RESUMO
We have investigated the suitability of proteomics for identification of tumor-associated antigens. First, we compared the proteomes of nontumorous kidney and renal cell carcinoma (RCC) by two-dimensional gel electrophoresis (2-DE) and silver staining. Protein patterns were markedly different (approximately 800 spots in RCCs versus approximately 1400 spots in kidney). 2-DE immunoblotting revealed five RCC-specific spots, reproducibly reactive with RCC-patient but not healthy donor control sera. Two of these antigens were isolated by preparative 2-DE, and identified by Edman sequencing of tryptic peptides. The first antigen, smooth muscle protein 22-alpha (SM22-alpha), is an actin-binding protein of unknown function predominantly expressed in smooth muscle cells. In situ hybridization revealed that SM22-alpha is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usually not expressed in kidney. Interestingly, a different isoform (CAXII) has previously been identified by serological expression cloning as an antigen overexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22-alpha were detected in sera from 3/11 or 5/11 RCC patients, respectively, whereas sera from 13 healthy individuals did not react. In conclusion, serological proteome analysis may be a new tool for the identification of tumor-associated antigens.
Assuntos
Antígenos de Neoplasias/análise , Carcinoma de Células Renais/química , Carcinoma de Células Renais/imunologia , Neoplasias Renais/química , Neoplasias Renais/imunologia , Proteoma/análise , Proteoma/imunologia , Testes Sorológicos/métodos , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Sequência de Bases , Northern Blotting , Anidrase Carbônica I/análise , Anidrase Carbônica I/genética , Anidrase Carbônica I/imunologia , Carcinoma de Células Renais/genética , DNA Complementar/genética , DNA de Neoplasias/genética , Eletroforese em Gel Bidimensional , Humanos , Rim/química , Rim/imunologia , Neoplasias Renais/genética , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/imunologia , Dados de Sequência Molecular , Proteínas Musculares/análise , Proteínas Musculares/genética , Proteínas Musculares/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Proteoma/genética , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
The CD44 protein family consists of isoforms, encoded by standard exons and up to nine alternatively spliced variant exons (v2-v10), which are expressed in a tissue-specific way. Expression of v6-containing variants (CD44v6) has been related to aggressive behavior of various tumor types and was shown to be particularly high in squamous cell carcinoma (SCC). Therefore, CD44v6 might be a suitable target for radioimmunoscintigraphy (RIS) and therapy. The present study evaluates the novel high-affinity murine anti-CD44v6 monoclonal antibody (MAb) BIWA 1 for its safety and targeting potential in patients with SCC of the head and neck (HNSCC). Twelve HNSCC patients, who had planned to undergo resection of the primary tumor and neck dissection, were included. Preoperatively, 2, 12, or 52 mg of 99nTc-labeled MAb BIWA 1 was administered. RIS results obtained 21 h after injection were compared with palpation, computed tomography, and magnetic resonance imaging, with histopathology as the gold standard. Moreover, biodistribution of BIWA 1 was evaluated by radioactivity measurement in blood and bone marrow and in biopsies from the surgical specimen obtained 40 h after injection. The distribution of BIWA 1 in tumor biopsies was analyzed by immunohistochemistry. BIWA 1 integrity in the blood was assessed by high-performance liquid chromatography and related to soluble CD44v6 levels in serum samples. No drug-related adverse events were observed. Human antimouse antibody responses were observed in 11 patients. The diagnostic efficacy of RIS appeared to be comparable for the three BIWA 1 dose levels and for the four diagnostic methods. Besides activity uptake in tumor tissue, minimal accumulation of activity was observed in mouth, lungs, spleen, kidney, bone marrow, and scrotal area. Analysis of tissue biopsies revealed high uptake in tumors, with a mean value of 14.2+/-8.4% of the injected dose/kg tumor tissue and a mean tumor:blood ratio of 2.0+/-1.4 at 40 h after injection. Differences among the three dose groups were not statistically significant, although a trend toward lower uptake in the highest dose group was noted. Distribution of BIWA 1 throughout the tumor was heterogeneous for all dose groups, which might be related to the high affinity of the MAb. The mean biological half-life in blood (34.5+/-6.1 h) was not dose dependent. Extensive complex formation of BIWA 1 was observed in the 2-mg group, most probably with soluble CD44v6 present in the blood, and complex formation relatively diminished upon increase of the MAb dose. BIWA 1 is a promising MAb for targeting HNSCC because it can be safely administered to HNSCC patients, while it shows high and selective tumor uptake. However, BIWA 1 is immunogenic, and therefore a chimerized or humanized derivative of BIWA 1 with intermediate affinity will be used in future clinical trials.