RESUMO
BACKGROUND: Tumor cell lines have enormous value for the study of different aspects of cancer biology and have also recently gained great importance in autologous cell-based anti-tumor therapies. However, the use of these cells is still limited because in vitro growth is hampered by suboptimal culture conditions and current media contain fetal bovine serum (FBS), which poses serious safety concerns regarding clinical application. METHODS: To address this drawback, we aimed to develop a strategy for optimization of the culture medium for human medullary thyroid carcinoma (MTC) cell lines as a model system. We combined the general cell screening system (GCSS), which continuously measured the growth behavior of cells in a 96-well plate format, with statistically based experimental designs. RESULTS: The results obtained clearly demonstrated that, just by changing the composition of the basal medium, a significantly enhanced growth rate could be observed, and by subsequent addition of several substances a serum-free cell culture medium could be developed. This medium allowed the propagation of two MTC cell lines comparable with conventionally used serum-supplemented medium. DISCUSSION: We present a fast and easy way to screen for substances that are essential for tumor cell growth in vitro. Furthermore, these tumor cells can be adapted to culture conditions that allow the use of the cells in safe cell-based therapies. This is of utmost importance because of increasing regulatory requirements.
Assuntos
Carcinoma/metabolismo , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Neoplasias da Glândula Tireoide/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Meios de Cultura Livres de Soro/normas , HumanosRESUMO
Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.
Assuntos
Lipossomos/química , Nebulizadores e Vaporizadores , Superóxido Dismutase/química , Vibração , Aerossóis , Sistemas de Liberação de Medicamentos , Estabilidade de Medicamentos , Estabilidade Enzimática , Humanos , Tamanho da Partícula , Estresse MecânicoRESUMO
We generated several attenuated recombinant influenza A vectors expressing the Mycobacterium tuberculosis early secretory antigenic target (ESAT-6) protein. The ESAT-6 protein was recently identified as one of the most promising protective antigens for cell-mediated immunity. The obtained vectors appeared to be capable of inducing ESAT-6 specific Th1 immune response in mice after intranasal immunization. We found that double immunization with two influenza vectors of different subtypes provided a significant level of protection in mice, when applied as prophylactic vaccine, as well as substantial therapeutic effect in mice with pre-established tuberculosis infection. Moreover, we found a strong synergistic effect when vaccination with Flu/ESAT-6 vectors was combined with isoniazid treatment, resulting in a dramatic reduction of bacterial load in the lungs of infected mice.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Administração Intranasal , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/uso terapêutico , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/uso terapêutico , Terapia Combinada , Vetores Genéticos/administração & dosagem , Imunidade Celular , Imunização/métodos , Vírus da Influenza A/genética , Isoniazida/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Células Th1/imunologia , Tuberculose/imunologia , Tuberculose/terapia , Vacinas contra a Tuberculose/uso terapêuticoRESUMO
Establishment of tumor cell lines as model systems for studying tumor biology or as a part of immunotherapeutic anti-cancer strategies is of high importance, whereby the highest possible preservation of the original tumor cell phenotype is a prerequisite for these aims. Since overexpression of the catalytic subunit of human telomerase (hTERT) is known to minimally alter the cellular phenotype, we focused on the establishment of cell lines derived from human fibroma from a MEN1 patient by ectopic expression of hTERT. Additionally, a cell line was generated by introduction of the early region of SV40 (SV40 ER). Both approaches resulted in continuous cell lines, and neither T1-LOHG (hTERT) nor SV1-LOHG (SV40 ER) showed a transformed phenotype. While SV40 ER-transfected cells underwent dramatic changes in morphology and growth characteristics, hTERT-expressing cells indeed retained a phenotype highly similar to the parental cells. Nevertheless, hTERT overexpression resulted in increased growth rates after about 70 population doublings (PD) and alterations of mRNA levels of genes associated with tumor pathogenesis. Thus, our data suggest that ectopic hTERT expression leads to immortalization of LOHG-F, sustaining many characteristics of the non-transfected counterparts, but continuous growth in vitro is associated with changes of the cellular phenotype.