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1.
Vopr Virusol ; 66(2): 91-102, 2021 May 15.
Artigo em Russo | MEDLINE | ID: mdl-33993679

RESUMO

The Lassa virus one of the main etiological agent of hemorrhagic fevers in the world: according to WHO estimates, it affects 100,000 to 300,000 people annually, which results in up to 10,000 deaths [1]. Although expansion of Lassa fever caused by this pathogen is mostly limited to the West African countries: Sierra Leone, Liberia, Guinea and Nigeria, imported cases have been historically documented in Europe, the United States of America (USA), Canada, Japan, and Israel [2]. In 2017, WHO included the Lassa virus in the list of priority pathogens in need of accelerated research, development of vaccines, therapeutic agents and diagnostic tools regarding infections they cause [3]. This review describes main technological platforms used for the development of vaccines for the prevention of Lassa fever.


Assuntos
Febre Lassa , África Ocidental , Europa (Continente) , Humanos , Febre Lassa/epidemiologia , Febre Lassa/prevenção & controle , Vírus Lassa/imunologia , Vacinas Virais
2.
Acta Naturae ; 13(4): 53-63, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35127147

RESUMO

Ebola fever is an acute, highly contagious viral disease with a mortality rate that can reach 90%. There are currently no licensed therapeutic agents specific to Ebola in the world. Monoclonal antibodies (MAbs) with viral-neutralizing activity and high specificity to the Ebola virus glycoprotein (EBOV GP) are considered as highly effective potential antiviral drugs. Over the past decade, nanobodies (single-domain antibodies, non-canonical camelid antibodies) have found wide use in the diagnosis and treatment of various infectious and non-infectious diseases. In this study, a panel of nanobodies specifically binding to EBOV GP was obtained using recombinant human adenovirus 5, expressing GP (Ad5-GP) for alpaca (Vicugna pacos) immunization, for the first time. Based on specific activity assay results, affinity constants, and the virus-neutralizing activity against the recombinant vesicular stomatitis virus pseudotyped with EBOV GP (rVSV-GP), the most promising clone (aEv6) was selected. The aEv6 clone was then modified with the human IgG1 Fc fragment to improve its pharmacokinetic and immunologic properties. To assess the protective activity of the chimeric molecule aEv6-Fc, a lethal model of murine rVSV-GP infection was developed by using immunosuppression. The results obtained in lethal model mice have demonstrated the protective effect of aEv6-Fc. Thus, the nanobody and its modified derivative obtained in this study have shown potential protective value against Ebola virus.

3.
Acta Naturae ; 12(3): 114-123, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33173601

RESUMO

The Middle East Respiratory Syndrome (MERS) is an acute inflammatory disease of the respiratory system caused by the MERS-CoV coronavirus. The mortality rate for MERS is about 34.5%. Due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of MERS beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of MERS and reduce its death toll. We have developed a combined vector vaccine for the prevention of MERS based on recombinant human adenovirus serotypes 26 and 5. Studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. Studies of the cellular immune response in mice after vaccination showed the emergence of a specific CD4+ and CD8+ T cell response. A study of the vaccine protectivity conducted in a model of transgenic mice carrying the human DPP4 receptor gene showed that our vaccination protected 100% of the animals from the lethal infection caused by the MERS-CoV virus (MERS-CoV EMC/2012, 100LD50 per mouse). Studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing.