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Carcinoma de Células Escamosas/metabolismo , Glicoproteínas/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Receptores de Hialuronatos/imunologia , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacocinética , Compostos Radiofarmacêuticos/efeitos adversos , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio , Adulto , Idoso , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Antineoplásicos/sangue , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/imunologia , Feminino , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/imunologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Radioimunodetecção , Tecnécio/efeitos adversos , Tecnécio/farmacocinética , Distribuição TecidualRESUMO
The expression of certain isoforms of CD44 was shown to correlate with aggressiveness and metastatic potential of various tumour types. We analysed the expression of the adhesion molecule CD44 and its variant domains (v6, v7, v7/8, v10) on isolated bone marrow (BM) plasma cells and peripheral blood (PBL) CD19+ B cells of 21 patients with MM and 15 healthy donors. B cells and plasma cells were isolated by immunomagnetic sorting and analysed by two-colour flow cytometry. The expression of CD44 isoforms was significantly higher on PBL B cells of patients with MM than in healthy controls. The elevated expression of CD44 isoforms (v6, v7/8, v10) on PBL B cells correlated with reduced overall survival in MM. CD44 isoforms were more strongly expressed on "larger", activated B cells. Furthermore, CD44 isoforms were found to be simultaneously expressed with CD38hi and CD56 on both, B lymphocytes and plasma cells of patients with MM. The determination of CD44 isoforms on circulating B cells may be helpful in defining prognostically unfavourable subgroups in MM.
Assuntos
Antígenos CD19/biossíntese , Linfócitos B/metabolismo , Células da Medula Óssea/metabolismo , Receptores de Hialuronatos/biossíntese , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Plasmócitos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Tamanho Celular/fisiologia , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Prognóstico , Isoformas de Proteínas/biossínteseRESUMO
BACKGROUND: Increased expression of CD44v6 on colonic crypt epithelial cells in ulcerative colitis has been suggested as a diagnostic tool to distinguish ulcerative colitis from colonic Crohn's disease. AIMS: To investigate colonic CD44v6 expression and serum concentrations of soluble CD44v6 (sCD44v6) in patients with ulcerative colitis and Crohn's disease. METHODS: Colonic biopsy samples were obtained from 16 patients with ulcerative colitis, 13 with ileocolonic Crohn's disease, and 10 undergoing polypectomy. Serum samples were obtained from 15 patients with active ulcerative colitis, 20 with active Crohn's disease, and 20 healthy donors. Colonic CD44v6 expression was evaluated immunohistochemically by monoclonal antibody 2F10 and the higher affinity monoclonal antibody VFF18. Serum sCD44v6 concentrations were measured by ELISA. RESULTS: 2F10 stained colonic epithelium of inflamed ulcerative colitis and Crohn's disease samples in 80% and 40% of cases, respectively, and VFF18 in 95% and 87%, respectively. Both monoclonal antibodies displayed a sensitivity and specificity of 60% and 87% to differentiate ulcerative colitis from colonic Crohn's disease. Serum concentrations of sCD44v6 were lower in patients with ulcerative colitis (median 153 ng/ml; interquartile range (IQR) 122-211) compared with Crohn's disease (219; IQR 180-243) and healthy donors (221; IQR 197-241 (p = 0.002)). Its sensitivity and specificity to discriminate ulcerative colitis from Crohn's disease was 75% and 71%, respectively. CONCLUSION: Colonic CD44v6 and serum sCD44v6 concentrations do not facilitate reliable differential diagnosis between ulcerative colitis and Crohn's disease.
