RESUMO
In this research we investigate neuroblastoma cells cultivated on single-walled carbon nanotubes networks made by CVD method on silicon substrates. The complex analysis of grown cells made by atomic force, electron microscopy and Raman spectroscopy was carried out and the effect of nanotube growth process on proliferation factor was investigated. It is shown that despite of a weak decrease in proliferation, cell morphology remains unchanged and no physical or chemical interaction between carbon nanotubes and cells is observed. The results of the research can be used to investigate the interaction between conductive nanomaterials and cells for the development of neural replacement implants. Also they can be useful in bio-electronic interface investigation of signal propagation in neurons.
Assuntos
Proliferação de Células , Nanotubos de Carbono/química , Neuroblastoma/metabolismo , Neurônios/metabolismo , Linhagem Celular Tumoral , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Neuroblastoma/ultraestrutura , Neurônios/ultraestrutura , Análise Espectral RamanRESUMO
Different cell tissue cultures and commercial fetal calf sera (FTS) used in biological and virological research were screened for the bovine viral diarrhea virus (BVDV, Pestivirus genus, Flaviviridae family) and mycoplasma contamination. BVDV was detected using RT-PCR and Indirect immunofluorescence (with monoclonal antibodies) methods in 33% cases of the studied cell lines and in > 60% cases of FCS. BVDV was shown to present and reproduce in high spectra of human cell lines, as well as in monkey, pig, rabbit, goat, dog, and cat cells at high levels (up to 100-1000 genome-equivalent copies per cell) and reached up to 10(3)-10(7) genome-equivalent copies per serum ml. The molecular mechanisms of the long virus persistence without definite signs of destruction should be studied.
Assuntos
Linhagem Celular/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Mycoplasma/isolamento & purificação , RNA Viral/isolamento & purificação , Animais , Bovinos , Técnicas de Cultura de Células , Sangue Fetal/virologia , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Soro/virologiaRESUMO
It was demonstrated that the three studied samples of carbon nanotubes of domestic production fixed on the substrate surface did not have toxic effect and could be used for cell cultivation. A biocompatible conductive coating based on carbon nanotubes and bovine serum albumin was developed. The efficacy of the coating for growing in vitro cell cultures was tested. A device was developed for electric stimulation of the cells. Local electric potential was applied to the cells using nanoscale electrodes. The results of human embryonic fibroblast cultivation in a pulsed electric field on conductive nanocomposite substrates were presented. An 26% increase in the proliferative activity of cells was observed at potentials up to 100 mV.
Assuntos
Estimulação Elétrica , Nanotubos de Carbono , Soroalbumina Bovina/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/toxicidade , Bovinos , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Eletricidade , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidadeRESUMO
The reproduction of the metapneumovirus was comparatively studied in 19 human and animal cell lines. The most sensitive transplanted cell lines were found to be human Chang Conjunctiva (clone 1-5C4) and animal cell lines of feline kidney CRFK.
Assuntos
Técnicas de Cultura de Células/métodos , Linhagem Celular/virologia , Metapneumovirus/crescimento & desenvolvimento , Replicação Viral/genética , Animais , Gatos , Cães , Humanos , Metapneumovirus/genética , Camundongos , Infecções por Paramyxoviridae/virologiaRESUMO
The currently used tick-borne encephalitis virus vaccines are based on the inactivation of tick-borne encephalitis virus (TBEV) of Far Eastern or West European genetic types from the primary cultures of chick embryo fibroblasts. Since the WHO recommends that vaccines should be designed using continuous cell cultures rather than chick embryos as a substrate, this investigation has compared the infection of continuous monolayer SPEV, Vero E6, and vaccine line Vero (B) cell cultures with TBEV strains of the Siberian and Far Eastern genetic types dominating in the endemic regions of Russia. After cell infection with Far Eastern (Sofyin and 205 strains) or Siberian (Aina, 2530, 2689, and 2703 strains) TBEV genetic types, the viable TBEV titers reached 2.8 Ig CPD50 for Vero (B) cells, 5.5 Ig CPD50 for Vero E6 cells, and up to 9 Ig CPD50 for SPEV cells. The quantitative scores of TBEV E antigen in enzyme immunoassay (EIA) and genome equivalents by reverse-transcription polymerase chain reaction (PCR), followed by real-time PCR, permitted one to estimate as high as 108 virions in 1 ml of culture fluid, which corresponded to those of the microscopic observations of CPD for SPEV cells and substantially exceeded the values for Vero E6 cells, and for Vero (B) cells in particular. The data of TBEV strain titration, EIA, and realtime reverse-transcription PCR suggest that the Russian vaccine Vero (B) cell line defined as meeting the WHO requirements, as well as Vero E6 cells may be used to design tick-borne encephalitis vaccine.