4.
Acta Naturae ; 11(1): 38-47, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024747

RESUMO

The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in 2012 during the first Middle East respiratory syndrome (MERS) outbreaks. MERS-CoV causes an acute lower-respiratory infection in humans, with a fatality rate of ~35.5%. Currently, there are no registered vaccines or means of therapeutic protection against MERS in the world. The MERS-CoV S glycoprotein plays the most important role in the viral life cycle (virus internalization). The S protein is an immunodominant antigen and the main target for neutralizing antibodies. In the present study, the immunogenicities of five different forms of the MERS-CoV S glycoprotein were compared: the full-length S glycoprotein, the full-length S glycoprotein with the transmembrane domain of the G glycoprotein of VSV (S-G), the receptor-binding domain (RBD) of the S glycoprotein, the membrane-fused RBD (the RBD fused with the transmembrane domain of the VSV G glycoprotein (RBD-G)), and the RBD fused with Fc of human IgG1 (RBD-Fc). Recombinant vectors based on human adenoviruses type 5 (rAd5) were used as delivery vehicles. Vaccination with all of the developed rAd5 vectors elicited a balanced Th1/Th2 response in mice. The most robust humoral immune response was induced after the animal had been vaccinated with the membrane-fused RBD (rAd5-RBD-G). Only immunization with membrane forms of the glycoprotein (rAd5-S, rAd5-S-G, and rAd5-RBD-G) elicited neutralizing antibodies among all vaccinated animals. The most significant cellular immune response was induced after vaccination of the animals with the full-length S (rAd5-S). These investigations suggest that the full-length S and the membrane form of the RBD (RBD-G) are the most promising vaccine candidates among all the studied forms of S glycoprotein.

5.
Acta Naturae ; 9(3): 4-11, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29104771

RESUMO

The Ebola virus disease (EVD) is one of the most dangerous infections affecting humans and animals. The first EVD outbreaks occurred in 1976 in Sudan and Zaire. Since then, more than 20 outbreaks have occurred; the largest of which (2014-2016) evolved into an epidemic in West Africa and claimed the lives of more than 11,000 people. Although vaccination is the most effective way to prevent epidemics, there was no licensed vaccine for EVD at the beginning of the latest outbreak. The development of the first vaccines for EVD started in 1980 and has come a long technological way, from inactivated to genetically engineered vaccines based on recombinant viral vectors. This review focuses on virus-vectored Ebola vaccines that have demonstrated the greatest efficacy in preclinical trials and are currently under different phases of clinical trial. Particular attention is paid to the mechanisms of immune response development, which are important for protection from EVD, and the key vaccine parameters necessary for inducing long-term protective immunity against EVD.

6.
Hum Vaccin Immunother ; 13(3): 613-620, 2017 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-28152326

RESUMO

Ebola hemorrhagic fever, also known as Ebola virus disease or EVD, is one of the most dangerous viral diseases in humans and animals. In this open-label, dose-escalation clinical trial, we assessed the safety, side effects, and immunogenicity of a novel, heterologous prime-boost vaccine against Ebola, which was administered in 2 doses to 84 healthy adults of both sexes between 18 and 55 years. The vaccine consists of live-attenuated recombinant vesicular stomatitis virus (VSV) and adenovirus serotype-5 (Ad5) expressing Ebola envelope glycoprotein. The most common adverse event was pain at the injection site, although no serious adverse events were reported. The vaccine did not significantly impact blood, urine, and immune indices. Seroconversion rate was 100 %. Antigen-specific IgG geometric mean titer at day 42 was 3,277 (95 % confidence interval 2,401-4,473) in volunteers immunized at full dose. Neutralizing antibodies were detected in 93.1 % of volunteers immunized at full dose, with geometric mean titer 20. Antigen-specific response in peripheral blood mononuclear cells was also detected in 100 % of participants, as well as in CD4+ and CD8+ T cells in 82.8 % and 58.6 % of participants vaccinated at full dose, respectively. The data indicate that the vaccine is safe and induces strong humoral and cellular immune response in up to 100 % of healthy adult volunteers, and provide a rationale for testing efficacy in Phase III trials. Indeed, the strong immune response to the vaccine may elicit long-term protection. This trial was registered with grls.rosminzdrav.ru (No. 495*), and with zakupki.gov.ru (No. 0373100043215000055).