Assuntos
Colite Ulcerativa/diagnóstico , Colo/imunologia , Doença de Crohn/diagnóstico , Receptores de Hialuronatos/análise , Doença Aguda , Adulto , Anticorpos Monoclonais , Biomarcadores/análise , Biomarcadores/sangue , Proteína C-Reativa/análise , Colite Ulcerativa/sangue , Colite Ulcerativa/imunologia , Doença de Crohn/sangue , Doença de Crohn/imunologia , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Feminino , Humanos , Receptores de Hialuronatos/sangue , Imuno-Histoquímica , Isomerismo , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (Kd = 1.7 x 10(-10) M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans.
Assuntos
Anticorpos Monoclonais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Receptores de Hialuronatos/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Especificidade de Anticorpos , Carcinoma de Células Escamosas/imunologia , Epitélio/imunologia , Epitélio/metabolismo , Feminino , Humanos , Receptores de Hialuronatos/imunologia , Imuno-Histoquímica , Imunoterapia , Radioisótopos do Iodo , Isomerismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Neoplasias/imunologia , Radioimunodetecção , Transplante HeterólogoRESUMO
In animal models, isoforms of CD44 (CD44v) containing sequences encoded by one or several of ten different exons (v1-v10) contribute to tumour metastasis. In certain human cancers, CD44v6 expression is associated with poor prognosis. This paper examines CD44v expression in skin carcinogenesis and skin cancer metastasis. CD44v expression was studied in basal cell carcinoma (BCC), squamous cell carcinoma (SCC), primary malignant melanoma (PMM), metastases of MM (MMM), benign melanocytic naevi (BMN) and normal skin (NS) by immunohistochemistry and reverse transcript polymerase chain reaction (RT-PCR). BCC, SCC and NS expressed several CD44v, including v6, albeit in different distributions and intensities. PMM, MMM and BMN expressed isoforms containing v7/8 and v10, but failed to express epitopes encoded by v5 or v6. Thus, different CD44 isoforms are found in human skin cancers and are modulated during carcinogenesis. However, we did not observe a correlation of CD44v6 expression with metastatic potential.
Assuntos
Antígenos de Neoplasias/análise , Receptores de Hialuronatos/análise , Neoplasias Cutâneas/química , Pele/química , Antígenos de Neoplasias/genética , Carcinoma Basocelular/química , Carcinoma Basocelular/genética , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/genética , Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Técnicas Imunoenzimáticas , Melanoma/química , Melanoma/genética , Melanoma/secundário , Nevo Pigmentado/química , Nevo Pigmentado/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Neoplásico/genética , Neoplasias Cutâneas/genéticaRESUMO
A series of 27 renal cell carcinomas 4 oncocytomas and 7 samples of tumour free kidney parenchyma were analysed immunohistochemically using eight different CD44 isoform-specific monoclonal antibodies. In normal kidney expression of CD44 isoforms (containing variant exons v6, v7/8 and v10) was found predominantly at the distal tubules. The majority of clear cell carcinomas investigated showed expression of variant exons v5, v7/8 and v10, but not v6. Lack of CD44v6 expression was confirmed by reverse transcription-polymerase chain reaction analysis. Carcinomas of the chromophilic cell type were almost completely devoid of CD44 expression, including the standard form CD44s. This study shows that there are statistically significant differences in the CD44 expression pattern of the two major histological subtypes of renal cell carcinomas (clear cell and chromophilic carcinomas). Moreover, the almost complete lack of CD44 expression in chromophilic carcinomas contrasts with carcinomas of other histogenetic origin investigated including stomach, breast and lung which express various CD44 isoforms abundantly.