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Proliferação de Células , Fibroma/genética , Fibroma/patologia , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasia Endócrina Múltipla Tipo 1/patologia , Telomerase/biossíntese , Telomerase/farmacologia , Células Tumorais Cultivadas , Sobrevivência Celular , Proteínas de Ligação a DNA , Humanos , Fenótipo , RNA Mensageiro/biossíntese , TransfecçãoAssuntos
Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Antígenos CD/sangue , Antígenos de Diferenciação de Linfócitos T/sangue , Divisão Celular , Técnicas de Cocultura , Neoplasias Colorretais/imunologia , Humanos , Ativação Linfocitária , Células Estromais/imunologia , Células Tumorais CultivadasRESUMO
Simian-human immunodeficiency viruses (SHIV) allow the evaluation of antiviral strategies that target the envelope glycoproteins of the human immunodeficiency virus 1 (HIV-1) in macaques. We previously protected neonates from oral challenge with cell-free SHIV-vpu+ by passive immunization with synergistic human neutralizing monoclonal antibodies (mAbs) (Baba et al., Nat Med 6:200-206, 2000). mAbs were administered prenatally to pregnant dams and postnatally to the neonates. Here, we used solely postnatal or postexposure mAb treatment, thus significantly reducing the amount of mAbs necessary. All neonatal monkeys were also protected with these abbreviated mAb regimens. Our results are directly relevant for humans because we used mAbs that target HIV-1 envelope glycoproteins. Thus, the large-scale use of passive immunization with neutralizing mAbs may be feasible in human neonates. The mAbs, being natural human proteins, can be expected to have low toxicity. Passive immunization has promise to prevent intrapartum as well as milk-borne virus transmission from HIV-1-infected women to their infants.
Assuntos
Animais Recém-Nascidos/imunologia , HIV/imunologia , Imunização Passiva/métodos , Macaca mulatta/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Administração Oral , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Anticorpos Anti-HIV/imunologia , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Imunidade nas Mucosas , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Fatores de Tempo , Carga Viral , Proteínas Virais Reguladoras e Acessórias/fisiologiaRESUMO
Several reports have described the existence of synergy between neutralizing monoclonal antibodies (MAbs) against human immunodeficiency virus type 1 (HIV-1). Synergy between human MAbs b12, 2G12, 2F5, and 4E10 in neutralization of primary isolates is of particular interest. Neutralization synergy of these MAbs, however, has not been studied extensively, and the mechanism of synergy remains unclear. We investigated neutralization synergy among this human antibody set by using the classical approach of titrating antibodies mixed at a fixed ratio as well as by an alternative, variable ratio approach in which the neutralization curve of one MAb is assessed in the presence and absence of a fixed, weakly neutralizing concentration of a second antibody. The advantage of this second approach is that it does not require mathematical analysis to establish synergy. No neutralization enhancement of any of the MAb combinations tested was detected for the T-cell-line-adapted molecular HIV-1 clone HxB2 using both assay formats. Studies of primary isolates (89.6, SF162, and JR-CSF) showed neutralization synergy which was relatively weak, with a maximum of two- to fourfold enhancement between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations. Analysis of b12 and 2G12 binding to oligomeric envelope glycoprotein by using flow cytometry failed to demonstrate cooperativity in binding between these two antibodies. The mechanism by which these antibodies synergize is, therefore, not yet understood. The results lend some support to the notion that an HIV-1 vaccine that elicits moderate neutralizing antibodies to multiple epitopes may be more effective than hereto supposed, although considerable caution in extrapolating to a vaccine situation is required.