Assuntos
Vacinas contra Ebola/imunologia , Voluntários Saudáveis , Doença pelo Vírus Ebola/prevenção & controle , Adenoviridae/genética , Adolescente , Adulto , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Portadores de Fármacos/administração & dosagem , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Vacinas contra Ebola/administração & dosagem , Feminino , Humanos , Imunoglobulina G/sangue , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Dor/induzido quimicamente , Dor/epidemiologia , Federação Russa , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Vesiculovirus/genética , Voluntários , Adulto Jovem
7.
Acta Naturae ; 6(2): 95-105, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25093116

RESUMO

Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (aCEA-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying aCEA-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to "nanoantibody-modified RPAN" binding.

8.
Acta Naturae ; 6(4): 27-39, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25558392

RESUMO

Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling.

9.
Bull Exp Biol Med ; 154(4): 558-61, 2013 Feb.
Artigo em Inglês, Russo | MEDLINE | ID: mdl-23486603

RESUMO

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by progressive death of cerebral and spinal motorneurons. Using behavioral tests we studied the efficiency of gene-cell therapy in SOD1 G93A transgenic mice receiving xenotransplantation of human umbilical cord blood mononuclear cells genetically modified with adenoviral vectors encoding vascular endothelial growth factor (VEGF) and reporter green fluorescent protein (EGFP) genes. The cells were transplanted to mice on week 27 of life (preclinical stage of the disease). Behavioral tests (open field, grip strength test) showed that transplantation of umbilical cord blood mononuclear cells expressing VEGF significantly improved the parameters of motor and explorative activity, grip strength, and animal survival. Thus, gene-cell therapy based on genetically modified mononuclear cells expressing VEGF can be efficient for the treatment of amyotrophic lateral sclerosis.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Esclerose Amiotrófica Lateral/terapia , Superóxido Dismutase/metabolismo , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Transgênicos , Superóxido Dismutase/genética , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Acta Naturae ; 4(3): 82-7, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23150806

RESUMO

Non-thermal plasma (NTP) consists of a huge amount of biologically active particles, whereas its temperature is close to ambient. This combination allows one to use NTP as a perspective tool for solving different biomedical tasks, including antitumor therapy. The treatment of tumor cells with NTP caused dose-dependent effects, such as growth arrest and apoptosis. However, while the outcome of NTP treatment has been established, the molecular mechanisms of the interaction between NTP and eukaryotic cells have not been thoroughly studied thus far. In this work, the mechanisms and the type of death of human colon carcinoma HCT 116 cells upon application of non-thermal argon plasma were studied. The effect of NTP on the major stress-activated protein p53 was investigated. The results demonstrate that the viability of HCT116 cells upon plasma treatment is dependent on the functional p53 protein. NTP treatment caused an increase in the intracellular concentration of p53 and the induction of the p53-controlled regulon. The p53-dependent accumulation of active proapoptotic caspase-3 was shown in NTP-treated cells. The study was the first to demonstrate that treatment of human colon carcinoma cells with NTP results in p53-dependent apoptosis. The results obtained contribute to our understanding of the applicability of NTP in antitumor therapy.

11.
Acta Naturae ; 3(1): 77-84, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22649675

RESUMO

Pattern-recognition receptors (PRR) play a crucial role in the induction of the defense reactions of the immune system against pathogenic bacterial and viral infections. The activation of PRR by specific, highly conserved pathogen-associated molecular patterns (PAMPs) induces numerous immune reactions related both to innate and adaptive immunity. In addition to the well-studied Toll-like receptors, pathogens can be recognized by the receptors belonging to the other PRR families; including NOD-like receptors (NLR). Stimulation of members of NOD-like receptors (NOD1, 2) and Toll-like receptors results in the activation of the transcriptional factor NF-kB regulating gene expression in numerous molecules implicated in the development of proinflammatory reactions. As opposed to Toll-like receptors, the NF-kB-activating ability of NLRs has not been fully studied. In this work, we examine the ability of one member of the NLR family - NOD1 - to activate the main proinflammatory transcriptional factor NF-kB. We also compare the NF-kB-activating ability of NOD1 ligands of a different structure with TLR4,5 ligandsin vitroandin vivo.