Assuntos
Carcinoma de Células Renais/imunologia , Receptores de Hialuronatos/análise , Neoplasias Renais/imunologia , Adulto , Idoso , Anticorpos Monoclonais , Carcinoma de Células Renais/patologia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Certain isoforms of the CD44 glycoprotein family play an essential role in the metastatic spread of tumour cells. Protein expression of such CD44 isoforms has also been observed in a variety of human malignancies. In this study, we compared the expression of exon v5- and v6-containing CD44 isoforms in normal human and cynomolgus monkey (Macacca fasciculata) tissues. Cloning and sequencing of cynomolgus CD44 exons v5 and v6 revealed a homology of 97% and 95%, respectively, between man and monkey. Two monoclonal antibodies (MAbs) directed against an epitope encoded by human exon v5 (VFF8) and an epitope encoded by exon v6 (VFF18) were used to determine expression of CD44 isoforms in man and monkey. Immunohistochemical screening of a representative profile of normal human and cynomolgus tissues revealed that expression of exon v5- and v6-containing CD44 isoforms was almost identical in the two species. Exon v6 staining was observed only in a subset of epithelial tissues, whereas v5 staining was additionally detected on certain non-epithelial tissues. These data suggest that cynomolgus monkey could serve as a system to test the usefulness of antivariant CD44 MAbs with regard to antibody-based tumour therapy.
Assuntos
Processamento Alternativo , Epitopos/análise , Éxons/genética , Receptores de Hialuronatos/genética , Isoenzimas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Humanos , Receptores de Hialuronatos/análise , Receptores de Hialuronatos/imunologia , Isoenzimas/análise , Isoenzimas/imunologia , Macaca fascicularis , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
In 44 ovarian cancers, CD44 variant (CD44v) expression was investigated immunohistochemically using a variant-specific polyclonal antibody. Patients with CD44v-positive carcinomas had a significantly shorter disease-free survival than patients with CD44v-negative tumors. Overall survival was also significantly reduced for stages III and IV of the International Federation of Gynecology and Obstetrics. Furthermore, a highly significant inverse correlation was observed between CD44v expression and preoperative platelet count. Urinary neopterin concentration, a marker of cell-mediated immunostimulation, did not differ between CD44v-positive and -negative ovarian cancer patients. Moreover, in seven ovarian carcinoma cell lines, modulation of CD44v expression was analyzed by living cell radioimmunoassay. Interferon-alpha, interferon-gamma, tumor necrosis factor, transforming growth factor-beta, all-trans retinoic acid and cisplatin did not affect CD44v expression.
Assuntos
Carcinoma/imunologia , Proteínas de Transporte/análise , Neoplasias Ovarianas/imunologia , Receptores de Superfície Celular/análise , Receptores de Retorno de Linfócitos/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biopterinas/análogos & derivados , Biopterinas/urina , Antígeno Ca-125/sangue , Carcinoma/urina , Feminino , Secções Congeladas , Expressão Gênica , Humanos , Receptores de Hialuronatos , Pessoa de Meia-Idade , Neopterina , Neoplasias Ovarianas/urina , Contagem de Plaquetas , Prognóstico , Estatísticas não Paramétricas , Análise de Sobrevida , Células Tumorais CultivadasRESUMO
Colorectal tumorigenesis evolves through a series of molecular genetic changes, providing putative markers for tumour progression. This study investigated the relation between expression of the tumour suppressor gene p53 and splice variants v5 and v6 of the cell adhesion molecule CD44 by immunohistochemistry on tissue samples of early adenomas (n = 12), late adneomas (n = 12), Dukes's A and B carcinomas (n = 21), and Dukes's C and D carcinomas (n = 22) and compared these results with expression of these proteins in normal colonic mucosa (n = 17). A statistically significant trend of increasing expression was seen for both p53 (p < 0.005) and CD44 variant exon v6 (p < 0.0005) in subsequent stages of this tumour progression model. High expression of CD44 v5 was seen in most colorectal neoplasms (83%-96%), independent of stage. A statistically significant correlation was present between p53 expression and expression of variant v6 of CD44 (p < 0.01). Both p53 expression and CD44 v6 expression in adenomas increased with the degree of dysplasia (p < 0.05). The results of this study show that mutant p53 protein and variant v6 of the CD44 glycoprotein are markers of tumour progression in colorectal cancer.
Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Transporte/biossíntese , Neoplasias Colorretais/metabolismo , Receptores de Superfície Celular/biossíntese , Receptores de Retorno de Linfócitos/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Adenoma/genética , Adenoma/metabolismo , Proteínas de Transporte/genética , Neoplasias Colorretais/genética , Progressão da Doença , Éxons , Expressão Gênica , Genes p53 , Humanos , Receptores de Hialuronatos , Técnicas Imunoenzimáticas , Mutação , Receptores de Superfície Celular/genética , Receptores de Retorno de Linfócitos/genéticaRESUMO
A single functional gene in the human genome codes for interferon omega (IFN-omega), a monomeric glycoprotein distantly related in structure to IFN-alpha and IFN-beta, but unrelated to IFN-gamma. IFN-omega is secreted by virus-infected leukocytes as a major component of human leukocyte interferon. The human class I IFN receptor complex which mediates the biological activity of IFN-alpha and IFN-beta also binds IFN-omega. Its specific activity in a standard in vitro antiviral assay system is 4 x 10(8) U mg-1; potent antiviral activity against several DNA and RNA viruses has been demonstrated. IFN-omega inhibits proliferation of a variety of tumor cell lines in vitro. The protein stimulates natural killer cell activity, enhances expression of major histocompatibility complex class I (but not class II) antigens and inhibits proliferation of lymphocytes stimulated with mitogens or allogeneic cells. IFN-omega is unrelated to IFN-alpha, -beta and -gamma in its antigenic properties, as it does not cross-react with antisera or monoclonal antibodies in immunoassays or antiviral neutralization bioassays. Antibodies induced in patients by long-term IFN-alpha 2 therapy that block IFN-alpha 2 activity do not inactivate IFN-omega. As IFN-omega, like other human IFNs, has a species-restricted biological activity, evaluation of its therapeutic potential will have to await clinical trials.
Assuntos
Interferon Tipo I/genética , Interferon Tipo I/imunologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência MolecularRESUMO
We have tested the hypothesis that the tumor necrosis factor (TNF) plays a significant role in vivo in TNF receptor shedding and studied the release of TNF-binding protein 1 (TNF-BP1), the soluble fragment of the 55- to 60-kDa TNF alpha (TNF) receptor, in a baboon model of Escherichia coli bacteremia, using three different doses of bacteria in acute infection (8 h) experiments (n [animals] = 11) and a single dose in a subchronic infection (72 h) experiment. In the subchronic infection study, one group of animals (n = 6) was pretreated with a neutralizing murine monoclonal antibody to TNF (CB0006). Concentrations of TNF and TNF-BP1 in plasma were determined in specific, monoclonal antibody-based immunoassays. Untreated animals (n = 6) showed undetectable TNF concentrations (< 10 pg/ml at baseline), whereas TNF-BP1 levels in plasma were in the range of 2 ng/ml, similar to concentrations observed in humans. Infusion of bacteria resulted in a rapid, dose-dependent increase in plasma TNF concentrations that reached a maximal level after 2 h and returned to baseline within 6 h. TNF-BP1 concentrations also showed a dose-dependent increase to peak concentrations three- to fivefold above baseline within 2 h but, in contrast to TNF levels, remained significantly elevated for up to 48 h. In animals pretreated with antibody CB0006, circulating TNF was completely neutralized, and TNF-BP1 was significantly reduced. We conclude that TNF-BP1 is released in bacteremia and that release in vivo is partially dependent on the presence of TNF.
