RESUMO
Edwardsiella tarda (E. tarda), an intracellular pathogen, has caused severe economic losses in aquaculture. Effective vaccine development for E. tarda prevention is urgently needed. A previous study indicates that cell-mediated immunity (CMI) might play an important role in E. tarda infection. We believe that the involvement of allograft rejection and CMI has now been well documented in mammals and some fishes. However, there is still little research on the application of blood allograft rejection in vaccine development. In the current study, we investigate the immune response and vaccine effect in fish vaccinated with allogeneic blood + formalin-killed cells vaccine (FKC), allogeneic blood + phosphate-buffered saline (PBS), PBS + FKC and PBS + PBS. In the challenge test, the relative percentage survival (RPS) of the allogeneic + FKC, the allogeneic blood + PBS and the PBS + FKC group was 61.46, 35.41, and 30.63 % respectively. The up-regulated expression of Th1-related genes IFN-γ 1, IFN-γ 1rel2, IL-12p35 and T-bet suggests the protection is via CMI induction. Only in the allogeneic + FKC group, gene expression of IFN-γ 1, IL-12p35 and T-bet is significantly higher, indicating synergy between the two substances. Furthermore, among the fish injected with the allogeneic blood cells, syngeneic blood cells and PBS group, only in the fish of the allogenic blood cells injection group, did expression of IFN-γ 1, IFN-γ 2 and IFN-γ rel2 gene expression significantly increased. The results indicate that the rejection was induced by allogeneic components. Thus, our findings might provide essential information and insights into vaccine development in aquaculture.
Assuntos
Carpas , Infecções por Enterobacteriaceae , Doenças dos Peixes , Transplante de Células-Tronco Hematopoéticas , Animais , Carpa Dourada , Subunidade p35 da Interleucina-12 , Adjuvantes Imunológicos , Vacinas de Produtos Inativados , Doenças dos Peixes/prevenção & controle , Vacinas Bacterianas , Edwardsiella tarda , MamíferosRESUMO
Unlike most mammalian cell lines, fish cell lines are immortal and resistant to cellular senescence. Elevated expression of H-Ras contributes to the induction of senescence in a fish cell line, EPC, but is not sufficient to induce full senescence. Here, we focused on the absence of a p16INK4a/ARF locus in the fish genome, and investigated whether this might be a critical determinant of the resistance of EPC cells to full senescence. We found that transfected EPC cells constitutively overexpressing p16INK4a exhibited large size and flat morphology characteristic of prematurely senescent cells; the cells also showed p53-independent senescence-like growth arrest and senescence-associated ß-galactosidase (SA-ß-gal) activity. Furthermore, the mRNA levels of proinflammatory senescence-associated secretory phenotype (SASP) factors increased in EPC cells constitutively overexpressing p16INK4a. These results suggest that the lack of p16INK4a in the fish genome may be a critical determinant of senescence resistance in fish cell lines.
Assuntos
Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina , Animais , Linhagem Celular , Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Peixes/genética , Peixes/metabolismo , MamíferosRESUMO
We performed several experiments using three strains of Virgibacillus salexigens, namely, P2, NT N53, and C-20MoT (DSM 11483T), which were isolated from completely different sources, in relation to bacteriocin production ability. Results of whole-genome sequencing analysis revealed that all strains have very similar sequences encoding class IId bacteriocin. Although a partial amino acid sequence of the purified bacteriocin produced by strain P2 isolated from fermented food was previously reported, whole-genome sequencing and the N-terminal sequencing results in this study showed that its complete amino acid sequence consisted of 48 residues, which corresponded to that of the hypothetical bacteriocin encoded by the gene in Virgibacillus massiliensis strain Vm-5T (DSM 28587T) isolated from the human gut. From the results of 16S rRNA gene sequencing and whole-genome sequencing analyses, we taxonomically confirmed Vm-5T to be a strain of V. salexigens, and its broth culture showed antibacterial activity. Strain NT N53 isolated from the deep-sea floor produced two bacteriocins, namely, NTN-A and NTN-B. The results of N-terminal sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and whole-genome sequencing analyses showed that their amino acid sequences differed in only one residue, and NTN-A showed the same sequence as the bacteriocin produced by strain P2. Although strain C-20MoT isolated from a solar saltern had the coding sequence very similar to that of NTN-A, its broth culture showed no antibacterial activity. This finding suggests that class IId bacteriocin-producing or bacteriocin-gene-encoding V. salexigens strains are widely distributed in distinct environment sources with different geographical and material properties.