12.
Acta Naturae ; 3(3): 100-6, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22649700

RESUMO

Recombinant human adenovirus serotype 5 (Ad5/35F-IL2) with modified fibres containing the C-terminal domain fiber-knob of human adenovirus serotype 35, carrying the gene of recombinant human IL-2, has been designed. As a result of the fiber modification, the adenovirus can efficiently deliver the genetic information to bone marrow leukocytes and the tumor blood cells KG-1A (human myeloblastic leukemia cells) and U937 (human histiocytic lymphoma cells), which are normally resistant to Ad5 infection. The flow cytometry data reveal that the modified Ad5/35F penetrates into a population of monocytes, granulocytes, and blast cells of human bone marrow. The expression of interleukin-2 in CAR-negative bone marrow leukocytes (3682.52 ± 134.21 pg/ml) and the cell lines KG-1A (748.3 ± 32.8 pg/ml) and U937 (421.5 ± 59.4 pg/ml) transduced with adenovirus Ad5/35F-IL2 is demonstrated. The fiber-modified adenovirus can be used as a vector for the efficient gene delivery of interleukin-2 to human normal and tumor hematopoietic cells.

13.
Acta Naturae ; 3(4): 83-93, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22649707

RESUMO

Toll-like receptors are the essential components of innate immunity. It is shown that TLRs play an essential role in the immune resistance of an organism to bacterial and viral infections. The binding of TLR to its own ligands results in the activation of several adapter molecules and kinases, inducing the activation of the main pro-inflammatory transcriptional factors, which in turn induce the activation of the main pro-inflammatory transcriptional factors. This activation results in the development of both the innate immune response triggered by the enhanced expression of a number of pro-inflammatory cytokines and antimicrobial peptides and that of the adaptive immune response, via the activation of dendritic cells and enhancement of antigen presentation, etc. The ability of TLR agonists to bolster the immune reaction makes them promising for use in the therapy of infectious diseases and in the chemotherapy of malignant neoformations. However, different TLR ligands may have either antitumor activity (lipopolysaccharide, imiquimod, CpG) or, conversely, could beef up the resistance of tumor cells to apoptosis, stimulating their proliferation under certain conditions (lipopolysaccharide, lipopeptide). It has been shown that the TLR2-dependent signalling pathway in the myelomonocytic mouse leukaemia cell line WEHI-3B leads to the constitutive activation of the transcriptional factor NF-kB, suppression of apoptosis in tumor cells, and progression of myelomonocytic mouse leukaemiain vivo, upon the addition of TLR2 agonist (synthetic lipopeptide Pam2CSK4) or following the infection of tumor cells withMycoplasma arginini.

14.
Biochemistry (Mosc) ; 75(9): 1098-114, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21077829

RESUMO

Toll-like receptors (TLR) are among key receptors of the innate mammalian immune system. Receptors of this family are able to recognize specific highly conserved molecular regions (patterns) in pathogen structures, thus initiating reactions of both innate and acquired immune response finally resulting in the elimination of the pathogen. In this case every individual TLR type is able to bind a broad spectrum of molecules of microbial origin characterized by different chemical properties and structures. Recent data demonstrate the existence of a multistep mechanism of the TLR recognition of the pathogen in which, in addition to receptors proper, the involvement of different adapter molecules is necessary. However, functions of separate adapter molecules as well as the principles of formation of a multicomponent system of ligand-specific recognition are still not quite understandable. We describe all identified as well as possible (candidate) adapter TLR molecules by giving their brief characteristics, and we also propose generalized possible variants of the TLR ligand-specific recognition with involvement of adapter molecules.


