health Science Center (UNTHSC) were reluctant to enroll in dissection-based human gross anatomy courses. Furthermore, few graduate faculty mentors would allow their students to enroll in these courses. The significant amount of time allotted to courses such as anatomy and its effect on students' research programs have been identified by faculty as the primary reason for this lack of enthusiasm. For example, prior to 1999, graduate students taking human gross anatomy at UNTHSC registered for a 13-semester credit hour (SCH) course that was offered only in the fall semester. In the last 5 years, the anatomy teaching faculty in the Department of Cell Biology and Genetics (CGEN) restructured the human gross anatomy course for graduate students. A series of small, compact anatomy courses, ranging from 3-7 SCHs, are now offered throughout the school year to replace the single anatomy course. The CGEN faculty designed courses based on single or multiple body systems that varied in length from a few weeks to an entire semester. This change was initiated with the implementation of a system-based approach to anatomy instruction in our medical school curriculum and the elimination of our graduate anatomy course. With the development of six anatomy courses covering the entire human body, we have had a significant increase in graduate student participation. Moreover, the shorter duration of the courses has made them more appealing to graduate faculty mentors who want to keep graduate students focused on their research.
Assuntos
Centros Médicos Acadêmicos , Anatomia/educação , Educação de Pós-Graduação/métodos , Anatomia/tendências , Educação de Pós-Graduação/tendências , Humanos , TexasRESUMO
Asparaginase of Escherichia coli, a tetramer of identical subunits, was tested as a vector to display linear peptides on the surface of each enzyme subunit. A recombinant gene encoding a chimeric protein composed of asparaginase, a tetanus toxin peptide (TTP) spacer (831-854 fragment), and the foreign cholesteryl ester transfer protein C-terminal fragment (CETPC) was expressed and targeted to the periplasm of E. coli. The purified chimeric enzyme exhibited approximately 83% activity of the native enzyme, allowing the rapid screening of recombinant clones. In contrast, an asparaginase-CETPC fusion protein without the TTP spacer produced only about 23% activity of the native enzyme. Rats immunized with bacterial cells containing the chimeric enzymes induced CETP-specific immunoresponse. In contrast, rats inoculated with the cells expressing asparaginase only did not generate specific anti-CETP antibodies. Our study showed that asparaginase of E. coli was an effective carrier for displaying foreign peptides or epitopes. Moreover, the use of the TTP spacer appeared to play a critical role in maintaining the catalytic activity of the chimeric enzymes by redirecting the foreign CETPC peptide to the surface of the enzyme. The chimeric enzyme constructs fusing asparaginase with foreign peptides via a TTP spacer could be utilized as a rapid pepscan technique for antigen epitope mapping. The fusion protein of asparaginase-TTP-CETPC could also be useful for the development of a vaccine against atherosclerosis.
Assuntos
Asparaginase/genética , Proteínas de Transporte/imunologia , Mapeamento de Epitopos/métodos , Epitopos/análise , Escherichia coli/enzimologia , Glicoproteínas/imunologia , Biblioteca de Peptídeos , Periplasma/química , Animais , Anticorpos/imunologia , Asparaginase/análise , Asparaginase/química , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transferência de Ésteres de Colesterol , Glicoproteínas/química , Glicoproteínas/genética , Peptídeos/genética , Peptídeos/imunologia , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Toxina Tetânica/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
PURPOSE: Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium. METHODS: RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-kappaB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5', 6,6'-tetrachloro 1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c. RESULTS: Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-kappaB (NF-kappaB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells. CONCLUSIONS: Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.
Assuntos
Apoptose , Mitocôndrias/metabolismo , Células Ganglionares da Retina/patologia , Transdução de Sinais/fisiologia , Animais , Benzimidazóis/metabolismo , Carbocianinas/metabolismo , Caspases/metabolismo , Linhagem Celular Transformada , Meios de Cultura Livres de Soro , Citocromos c/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Immunoblotting , Potenciais da Membrana/fisiologia , Microscopia Confocal , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Células Ganglionares da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2RESUMO
The heat shock protein 65 kDa (Hsp65) of Mycobacterium tuberculosis var. bovis was fused with the linear polypeptide epitope of cholesteryl ester transfer protein C-terminal fragment (CETPC) and expressed as soluble protein in Escherichia coli. The fusion protein Hsp65-CETPC was purified by anion exchange column and eluted at 100-130 mM NaCl in 10mM phosphate buffer (pH 8.0), and then used to immunize mice via subcutaneous injection or intranasal delivery in the absence of adjuvants. Antibodies against CETPC were detected in immunized mice sera by enzyme-linked immunosorbent assay (ELISA) and verified by Western blot analysis. Specific antibodies were successfully induced and lasted for more than 12 weeks in animals immunized with the fusion protein via both subcutaneous and intranasal routes even in the absence of adjuvants. Results showed that Hsp65 could be used as a convenient carrier molecule for presenting foreign polypeptide epitopes, such as CETPC, to the immune system in vivo. Antibodies induced by Hsp65-CETPC could partially inhibit the excessive activity of CETP to normal level. Therefore, Hsp65-CETPC might be further developed to a vaccine against atherosclerosis.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Chaperoninas/imunologia , Epitopos/imunologia , Glicoproteínas/imunologia , Imunidade nas Mucosas/imunologia , Proteínas Recombinantes/imunologia , Adjuvantes Imunológicos , Administração Intranasal , Sequência de Aminoácidos , Animais , Anticorpos/análise , Vacina BCG/imunologia , Proteínas de Bactérias/administração & dosagem , Western Blotting , Proteínas de Transporte/administração & dosagem , Chaperonina 60 , Chaperoninas/administração & dosagem , Proteínas de Transferência de Ésteres de Colesterol , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Feminino , Vetores Genéticos , Glicoproteínas/administração & dosagem , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Recombinantes/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/sangue , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