Assuntos
Bacteriocinas/genética , Virgibacillus/classificação , Virgibacillus/genética , Sequência de Aminoácidos , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Microbiologia Ambiental , Humanos , RNA Ribossômico 16S , Água do Mar/microbiologia , Análise de Sequência de DNA , Virgibacillus/metabolismo , Sequenciamento Completo do GenomaRESUMO
In contrast to most mammals including human, fish cell lines have long been known to be immortal, with little sign of cellular senescence, despite the absence of transformation. Recently, our laboratory reported that DNA demethylation with 5-aza-2'-deoxycytidine (5-Aza-dC) induces telomere-independent cellular senescence and senescence-associated secretory phenotype (SASP) in an immortal fish cell line, EPC (Epithelioma papulosum cyprini). However, it is not known how fish derived cultured cells are usually resistant to aging in vitro. In this study, we focused on Ras, which carries out the main role of Ras-induced senescence (RIS), and investigated the role of Ras in the regulation of senescence in EPC cells. Our results show that 5-Aza-dC induced the expression of the ras (hras, kras, nras) gene in EPC cells. EPC cells overexpressing HRas or its constitutively active form (HRasV12) showed p53-dependent senescence-like growth arrest and senescence-associated ß-galactosidase (SA-ß-gal) activity with a large and/or flat morphology characteristic of cell senescence. On the other hand, the SASP was not induced. These results imply that the increased expression of HRas contributes to early senescence in EPC cells, but it alone may not be sufficient for the full senescence, even if HRas is aberrantly activated. Thus, the limited mechanism of RIS may play a role in the senescence-resistance of fish cell lines.
Assuntos
Senescência Celular/genética , Genes ras/genética , Genes ras/fisiologia , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Senescência Celular/fisiologia , Peixes/genética , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Fish cell lines are known to be immortal and do not show the signs of cellular senescence despite the absence of transformation. Furthermore, high telomerase activities responsible for maintenance of telomere length are detected in many organs in live fish, irrespective of fish age. On the other hand, although it is reported that cytosine methylation at CpG island shores decreases as zebrafish age, the relationship between DNA methylation and cellular senescence in fish has not been explored. In this study, we investigated the induction of cellular senescence and senescence-associated secretory phenotype (SASP) in a fathead minnow Pimephales promelas immortal cell line, Epithelioma papulosum cyprini (EPC) treated with the DNA demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). DNA demethylation by 10⯵M of 5-Aza-dC caused cell growth arrest, morphological senescence-like phenotypes and induction of senescence-associated ß-galactosidase (SA-ß-gal) activity, likely due to a mitotic catastrophe caused by disruption of chromosome segregation. Furthermore, RT-qPCR analyses revealed significant up-regulation of senescence markers such as p53-p21 and p16-Rb pathways as well as several SASP factors in 5-Aza-dC treated cells. Meanwhile, although DNA demethylation suppressed the transcription of myc and its downstream target, telomerase reverse transcriptase (tert), telomerase activity was no more than modestly decreased. These results suggest that although DNA methylation may be involved in the suppression of cellular senescence, it not critical for the immortalization of the fish cell line.
Assuntos
Senescência Celular/genética , Cyprinidae/genética , Envelhecimento/genética , Animais , Antimetabólitos Antineoplásicos , Linhagem Celular Tumoral , Ilhas de CpG/genética , Desmetilação do DNA , Metilação de DNA/genética , Decitabina/farmacologia , Neoplasias Epiteliais e Glandulares , Telomerase/fisiologia , Telômero/genética , beta-GalactosidaseRESUMO
Hypoxia is known as a potential immunomodulator in fish. This study therefore assesses the impact of chronic, moderate hypoxia on vaccine efficacy in Oreochromis niloticus. Serum antibody titer was used as a surrogate marker to detect vaccine efficacy. The fish were acclimatized to either moderate hypoxia (55 ± 5% DO) or normoxia (85 ± 5%DO) and immunized with formalin inactivated Vibrio anguillarum. Significantly, a higher antibody titer was found in normoxic fish than in moderate hypoxia. The normoxic group titer peaked at 14th dpv (days post vaccination) while the moderate hypoxic group peaked at 21st or 28th dpv. The absolute blood lymphocyte counts and serum bactericidal activities against V. anguillarum were significantly higher in normoxic fish. Serum killing of V. anguillarum appeared to be mainly via antibody-dependent classical complement pathway. Furthermore, the first week following vaccination appears critical for antibody production. This view was further supported by results obtained from gene expression assay, where the transcription level of all the detected immune related genes (IgM, IL-1 ß, TCR-ß, MHC-II ß), except B cell activating factor, were significantly suppressed following exposure to moderate hypoxia. The overall results highlight that even though moderate hypoxia is not easily detectable in Oreochromis niloticus, it negatively affects antibody production by suppressing and delaying antibody response, ultimately affecting vaccine efficacy.