Assuntos
Ligantes , Receptores Toll-Like/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antígenos CD36/metabolismo , Drosophila , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Receptores Toll-Like/análise , Receptores Toll-Like/química
15.
Acta Naturae ; 2(1): 111-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22649637

RESUMO

Influenza viruses are characterized by a high degree of antigenic variability, which causes the annual emergence of flu epidemics and irregularly timed pandemics caused by viruses with new antigenic and biological traits. Novel approaches to vaccination can help circumvent this problem. One of these new methods incorporates genetic vaccines based on adenoviral vectors. Recombinant adenoviral vectors which contain hemagglutinin-encoding genes from avian H5N1 and H5N2 (Ad-HA5-1 and Ad-HA5-2) influenza viruses were obtained using the AdEasy Adenoviral Vector System (Stratagene). Laboratory mice received a double intranasal vaccination with Ad-HA5-1 and Ad-HA5-2. This study demonstrates that immunization with recombinant adenoviruses bearing the Н 5 influenza virus hemagglutinin gene induces a immune response which protects immunized mice from a lethal dose of the H5 influenza virus. Moreover, it also protects the host from a lethal dose of the H1 virus, which belongs to the same clade as H5, but does not confer protection from the subtype H3 influenza virus, which belongs to a different clade.

16.
Acta Naturae ; 2(3): 21-9, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22649649

RESUMO

Toll-like receptors (TLRs) are major components of the innate immune system that recognize the conserved molecular structures of pathogens (pathogen-associated molecular patterns; PAMPs). TLRs are found in many different cell types, ranging from epithelial to immunocompetent cells. TLR binding triggers the expression of several adapter proteins and downstream kinases, leading to the induction of key pro-inflammatory mediators. This results in the activation of both the innate immune response (elevated expression of antiapoptotic proteins, proinflammatory cytokines, and antibacterial proteins), as well as the adaptive immune response (maturation of the dendritic cells, antigen presentation, etc.). In consequence of their ability to enhance the specific and nonspecific immune reactions of an organism, TLR agonists are widely used in the therapy of infectious diseases and, as adjuvants, in the therapy of malignant neoplasia. However, to date, TLRs have had the opposite effects on tumor progression. On the one hand, TLR ligands can suppress tumor growth. On the other hand, TLR agonists can promote the survival of malignant cells and increase their resistance to chemotherapy. The purpose of this review is to summarize the available data on the effects of TLRs and their agonists on tumor progression, as well as the mechanisms underlying the differences in the effects of TLRs on tumor growth.

17.
Bull Exp Biol Med ; 145(4): 460-3, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19110594

RESUMO

The effects of gram-positive bacterial strains (Lactobacillus acidophilus and Lactobacillus rhamnosus) and their subcellular components on the survival of hemopoietic clonogenic cells were evaluated by the formation of endogenous splenic colonies. The effects of these preparations on NO production were studied by the spin-trap paramagnetic resonance spectroscopy. Bacterial preparations from gram-positive bacteria stimulated survival of hemopoietic clonogenic cells, but did not induce NO production in contrast to E. coli LPS.


Assuntos
Bactérias Gram-Positivas/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Lipopolissacarídeos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Feminino , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/citologia , Células-Tronco Hematopoéticas/microbiologia , Lactobacillus acidophilus/química , Lactobacillus acidophilus/citologia , Lactobacillus acidophilus/fisiologia , Lactobacillus rhamnosus/química , Lactobacillus rhamnosus/citologia , Lactobacillus rhamnosus/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Frações Subcelulares/fisiologia
18.
Oncogene ; 27(33): 4521-31, 2008 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-18408766