A chimeric polypeptide of TTP-CETPC was successfully expressed as inclusion bodies in Escherichia coli by fusing it with the C-terminus of asparaginase and a basic amino acid-rich peptide (KR). After partially purified by washing with 0.5% (v/v) Triton X-100 in 10 mM PB, the pellet was solubilized in 8 M urea. The solution was precipitated with single volume and double volumes of cold ethanol for removing impurities. The fusion protein in solution was precipitated with triple volumes of ethanol to increase purity and then hydrolyzed with 50 mM hydrochloric acid at 55 degrees C for 72 h. The TTP-CETPC polypeptide was released after the unique acid-labile aspartylprolyl bond in the fusion protein was cleaved by acid. After impurities were removed by adjusting the hydrolysis solution pH to 9.45 and then to 8.37, the TTP-CETPC polypeptide was further purified by DEAE-cellulose column. The TTP-CETPC containing fractions were eluted at 60-80 mM NaCl. The purified TTP-CETPC cysteines were oxidized to form into intermolecular disulfide bonded dimers for immunizing mice. Specific anti-CETP antibodies in mice serum were assayed by ELISA and Western blot to verify that antibodies against CETP had been successfully induced and lasted for more than seventeen weeks in vivo.
Assuntos
Arteriosclerose/imunologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte/isolamento & purificação , Glicoproteínas/isolamento & purificação , Toxina Tetânica/isolamento & purificação , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Ácido Aspártico/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transferência de Ésteres de Colesterol , Dimerização , Escherichia coli/química , Escherichia coli/genética , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Ácido Clorídrico/química , Imunização , Corpos de Inclusão/química , Corpos de Inclusão/genética , Camundongos , Dados de Sequência Molecular , Prolina/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Toxina Tetânica/genética , Toxina Tetânica/imunologia , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificaçãoRESUMO
Reports implicating microglia-derived nerve growth factor (NGF) during programmed cell death in the developing chick retina led us to investigate its possible role in degenerative retinal disease. Freshly isolated activated retinal microglia expressed high molecular weight forms of neurotrophins including that of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. Conditioned media from cultured retinal microglia (MGCM) consistently yielded a approximately 32-kDa NGF-reactive band when supplemented with bovine serum albumin (BSA) or protease inhibitors (PI); and promoted cell death that was suppressed by NGF immunodepletion in a mouse photoreceptor cell line (661w). The approximately 32 kDa protein was partially purified (MGCM/p32) and was highly immunoreactive with a polyclonal anti-pro-NGF antibody. Both MGCM/p32 and recombinant pro-NGF protein promoted cell death in 661w cultures. Increased levels of pro-NGF mRNA and protein were observed in the RCS rat model of retinal dystrophy. MGCM-mediated cell death was reversed by p75NTR antiserum in p75NTR(+)/trkA(-) 661w cells. Our study shows that a approximately 32 kDa pro-NGF protein released by activated retinal microglia promoted degeneration of cultured photoreceptor cells. Moreover, our study suggests that defective post-translational processing of NGF might be involved in photoreceptor cell loss in retinal dystrophy.
Assuntos
Microglia/química , Fator de Crescimento Neural/farmacologia , Células Fotorreceptoras/citologia , Receptor de Fator de Crescimento Neural/fisiologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Microglia/citologia , Microglia/metabolismo , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Células Fotorreceptoras/química , RNA Mensageiro/análise , Ratos , Receptor de Fator de Crescimento Neural/análise , Receptor de Fator de Crescimento Neural/genética , Degeneração Retiniana/etiologia , Degeneração Retiniana/patologiaRESUMO
The need to increase the efficiency of dissection in the gross anatomy laboratory has been the driving force behind the technologic changes we have recently implemented. With the introduction of an integrated systems-based medical curriculum and a reduction in laboratory teaching hours, anatomy faculty at the University of North Texas Health Science Center (UNTHSC) developed a computer-based dissection manual to adjust to these curricular changes and time constraints. At each cadaver workstation, Apple iMac computers were added and a new dissection manual, running in a browser-based format, was installed. Within the text of the manual, anatomical structures required for dissection were linked to digital images from prosected materials; in addition, for each body system, the dissection manual included images from cross sections, radiographs, CT scans, and histology. Although we have placed a high priority on computerization of the anatomy laboratory, we remain strong advocates of the importance of cadaver dissection. It is our belief that the utilization of computers for dissection is a natural evolution of technology and fosters creative teaching strategies adapted for anatomy laboratories in the 21st century. Our strategy has significantly enhanced the independence and proficiency of our students, the efficiency of their dissection time, and the quality of laboratory instruction by the faculty.