Assuntos
Ciclídeos/imunologia , Doenças dos Peixes/prevenção & controle , Oxigênio/análise , Vacinação , Vibrioses/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Ciclídeos/sangue , Doenças dos Peixes/imunologia , Contagem de Linfócitos , Muramidase/sangue , Teste Bactericida do Soro , Baço/imunologia , Vibrio , Vibrioses/imunologia , Vibrioses/veterináriaRESUMO
The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of genetically defined animal models. The FLCN gene that codes for the protein folliculin, mutated in Birt-Hogg-Dubé syndrome, presents a new target for mouse modeling of kidney cancer. Here we developed a kidney-specific knockout model by disrupting the mouse Flcn in the proximal tubules, thus avoiding homozygous embryonic lethality or neonatal mortality, and eliminating the requirement of loss of heterozygosity for tumorigenesis. This knockout develops renal cysts and early onset (6 months) of multiple histological subtypes of renal neoplasms featuring high tumor penetrance. Although the majority of the tumors were chromophobe renal cell carcinomas in affected mice under 1 year of age, papillary renal cell carcinomas predominated in the kidneys of older knockout mice. This renal neoplasia from cystic hyperplasia at 4 months to high-grade renal tumors by 16 months represented the progression of tumorigenesis. The mTOR and TGF-ß signalings were upregulated in Flcn-deficient tumors, and these two activated pathways may synergetically cause renal tumorigenesis. Treatment of knockout mice with the mTOR inhibitor rapamycin for 10 months led to the suppression of tumor growth. Thus, our model recapitulates human Birt-Hogg-Dubé kidney tumorigenesis, provides a valuable tool for further study of Flcn-deficient renal tumorigenesis, and tests new drugs/approaches to their treatment.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Cistos/patologia , Modelos Animais de Doenças , Neoplasias Renais/genética , Neoplasias Renais/patologia , Túbulos Renais Proximais/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Animais , Antibióticos Antineoplásicos/uso terapêutico , Carcinogênese/genética , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Cistos/genética , Hiperplasia/patologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Lactococcicosis is an infection caused by the bacterium Lactococcus garvieae and creates serious economic damage to cultured marine and fresh water fish industries. The use of the assay currently applied to evaluate the potency of the lactococcicosis vaccine is contingent upon meeting specific parameters after statistical analysis of the percent survival of the vaccinated yellowtail or greater amberjack fish after challenge with a virulent strain of L. garvieae. We found that measuring the serological response with a quantitative agglutinating antibody against the L. garvieae antigen (phenotype KG+) was an effective method of monitoring the potency of lactococcicosis vaccines. Vaccinated fish had significantly higher antibody titers than control fish when the L. garvieae Lg2-S strain was used as an antigen. Furthermore, the titer of the KG + agglutinating antibody was correlated with vaccine potency, and the cut-off titer was determined by comparing the data with those from the challenge test. An advantage of the proposed serology-based potency assay is that it will contribute to reduced numbers of animal deaths during vaccine potency evaluations.
Assuntos
Vacinas Bacterianas/imunologia , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Positivas/prevenção & controle , Lactococcus , Animais , PeixesRESUMO
The Birt-Hogg-Dubé (BHD) disease is a genetic cancer syndrome. The responsible gene, BHD, has been identified by positional cloning and thought to be a novel tumor suppressor gene. BHD mutations cause many types of diseases including renal cell carcinomas, fibrofolliculomas, spontaneous pneumothorax, lung cysts, and colonic polyps/cancers. By combining Gateway Technology with the Ksp-Cre gene knockout system, we have developed a kidney-specific BHD knockout mouse model. BHD(flox/flox)/Ksp-Cre mice developed enlarged kidneys characterized by polycystic kidneys, hyperplasia, and cystic renal cell carcinoma. The affected BHD(flox/flox)/Ksp-Cre mice died of renal failure at approximate three weeks of age, having blood urea nitrogen levels over tenfold higher than those of BHD (flox/+)/Ksp-Cre and wild-type littermate controls. We further demonstrated that these phenotypes were caused by inactivation of BHD and subsequent activation of the mTOR pathway. Application of rapamycin, which inhibits mTOR activity, to the affected mice led to extended survival and inhibited further progression of cystogenesis. These results provide a correlation of kidney-targeted gene inactivation with renal carcinoma, and they suggest that the BHD product FLCN, functioning as a cyst and tumor suppressor, like other hamartoma syndrome-related proteins such as PTEN, LKB1, and TSC1/2, is a component of the mTOR pathway, constituting a novel FLCN-mTOR signaling branch that regulates cell growth/proliferation.
Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Rim/metabolismo , Doenças Renais Policísticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Animais , Animais Recém-Nascidos , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Proteínas de Transporte/metabolismo , Clonagem Molecular , Embrião de Mamíferos , Genes Letais , Genes Supressores de Tumor/fisiologia , Rim/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Especificidade de Órgãos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Proteínas Proto-Oncogênicas/metabolismo , Serina-Treonina Quinases TOR , Proteínas Supressoras de Tumor/metabolismoRESUMO
The proto-oncogene c-myc is thought to be one of the most important genes in controlling cell proliferation. In a tetraploid fish, two c-myc genes (CAM1 and CAM2) were previously isolated from the common carp, Cyprinus carpio, and were shown to have different expression patterns in adult tissues. Here we found that CAM1 and CAM2 proteins had distinct properties in terms of their transcription regulation system, formation of the transcription activator complex Myc/Max, and transcriptional activation of the target gene. These results showed that the two carp c-Myc proteins have overlapping but distinct functions, suggesting that CAM1 and CAM2 are evolving to acquire different functions after an earlier tetraploidization event.
Assuntos
Carpas/genética , Duplicação Gênica , Genes myc , Poliploidia , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Primers do DNA , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , TransfecçãoRESUMO
Despite the moderate incidence of papillary renal cell carcinoma (PRCC), there is a disproportionately limited understanding of its underlying genetic programs. There is no effective therapy for metastatic PRCC, and patients are often excluded from kidney cancer trials. A morphologic classification of PRCC into type 1 and 2 tumors has been recently proposed, but its biological relevance remains uncertain. We studied the gene expression profiles of 34 cases of PRCC using Affymetrix HGU133 Plus 2.0 arrays (54,675 probe sets) using both unsupervised and supervised analyses. Comparative genomic microarray analysis was used to infer cytogenetic aberrations, and pathways were ranked with a curated database. Expression of selected genes was validated by immunohistochemistry in 34 samples with 15 independent tumors. We identified two highly distinct molecular PRCC subclasses with morphologic correlation. The first class, with excellent survival, corresponded to three histologic subtypes: type 1, low-grade type 2, and mixed type 1/low-grade type 2 tumors. The second class, with poor survival, corresponded to high-grade type 2 tumors (n = 11). Dysregulation of G1-S and G2-M checkpoint genes were found in class 1 and 2 tumors, respectively, alongside characteristic chromosomal aberrations. We identified a seven-transcript predictor that classified samples on cross-validation with 97% accuracy. Immunohistochemistry confirmed high expression of cytokeratin 7 in class 1 tumors and of topoisomerase IIalpha in class 2 tumors. We report two molecular subclasses of PRCC, which are biologically and clinically distinct and may be readily distinguished in a clinical setting.
Assuntos
Carcinoma Papilar/classificação , Carcinoma de Células Renais/classificação , Neoplasias Renais/classificação , Adulto , Idoso , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Aberrações Cromossômicas , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Smad4 is defined as the common-mediator Smad (Co-Smad) required for transducing signals for all transforming growth factor-beta (TGF-beta) superfamily members. In this study, we have isolated eight distinct Smad4 full-length cDNAs from the common carp (Cyprinus carpio). These cDNAs were classified into four types and each type consisted of two subtypes. The eight cDNAs encoded four distinct proteins ranging from 505aa to 568aa in size, with close similarities in the Mad homology 1 and 2 (MH1 and MH2, respectively), but with differences in the linker regions and the C-terminus as well as in the 5'- and 3'-untranslated regions. Genomic Southern blotting demonstrated the existence of at least six Smad4 gene loci in the carp genome, meaning that the multiple forms of the carp Smad4 cDNAs were not due to allelic variations. Reverse transcriptase polymerase chain reaction (RT-PCR)/Southern hybridizations showed different expression patterns among the four types of Smad4s. These results suggest that some of carp Smad4s have deviated from the original function of Smad4 through vertebrate evolution, and regulated the TGF-beta signaling pathway by changing the expression level in tissues.