Assuntos
HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Citometria de Fluxo , HIV-1/metabolismo , Humanos , Testes de NeutralizaçãoRESUMO
The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1(MN) virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Epitopos , Citometria de Fluxo , Proteína gp41 do Envelope de HIV/química , Humanos , Dados de Sequência Molecular , Testes de NeutralizaçãoRESUMO
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Mapeamento de Epitopos , Proteína gp41 do Envelope de HIV/química , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Testes de NeutralizaçãoRESUMO
Neonatal macaques were completely protected against oral challenge with SHIV-vpu+, a simian-human immunodeficiency virus that encodes the envelope gene of a laboratory-adapted HIV strain, by pre- and post-natal treatment with a triple combination of human neutralizing monoclonal antibodies (mAbs). The mAbs were directed either against the CD4 binding site, a glycosylation-dependent gp120 epitope, or against a linear epitope on gp41. This triple combination was highly synergistic in vitro and neutralized primary HIV completely. Subsequently, oral challenge was performed with pathogenic SHIV89.6P, an animal-passaged variant of a chimeric virus that encodes the envelope gene of the primary, dual-tropic HIV89.6. Only post-natal treatment with a similar triple mAb combination was used. One out of 4 mAb-treated infants was completely protected from infection. In the other 3 treated animals, there was a tendency towards lower peak viral RNA loads compared with untreated controls. Two out of 4 mAb-treated infants maintained normal CD4+ T-cell numbers, in contrast to all controls that had steep declines at 2 weeks post-challenge. We conclude that the triple mAb combination significantly protected the neonates, even against mucosal challenge with pathogenic SHIV89.6P. Passively administered synergistic human mAbs may play a role in preventing mother-infant transmission of HIV, both against intrapartum transmission as well as against infection through breast milk. As passive immunization is a tool to assess correlates of immune protection, we conclude that the epitopes recognized by the mAbs in our combinations are important for AIDS vaccine development. Future passive immunization studies may reveal other important conserved epitopes.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Anti-HIV/administração & dosagem , Infecções por HIV/prevenção & controle , HIV/imunologia , Imunização Passiva , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Vacinação , Vacinas contra a AIDS/imunologia , Administração Oral , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/imunologia , Contagem de Linfócito CD4 , Cesárea , Parto Obstétrico , Modelos Animais de Doenças , Feminino , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Humanos , Imunidade Materno-Adquirida , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Lactação , Macaca mulatta , Troca Materno-Fetal , Leite/virologia , Testes de Neutralização , Projetos Piloto , Gravidez , Complicações Infecciosas na Gravidez/virologia , Especificidade da Espécie , Montagem de Vírus , Eliminação de Partículas ViraisRESUMO
We have generated recombinant influenza A viruses belonging to the H1N1 and H3N2 virus subtypes containing an insertion of the 137 C-terminal amino acid residues of the human immunodeficiency virus type 1 (HIV-1) Nef protein into the influenza A virus nonstructural-protein (NS1) reading frame. These viral vectors were found to be genetically stable and capable of growing efficiently in embryonated chicken eggs and tissue culture cells but did not replicate in the murine respiratory tract. Despite the hyperattenuated phenotype of influenza/NS-Nef viruses, a Nef and influenza virus (nucleoprotein)-specific CD8(+)-T-cell response was detected in spleens and the lymph nodes draining the respiratory tract after a single intranasal immunization of mice. Compared to the primary response, a marked enhancement of the CD8(+)-T-cell response was detected in the systemic and mucosal compartments, including mouse urogenital tracts, if mice were primed with the H1N1 subtype vector and subsequently boosted with the H3N2 subtype vector. In addition, Nef-specific serum IgG was detected in mice which were immunized twice with the recombinant H1N1 and then boosted with the recombinant H3N2 subtype virus. These findings may contribute to the development of alternative immunization strategies utilizing hyperattenuated live recombinant influenza virus vectors to prevent or control infectious diseases, e.g., HIV-1 infection.
Assuntos
Produtos do Gene nef/imunologia , HIV-1/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Vacinas contra a AIDS , Animais , Vetores Genéticos , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Imunidade nas Mucosas , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Camundongos , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
To develop immunoprophylaxis regimens against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, we established a simian-human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al: AIDS Res Hum Retroviruses 10:351-357, 1994). We protected four neonates from oral SHIV-vpu+ challenge by ante- and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti-HIV-1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann-Lehmann et al: Nature Med 6:200-206, 2000), which recognize the CD4-binding site of Env, a glycosylation-dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV-vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb-treated neonates was protected from infection and two maintained normal CD4+ T-cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV-1 transmission in humans.