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Vacinas Virais/isolamento & purificação , Cultura de Vírus/métodos , Animais , Anticorpos Antivirais , Embrião de Galinha , Chlorocebus aethiops , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/prevenção & controle , Imunoensaio , Camundongos , Federação Russa , Suínos , Células Vero , Replicação ViralRESUMO
AIM: Comparative immunogenicity studies of experimental vaccines based onA/Aichi/2/68 neuraminidase peptide fragments (NA) and influenza virus A and B strains produced in MDCK cell culture. MATERIALS AND METHODS: Anti-hemagglutinin and virus neutralizing activity of mice sera was determined in MN and HI reactions in accordance with the WHO recommendations. RESULTS: Sera against peptides 136-147 and 154-164 from variable sites, as well as against peptide 314-328 from conservative region of the heavy chain of A/ Aichi/2/68 influenza virus NA showed distinct anti-hemagglutinin and neutralizing activity against homologous influenzavirus. Anti-(314-328) serum was also active in HI and MN reactions against other strains of the H3N2 subtype. Combined administration of peptide sample with an immunomodulator (Immunomax) increased the immunogenicity to the level of the cultural samples based on influenza A virus. CONCLUSION: The results show higher immunogenicity of cultural vaccines based on influenza virus in comparison to peptide samples. A possibility of peptide vaccine immunogenicity increase was demonstrated by combined administration with the immunomodulator.
Assuntos
Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Neuraminidase/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Linhagem Celular , Cães , Hemaglutininas Virais/imunologia , Humanos , Fenômenos Imunogenéticos , Vírus da Influenza A Subtipo H3N2/imunologia , Rim/citologia , Camundongos , Soro/imunologia , Soro/virologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
The paper provides the data of a comparative analysis of the indicators of immune and interferon states and cytokine profile and the results of virological studies in patients with different (acute and chronic) forms of mixed herpesvirus infection (with virus simplex herpes types 1 and 2, Epstein-Barr virus, human herpesvirus type 6, and others). Pronounced changes were found in immune responses in such patients. There were decreases in IFN-alpha and IFN-gamma values in 36 and 13%, respectively; 51% of the subjects showed a reduction in both IFN-alpha and IFN-gamma along with the high titers of antibodies to viruses of the Herpesviridae family and their infectious activity. There were changes in the cytokine profile, activation of IFN-alpha, IL-2, IL-8, IL-10, and IL-18 gene expression, and suppression of IL-2 gene transcription in the majority of the patients. Determination of IFN susceptibility revealed that 86% of the subjects responded to IFN-alpha therapy and only 11% of cases did to IFN-gamma one.
Assuntos
Anticorpos Antivirais/metabolismo , Herpesviridae/imunologia , Interferon-alfa , Interferon gama , Interleucinas/metabolismo , Adolescente , Adulto , Idoso , Feminino , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/terapia , Humanos , Interferon-alfa/metabolismo , Interferon-alfa/uso terapêutico , Interferon gama/metabolismo , Interferon gama/uso terapêutico , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Whether the MDCK cell line might adapt to grow in serum-free Hybris-2 medium and influenza viruses might be reproduced in the adapted cells was studied. Seventeen passages using the Hybris-2 medium yielded cells adapted to growth in this medium (MDCK-BS). The reproduction of influenza A (H1N1 and H3N2) and B viruses versus the cells cultured in Eagle's medium was studied. The laboratory strain of influenza A/Aichi/1/68 (H3N2) and the strain B/Ohio/01/05 of influenza B in equal titers were shown to be reproduced in both control cells on Eagle's medium and MDCK-BS cells adapted to growth in the Hybris-2 medium. The reproduction of the strains A/Brisbane/10/07 (H3N2) and A/Solomon Islands/3/06 (H1N1) was less active in the MDCK cells. Each strain of influenza viruses displayed varying infective activities. The developed serum-free Hybris-2 medium may be used for cultivation of monolayer continuous MDCK cells and for their reproduction of influenza A and B viruses.