RESUMO

Prokaryotes of the genus Mycoplasma are the smallest cellular organisms that persist as obligate extracellular parasites. Although mycoplasma infection is known to be associated with chromosomal instability and can promote malignant transformation, the mechanisms underlying these phenomena remain unknown. Since persistence of many cellular parasites requires suppression of apoptosis in host cells, we tested the effect of mycoplasma infection on the activity of the p53 and nuclear factor (NF)-kappaB pathways, major mechanisms controlling programmed cell death. To monitor the activity of p53 and NF-kappaB in mycoplasma-infected cells, we used a panel of reporter cell lines expressing the bacterial beta-galactosidase gene under the control of p53- or NF-kappaB-responsive promoters. Cells incubated with media conditioned with different species of mycoplasma showed constitutive activation of NF-kappaB and reduced activation of p53, common characteristics of the majority of human tumor cells, with M. arginini having the strongest effect among the species tested. Moreover, mycoplasma infection reduced the expression level and inducibility of an endogenous p53-responsive gene, p21(waf1), and inhibited apoptosis induced by genotoxic stress. Infection with M. arginini made rat and mouse embryo fibroblasts susceptible to transformation with oncogenic H-Ras, whereas mycoplasma-free cells underwent irreversible p53-dependent growth arrest. Mycoplasma infection was as effective as shRNA-mediated knockdown of p53 expression in making rodent fibroblasts permissive to Ras-induced transformation. These observations indicate that mycoplasma infection plays the role of a p53-suppressing oncogene that cooperates with Ras in cell transformation and suggest that the carcinogenic and mutagenic effects of mycoplasma might be due to inhibition of p53 tumor suppressor function by this common human parasite.


Assuntos
Transformação Celular Neoplásica/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Infecções por Mycoplasma/metabolismo , Mycoplasma/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Linhagem Celular , Transformação Celular Neoplásica/genética , Instabilidade Cromossômica/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/genética , Embrião de Mamíferos/microbiologia , Fibroblastos/microbiologia , Humanos , Camundongos , Infecções por Mycoplasma/genética , NF-kappa B/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Elementos de Resposta/genética , Proteína Supressora de Tumor p53/genética
19.
Virus Res ; 100(2): 257-61, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15019245

RESUMO

In our study, a recombinant adenovirus based on the avian adenovirus CELO genome, has been constructed that contains the human interleukin-2 gene. We have shown the production of biologically active recombinant interleukin-2 in vitro (LMH and 293 cells) and in ovo (chicken embryos) infected with recombinant virus CELO-IL2. An increase in the median survival time of C57BL/6 mice carrying B16 melanoma tumors has been demonstrated after multiple intra-tumors injections of the recombinant adenovirus CELO-IL2.


Assuntos
Adenovirus A das Aves/genética , Interleucina-2/genética , Melanoma Experimental/imunologia , Animais , Embrião de Galinha/virologia , Clonagem Molecular/métodos , Feminino , Humanos , Interleucina-2/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos , Análise de Sobrevida
20.
Gene Ther ; 11(1): 79-84, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14681700

RESUMO

Human adenovirus (Ad) vectors are extensively used as gene transfer vehicles. However, a serious obstacle for the use of these vectors in clinical applications is due to pre-existing immunity to human Ads affecting the efficacy of gene transfer. One of the approaches to circumvent host immune response could be the development of vectors based on non-human Ads that are able to transduce genes into human cells. In this study, we explored the possibility of using avian Ad CELO vectors as gene-transfer vehicles. For this purpose, we constructed a set of recombinant CELO viruses and demonstrated that they are able to deliver transgenes into various organs on the background of pre-existing immunity to human Ad5. The created CELO-p53 vector restored the function of the p53 tumor suppressor both in cultured human tumor cells in vitro and in their xenografts in nude mice in vivo. The latter effect was accompanied by inhibition of tumor growth. Noteworthily, the delivery of CELO-p53 led to activation of p53 target genes in cells showing inactivation of endogenous p53 by three different mechanisms, that is, in the human epidermoid carcinoma A431, lung adenocarcinoma H1299, and cervical carcinoma HeLa.


Assuntos
Aviadenovirus/genética , Genes p53 , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Neoplasias/terapia , Animais , Linhagem Celular Tumoral/metabolismo , Expressão Gênica , Humanos , Injeções , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias/metabolismo , Transgenes , Proteína Supressora de Tumor p53/análise
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