Assuntos
Síndrome da Imunodeficiência Adquirida/transmissão , Infecções por HIV/transmissão , HIV-1/patogenicidade , Imunização Passiva , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Vírus da Imunodeficiência Símia/fisiologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Animais , Anticorpos Monoclonais/uso terapêutico , Quimera , Modelos Animais de Doenças , Feminino , Infecções por HIV/prevenção & controle , Humanos , Imunoglobulina G/uso terapêutico , Recém-Nascido , Macaca mulatta , Masculino , Período Pós-Parto , GravidezRESUMO
To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351-357, 1994). Using this model, neonates were protected from mucosal SHIV-vpu(+) challenge by pre- and postnatal treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and 2F5 (Baba et al., Nat. Med. 6:200-206, 2000). In the present study, we used this MAb combination only postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu(+) challenge, while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination against the more pathogenic SHIV89.6P, a chimeric virus encoding env of the primary HIV89.6. Remarkably, the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4(+) T-cell decline. In contrast, all control animals had dramatic drops in their CD4(+) T cells by 2 weeks postexposure. We conclude that our triple MAb combination partially protected against mucosal challenge with the highly pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1.
Assuntos
Anticorpos Monoclonais/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Administração Oral , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/administração & dosagem , Sinergismo Farmacológico , Humanos , Imunidade nas Mucosas , Imunização Passiva , Macaca , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/transmissãoRESUMO
The overproduction of biochemical mediators, and activation of leukocytes and endothelial cells, generated in thermally injured tissue, gives rise to both local and distant effects. The formation of short-lived, highly reactive metabolites, such as oxygen free radicals, increases with increasing tissue ischemia, and causes further cell damage. Human recombinant Cu/Zn-superoxide dismutase (rh-Cu/Zn-SOD), an enzyme which captures these radicals, may have a beneficial effect on the postburn inflammation processes. In this study, the influence of rh-Cu/Zn-SOD application to thermally injured tissue of rabbit backskin was examined. Three different delivery strategies were compared, pure or liposomally encapsulated enzyme, or intralesionally injected rh-Cu/Zn-SOD. For control, one animal group was treated with plain gel and another group was kept untreated. At 24 h following trauma a statistically significant difference in lesion sizes between the enzyme treated and control groups was observed. After 72 h tissue swelling had diminished significantly more in the rh-Cu/Zn-SOD treated groups as compared to the control animals. The best results were achieved by spreading liposomes encapsulating the enzyme onto the wounds. Our results suggest that local treatment of burn wounds with enzymatic radical scavengers such as rh-Cu/Zn-SOD has a beneficial effect on the extent of the postburn damage.
Assuntos
Queimaduras/tratamento farmacológico , Sequestradores de Radicais Livres/administração & dosagem , Sequestradores de Radicais Livres/uso terapêutico , Superóxido Dismutase/administração & dosagem , Superóxido Dismutase/uso terapêutico , Animais , Queimaduras/patologia , Composição de Medicamentos , Edema/tratamento farmacológico , Edema/patologia , Escherichia coli/metabolismo , Meia-Vida , Lipossomos , Tamanho da Partícula , Coelhos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/uso terapêutico , Pele/patologia , Superóxido Dismutase/biossínteseRESUMO
OBJECTIVES: We investigated the ability of several human neutralizing monoclonal antibodies (mAbs), originally raised against human immunodeficiency virus (HIV) clade B isolates, to neutralize primary clade C isolates as single agents and in combination. STUDY DESIGN/METHODS: HIV clade C isolates from five different countries were tested for susceptibility to neutralization by anti-clade B mAbs in human peripheral blood mononuclear cells. Monoclonal antibody combinations were evaluated for possible synergy. RESULTS: All 20 primary HIV clade C isolates could be neutralized 97.5% to 100% by a quadruple combination of mAbs IgG1b12, 2G12, 2F5, and 4E10. These mAbs recognized conserved epitopes and were highly synergistic, resulting in strong cross-clade neutralization. CONCLUSIONS: In our previous experiment, a synergistic combination of human neutralizing mAbs protected all macaque neonates against oral challenge with a simian-human immunodeficiency virus encoding HIV env. Together, our data suggest that passive immunization with currently available anti-clade B mAbs could play a role in preventing HIV clade C transmission through breastfeeding.