Assuntos
Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza B/crescimento & desenvolvimento , Cultura de Vírus/métodos , Animais , Linhagem Celular , Meios de Cultura Livres de Soro , Cães , Replicação ViralRESUMO
Vitaherpavac, a dry inactivated herpes simplex virus (HSV) culture vaccine, has been obtained, by using the Vero B continuous cell line as a substrate for accumulation of herpes simplex virus types 1 (US strain) and 2 (VN strain). Vitaherpavac and the similar vaccine Herpovax made by the Research Institute of Vaccines and Sera, Saint Petersburg (for which preparation a primary trypsinized chick embryo cell culture used as a substrate for accumulation of HSV types 1 and 2), underwent comparative clinical trials. The tolerability and therapeutic effectiveness of the vaccine were tested in patients diagnosed as having chronic frequently recurring herpes. The trials have yielded positive results that suggest that it is expedient to introduce of the new vaccine Vitaherpavac into practice to treat chronic recurrent herpetic infection of various localizations. Vitaherpavac has been registered in the Russian Federation and permitted for medical application.
Assuntos
Vacinas contra o Vírus do Herpes Simples/uso terapêutico , Herpes Simples/terapia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Adolescente , Adulto , Animais , Chlorocebus aethiops , Doença Crônica , Vacinas contra o Vírus do Herpes Simples/administração & dosagem , Vacinas contra o Vírus do Herpes Simples/efeitos adversos , Humanos , Injeções Intradérmicas , Pessoa de Meia-Idade , Resultado do Tratamento , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/uso terapêutico , Células VeroRESUMO
The reproduction of highly pathogenic avian influenza (HPAI) A/tern/SA/61 H5N3 and A/ducklNovosibirsk/56/05 H5NI viruses was comparatively studied in 16 human and animal cell lines. The strain A/duck/Novosibirsk/56/05 was shown to have a wider range of hosts. The most sensitive transplanted cell lines were found to be feline fibroblasts (CC-81), primarily trypsin-treated cells of chick embryonic fibroblasts (CEF), the kidney of dogs (MDCK), pigs (SPEV), monkeys (Vero), the human conjunctiva (1-5C-4), and, to a lesser extent, the feline kidney (CRFK). Unlike the strain A/tern/South Africa/61, that A/duck/Novosibirsk/56105 replicated in the polecat brain cells (Mpf).
Assuntos
Vírus da Influenza A/fisiologia , Animais , Gatos , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , Cães , Humanos , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/virologia , Especificidade da Espécie , Suínos , Células Vero , Cultura de Vírus , Replicação ViralRESUMO
Interaction of the synthetic peptides corresponding to the regions 122-133, 136-147, 154-164 and 314-328 of the virus A/Aichi/2/68 hemagglutinin heavy chain with monoclonal antibodies specific for this hemagglutinin was assayed in a variety of tests, e.g., ELISA, competition RIA, hemagglutinin-inhibition and virus-neutralization assays. The monoclonal antibody 152 reacted with the area 136-147 (epitope A), three monoclonal antibodies 3, 19 and 63 reacted exclusively with the area (154-164) Glu (epitope B). Mapping of two monoclonal antibodies IV A1 and IV G6 specific for the influenza virus A/Dunedin/ 4/73 hemagglutinin heavy chain and cross-reacting with a number of the H3 subtype viruses was carried out. The specificity of the interaction of the conservative peptide H3(314-328) with monoclonal antibodies IV A1 and IV G6 was confirmed by competition RIA and by competition hemagglutinin inhibition and virus neutralization.
Assuntos
Anticorpos Monoclonais/química , Antígenos Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Animais , Reações Antígeno-Anticorpo , Testes de Inibição da Hemaglutinação , Vírus da Influenza B/imunologia , Mapeamento de Peptídeos , CoelhosRESUMO
Many in vitro and in vivo experiments using contemporary methods supported safety and efficiency of "VITA" device protecting humans and living beings from electromagnetic fields. Therefore, bioenergetic safety device "VITA" is advisable for use.
Assuntos
Telefone Celular , Campos Eletromagnéticos/efeitos adversos , Equipamentos de Proteção , Animais , Encéfalo/citologia , Células Cultivadas , Meios de Cultura , Embrião de Mamíferos/citologia , Metabolismo Energético , Glioblastoma , Humanos , Sistema Imunitário/fisiologia , Influenza Humana/imunologia , Pulmão/citologia , Masculino , Camundongos , Microcomputadores , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Two optimal variants of growth medium with different content of growth proteins obtained from cattle and porcine blood sera are recommended for culturing lymphoblastoid cells. Use of growth proteins helps optimize the conditions of cell culturing by ruling out the toxic effect and Mycoplasma contamination and ensures the standardization of virological experiments due to the absence of gamma-globulin fraction.