Assuntos
Antígenos CD , Bacteriemia/metabolismo , Infecções por Escherichia coli/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Choque Séptico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Doença Aguda , Animais , Doença Crônica , Modelos Animais de Doenças , Masculino , Testes de Neutralização , Papio , Receptores Tipo I de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) as well as embryonic equine dermal fibroblasts and the equine fibroblast line E. Derm which were used as controls, were treated with recombinant equine interferon-beta 1 (rEqIFN-beta 1) in vitro which induced the expression of different proteins in these cells. A 74 kDa protein was induced in PBMC and an 82 kDa protein was additionally found in the equine fibroblast E. Derm cell line following treatment with rEqFN-beta 1. Both proteins reacted with anti-mouse and anti-human Mx protein antisera in immunoblot tests. The 74 kDa and perhaps the 82 kDa components may thus represent equine 'Mxanalogous proteins'. The 74 kDa protein was only detected in PBMC of ten out of 20 horses examined. The induction of Mx protein in the horse by Type 1 interferon may therefore resemble that in the mouse, where Mx protein is involved in selective resistance to influenza virus. The influence of rEqIFN-beta 1 on protein expression in equine PBMC and PMN was monitored by metabolic labeling and 2-D gel electrophoresis. Proteins of 82, 74, 58 and 40 kDa were induced in PBMC following exposure to rEqIFN-beta 1. A constitutively expressed 35 kDa protein, however, was no longer demonstrable upon treatment with interferon. None of the proteins induced within PBMC was found in highly purified PMN treated with interferon. PMN exposed to rEqIFN-beta 1 synthesized four proteins in the range of 25 to 27 kDa. These proteins have not been described in interferon-treated PMN of any other species.
Assuntos
Antivirais/biossíntese , Proteínas de Ligação ao GTP , Cavalos/sangue , Interferon beta/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Biossíntese de Proteínas , Proteínas Recombinantes/farmacologia , Animais , Linhagem Celular , Feminino , Fibroblastos/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Proteínas de Resistência a Myxovirus , Neutrófilos/metabolismo , Pele/efeitos dos fármacosRESUMO
Recombinant equine interferon-beta 1 (reqIFN-beta 1) induces an antiviral state in blood mononuclear cells (BMC) of horses. Maximal protection against replication of vesicular stomatitis virus is achieved 6 hours after treatment with IFN in vitro and in vivo. Duration of the protective effect depends on the dose of IFN in vitro and in vivo. Availability of reqIFN-beta 1 in cultures of BMC for up to 48 hours does not prolong the antiviral state. The protective effect on BMC after treatment with IFN has similar duration in vivo and in vitro. Monitoring of the effect of IFN in vivo is, thus, simplified because the antiviral state may be recorded by testing cells twice (ie, before and 6 hours after application of interferon). All further tests may be performed in vitro. Multiple administration of reqIFN-beta 1 do not prolong duration of the protective phases after each administration. Duration of the antiviral state depends only on the dose of reqIFN-beta 1.
Assuntos
Interferon beta/farmacologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Animais , Temperatura Corporal , Feminino , Doenças dos Cavalos/terapia , Cavalos , Técnicas In Vitro , Interferon beta/administração & dosagem , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Masculino , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Infecções por Rhabdoviridae/terapia , Infecções por Rhabdoviridae/veterinária , Estomatite/terapia , Estomatite/veterinária , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
Annexin VIII is a calcium-dependent phospholipid-binding protein previously identified as a blood anticoagulant based on in vitro studies. However, the physiologic function of annexin VIII remains unknown. In acute promyelocytic leukemia (APL) the annexin VIII gene is highly expressed, but its expression is undetectable in the blasts of other acute leukemias. In the present investigation, we showed using the APL-derived NB4 cell line that expression of the annexin VIII gene is regulated at the transcription level during induced differentiation by all-trans retinoic acid (ATRA). The half-life of the annexin VIII mRNA is about 5 to 6 hours, as determined by using actinomycin D as a transcription inhibitor. Analysis of the expression of annexin VIII protein in NB4 cells and in APL samples showed a consistent expression of a predominant 36-kD protein and a weak 72-kD protein. After ATRA-induced differentiation of NB4 cells, the annexin VIII protein level reduced gradually, but a detectable level persisted even after 4 days of induction. Because annexin VIII mRNA becomes undetectable after 48 hours of ATRA induction, this result indicates that annexin VIII is a relatively stable protein. A multiple tissue Northern blot analysis was performed, and we found that annexin VIII is normally expressed in the placenta and the lung. Cellular localization of the annexin VIII protein was determined by immunofluorescence staining and subcellular fractionation. These results indicated that annexin VIII is predominantly localized to the plasma membrane. The annexin VIII is neither an extracellular protein nor associated with the cell surface suggesting that it does not play a role in blood coagulation in vivo. The plasma membrane localization and its property as a phospholipase inhibitor suggests that annexin VIII may have a role in the signal transduction pathway in the APL cells.