Assuntos
Anatomia/educação , Instrução por Computador , Dissecação/normas , Educação de Pós-Graduação em Medicina/tendências , Ensino/tendências , Cadáver , Currículo , Dissecação/instrumentação , Avaliação Educacional , Eficiência , Humanos , Manuais como Assunto/normas , Design de Software , Estudantes de MedicinaRESUMO
The uptake of excess extracellular glutamate and the secretion of neurotrophins by glial cells have been suggested to protect CNS neurons from glutamate-induced toxicity. In the retina, perturbation of glutamate transport and decreased retrograde transport of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) may contribute to ganglion cell death in experimental glaucoma. Although many studies show a clear relationship between glutamate and neurotrophic factors, such relationship has not been thoroughly investigated in the retinal environment. In the following study, we determined the effects of glutamate on early passaged rat Müller cells, specifically their expression of neurotrophic factors including BDNF, nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and glial-cell line derived neurotrophic factor (GDNF); and of glutamate receptors and transporters using immunoblots or enzyme-linked immunosorbent assays. Binding of BDNF to its cognate receptor TrkB was also determined using co-immunoprecipitation studies. Cultured Müller cells grown in the presence of glutamate were also assayed for survival using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Our study showed that while glutamate treatment did not promote cell death, it upregulated secretion of BDNF, NGF, NT-3, NT-4, and GDNF by Müller cells. While solitary bands at approximately 13-14 kDa were observed for NGF, NT-3, and NT-4; two BDNF-reactive bands were observed in immunoblots: a faster migrating band at the reported size of the BDNF monomer (approximately 13 kDa); and a more intense band at approximately 36 kDa. GDNF-reactive bands were observed at approximately 22, approximately 28, and approximately 55 kDa. Glutamate also induced significant changes in glutamate receptor and transporter proteins, as well maintained the association of BDNF to TrkB in Müller cells. The decreased N-methyl-D-aspartate receptor (NMDAR) levels and sustained activation of TrkB by BDNF could serve as protective mechanisms for Müller cell survival. Moreover, the increased secretion of neurotrophic factors and upregulation of L-glutamate/L-aspartate transporter (GLAST) expression in Müller cells may protect retinal neurons from glutamate toxicity.
Assuntos
Glaucoma/metabolismo , Ácido Glutâmico/metabolismo , Fatores de Crescimento Neural/metabolismo , Neuroglia/metabolismo , Neurotoxinas/metabolismo , Retina/metabolismo , Regulação para Cima/efeitos dos fármacos , Sistema X-AG de Transporte de Aminoácidos/efeitos dos fármacos , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Glaucoma/fisiopatologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Ácido Glutâmico/farmacologia , Modelos Biológicos , Fator de Crescimento Neural/metabolismo , Neuroglia/efeitos dos fármacos , Neurotoxinas/farmacologia , Neurotrofina 3/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor trkB/efeitos dos fármacos , Receptor trkB/metabolismo , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Sais de Tetrazólio , Regulação para Cima/fisiologiaRESUMO
Although a gene mutation in the Royal College of Surgeons (RCS) dystrophic rat results in defective phagocytosis and in accumulation of debris in the subretinal space, the molecular mechanisms leading to photoreceptor cell death remain unclear. In this study, the expression of p75(NTR), the low-affinity neurotrophin receptor incriminated in the apoptosis of developing neurons, was investigated at various stages of retinal degeneration in dystrophic rats using immunohistochemistry, in situ reverse transcription polymerase chain reaction (RT-PCR), Western blot, and relative RT-PCR. In normal adult retinas, p75(NTR) immunolabeling was observed mainly in the outer limiting membrane, with punctate labeling in the inner nuclear and ganglion cell layers. In 18- to 30-day-old dystrophic retinas, the immunostaining appeared to increase especially in the photoreceptor outer and inner segments. Dense staining was also observed in the retinal pigment epithelium (RPE) and choroid. In 60-day-old dystrophic rat retinas, the density of immunolabeling for p75(NTR) increased dramatically in the remaining inner retina, especially in the inner nuclear, inner plexiform, and ganglion cell layers. Post-embedding immunogold labeling of normal retinas verified the distribution of p75(NTR) in photoreceptor cells within the inner segments, cell bodies, and outer segments. The apparent increased intensity in p75(NTR) immunostaining in dystrophic retinas was verified by Western blots and densitometric analyses. In situ RT-PCR and relative RT-PCR further established increased synthesis of p75(NTR) in dystrophic retinas. The increased levels of p75(NTR) in the RPE and photoreceptor cells, the initial sites of injury, during retinal degeneration in dystrophic rats strongly suggest that altered expression of p75(NTR) may be directly involved in photoreceptor death.