Assuntos
Proteínas Sanguíneas/fisiologia , Meios de Cultura , Linfócitos T/citologia , Animais , Bovinos , Divisão Celular , Linhagem Celular , Humanos , SuínosRESUMO
Monospecific antibodies to three nonoverlapping sites of influenza A/Singapore/1/57 (H2N2) virus isolated from hyperimmune serum by specific adsorption were characterized by capacity to inhibit virus hemagglutination, immunochemically interact with hemagglutinin, and neutralize the virus (6-7.5 lg). Monoclonal antibodies to the same virus hemagglutinin displayed an even higher activity than monospecific antibodies in serological and immunochemical tests and were characterized by a rather low (1.0-4.5 lg) or null activity in biological neutralization of the virus.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos/imunologia , Antígenos Virais/imunologia , Hemaglutininas Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Soros Imunes , Testes de NeutralizaçãoRESUMO
The authors recommend to replace nonstandard native animal sera by growth-stimulating proteins isolated from the sera of cattle, northern deer, pigs, and sheep by fractionation with polyethylene glycol aqueous solutions (mol. m 6000 dalton). Growth-stimulating proteins are conducive to active proliferation of primary and continuous cell cultures at various methods of culturing. Growth-stimulating proteins were for the first time added to nutrient media during culturing of mammalian cells on microcarriers.
Assuntos
Sangue , Substâncias de Crescimento/farmacologia , Proteínas/farmacologia , Animais , Células Cultivadas , Meios de Cultura , Humanos , MamíferosRESUMO
Conditions for pseudosuspension culturing of continuous cells of the kidneys of dogs (MDCK), green marmosets (CV-1), and swine (SPEV) on two types of microcarriers (Cytogel-3 and cytolar-2) under semiproduction conditions (50 liter bioreactor) were developed on a model of reassortants of influenza viruses A and B. To standardize the conditions, native sera were replaced by growth-stimulating proteins isolated from the sera of various animals (cattle, northern deer, swine). A better adhesive capacity and more active cell proliferation on porous microcarrier cytogel-3 were observed. The highest proliferative activity of cells was observed when porcine growth-stimulating proteins were used. Influenza A virus reassortant (H3N1) was actively reproduced in MDCK cells. The reproduction of influenza B virus reassortant was similarly higher in MDCK cells, in comparison with SPEV cells; at the same time, no differences in hemagglutinin titers in the two cell cultures were observed. The pseudosuspension method of cell culturing is recommended for the preparation of influenza antigens.
Assuntos
Antígenos Virais/biossíntese , Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Vacinas Virais/biossíntese , Animais , Divisão Celular , Células Cultivadas , Chlorocebus aethiops , Cães , Fermentação , Hemaglutininas Virais/análise , Vírus da Influenza A/fisiologia , Suínos , Replicação ViralRESUMO
Collase, an enzymatic preparation made from crab hepatopancreas, was used as a dissociative reagent in preparation of primary cell cultures and for detachment of continuous cells from the substrate during reinoculation. Collase was found to increase viable cell harvest by 2 to 2.5 times in comparison with trypsin and had a less detrimental effect on the cells which retained their proliferative activity, morphology, productivity, and sensitivity to viruses.
Assuntos
Meios de Cultura , Serina Endopeptidases , Animais , Braquiúros , Bovinos , Adesão Celular , Divisão Celular , Linhagem Celular , Embrião de Galinha , Cães , Células HeLa , Humanos , Fígado/enzimologia , Pâncreas/enzimologia , TripsinaRESUMO
Reproduction of parental strains and reassortants (with known genome composition) of influenza A and B viruses was studied in chick embryos (CE) and in different cell lines (SPEV, MDCK, BHK-21, M22, etc.). The results agree with the concept that the yield of influenza A virus in CE depends on its M-gene. At the same time, the experimental results suggest that reproduction of influenza B virus in the same system is not determined by M-gene. Reproduction (hr-phenotype) of influenza A and B viruses in cell cultures was shown to be determined not only by the gene coding for hemagglutinin but also by other virus genes, the reproduction level being dependent on different genes in different cell systems.
Assuntos
Genoma Viral , Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Vírus Reordenados/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Humanos , Vírus da Influenza A/genética , Vírus da Influenza B/genética , Vírus Reordenados/genética , Cultura de Vírus , Replicação Viral/genéticaRESUMO
Immunological analysis of the antigenic structure of hemagglutinin of newly isolated variants of influenza (H3N2) virus carried out using monoclonal and monospecific antibodies to individual antigenic sites of hemagglutinin showed the 1989-1990 isolates to be markedly different in their antigenic properties from the variants isolated in previous years. Sites with new antigenic properties were determined in hemagglutinin of the isolates. Wide variability was found in the region of three immunodominant sites. The fact of circulation in the human population of influenza viruses of one subtype with different antigenic structures within the limits of one epidemic season was established.