Assuntos
Anexinas/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Promielocítica Aguda/metabolismo , Anexinas/análise , Diferenciação Celular/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/patologia , Especificidade de Órgãos , Fosfolipases A/fisiologia , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.
Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais/imunologia , Variação Genética , Ativação Linfocitária , Receptores de Superfície Celular/genética , Receptores de Retorno de Linfócitos/genética , Processamento Alternativo , Animais , Biomarcadores Tumorais , Neoplasias Colorretais/genética , Neoplasias Colorretais/secundário , Humanos , Receptores de Hialuronatos , Imuno-Histoquímica , Prognóstico , Ratos , Linfócitos T/imunologiaRESUMO
Specific CD44 variant glycoproteins are overexpressed at particular stages of colorectal tumor progression. Some variants of the CD44 glycoprotein without exon v6 sequences appear at the earliest stage of tumorigenesis, i.e., in early adenomas. Expression of variants containing exon v6 sequences is largely restricted to the advanced stages of tumor development and in addition is more prevalent and intense in metastatic (Dukes C/D) than in nonmetastatic (Dukes A/B) carcinomas. The observation that CD44 variants containing a protein domain of CD44 that confers full metastatic potential to rat carcinoma and sarcoma cell lines is increasingly expressed during colorectal tumor progression indicates that this domain may have an important role in tumor progression and metastasis in humans. Information on v6 expression, which can be obtained by routine immunohistochemistry, may prove of important prognostic value, particularly in carcinomas (Dukes A and B) that have not yet given rise to detectable metastases.
Assuntos
Antígenos CD/biossíntese , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Expressão Gênica , Variação Genética , Receptores de Retorno de Linfócitos/biossíntese , Adenoma/metabolismo , Adenoma/patologia , Antígenos CD/análise , Biomarcadores Tumorais/análise , Southern Blotting , Colo/citologia , Colo/metabolismo , Neoplasias Colorretais/imunologia , DNA/análise , DNA de Neoplasias/análise , Éxons , Humanos , Receptores de Hialuronatos , Imuno-Histoquímica , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Metástase Neoplásica , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Receptores de Retorno de Linfócitos/análiseRESUMO
A recently described splice variant of CD44 has been shown to confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we have explored the expression of variant CD44 glycoproteins in human lymphoid cells and tissues, in non-Hodgkin's lymphomas, and in colorectal neoplasia. Normal lymphohematopoietic cells express barely detectable low levels of variant CD44 glycoproteins, while T lymphocytes, upon activation by mitogen or antigen, transiently upregulate expression of specific CD44 variant glycoproteins. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that in the rat confers metastatic capability. Interestingly, overexpression of v6 was also found in several aggressive, but not in low-grade, non-Hodgkin's lymphomas (NHL). In human colorectal neoplasia we also observed strong overexpression of CD44 splice variants in all invasive carcinomas and carcinoma metastasis. Interestingly, focal expression was already observed in adenomatous polyps, expression being related to areas of dysplasia. The findings establish CD44 variants as tumor progression markers in colorectal cancer.
Assuntos
Processamento Alternativo , Variação Genética , Ativação Linfocitária , Neoplasias/patologia , Receptores de Retorno de Linfócitos/biossíntese , Animais , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Humanos , Receptores de Hialuronatos , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/patologia , Neoplasias/imunologia , Ratos , Receptores de Retorno de Linfócitos/análise , Receptores de Retorno de Linfócitos/genéticaRESUMO
A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors, has been shown to confer metastatic potential to nonmetastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we have explored the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and on non-Hodgkin's lymphomas. Normal lymphohematopoietic cells express barely detectable low levels of variant CD44 glycoproteins, whereas T lymphocytes, upon activation by mitogen or antigen, transiently upregulate expression of specific CD44 variant glycoproteins. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that in the rat confers metastatic capability. It is interesting that overexpression of v6 was also found in several aggressive, but not low-grade, non-Hodgkin's lymphomas.