Assuntos
Anticorpos Monoclonais/imunologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Sinergismo Farmacológico , Anticorpos Anti-HIV/imunologia , Humanos , Leucócitos Mononucleares/virologia , Testes de NeutralizaçãoRESUMO
Dihydrofolate reductase (DHFR) based amplification of recombinant genes using increasing concentrations of methotrexate (MTX) is a common method to establish CHO cell lines producing high amounts of the desired protein. Once, cell lines with highly amplified target genes and good expression rates are isolated, further characterization of their transcriptional pattern is intended to clarify the question what other factors are elevated, as a prerequisite or consequence of recombinant protein production. In order to define genes which are upregulated in a cell line that shows high production rates, we have investigated alterations in gene expression which occur beside amplification of the recombinant genes. For this purpose, the suppression subtractive hybridization method was used to create a cDNA library enriched for differentially expressed sequences in the recombinant antibody producing CHO cell line versus the original counterpart. Differential expression was confirmed by Northern blotting and Northern ELISA. In addition to the expected recombinant genes, we have identified 5 transcripts which are upregulated in the recombinant cell line. One sequence has not been found in existing data bases, the others revealed to be genes involved in protein synthesis and regulation of transcription. Furthermore, an alternatively spliced, non-functional form of the DHFR mRNA was detected, suggesting a dramatic increase of the selection pressure exerted by MTX.
Assuntos
Proteínas Recombinantes/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Aspartato Aminotransferases/genética , Células CHO , Cricetinae , Perfilação da Expressão Gênica , Humanos , Hibridização In Situ/métodos , Lisina-tRNA Ligase/genética , Dados de Sequência Molecular , Proteínas/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Transcrição Gênica , Regulação para CimaRESUMO
We have established a panel of human monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1). The antibodies 2F5 and 2G12 have been identified to be two of the most potently in vitro neutralizing antibodies against HIV-1. Here we report on a further antibody, 4E10, of similar in vitro neutralizing potency. 4E10 binds to a novel epitope C terminal of the ELDKWA sequence recognized by 2F5, which has been so far the only described broadly neutralizing anti-gp41 antibody. Both 4E10 and 2F5 bind only weakly to infected cells compared with gp120-specific 2G12 and polyclonal anti-HIV-1 immunoglobulin (HIVIG), but show potent in vitro neutralizing properties. 4E10 neutralizes potently not only tissue culture-adapted strains but also primary isolates of different clades, including A, B, C, D, and E. Viruses that were found to be resistant to 2F5 were neutralized by 4E10 and vice versa; none of the tested isolates was resistant to both anti-gp41 antibodies. This confirms that the region recognized by 2F5 and 4E10 is essential for viral infectivity and may be important for vaccine design. Moreover, our results suggest that 4E10 should be further investigated for passive anti-HIV immunotherapy.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Testes de Neutralização , Sequência de Aminoácidos , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Células Gigantes , HIV-1/classificação , Humanos , Dados de Sequência Molecular , Especificidade da EspécieRESUMO
A new approach was applied with the aim at producing plant protein hydrolysates less heterogeneous and less contaminated with nonpeptide substances than are the presently available digests. A significant reduction of nonprotein contaminants was achieved by extraction of the plant material, soy flour or wheat flour, with acetone prior to isolation of the protein. Enzymes of nonanimal origin, papain or Pronase, were used for protein hydrolysis. The components of the hydrolysates were resolved by low-pressure liquid chromatography. Separation of peptide fractions and of remaining nonpeptide contaminants was achieved using small-pore size-exclusion chromatography matrices, Sephadex G-15 or Biogel P-2. Individual peptide fractions, both from soy protein and from wheat gluten, varied substantially in their growth-promoting and production-enhancing activities when tested on a mouse hybridoma culture in protein-free medium. The highest enhancement of viable cell density in batch cultures was 180% of control, and the highest enhancement of final immunoglobulin concentration was more than 230% of control. The existence of marked differences in activity of individual peptide fractions leads to a suggestion that the hydrolysates may provide peptides exerting specific positive effects on cultured animal cells.
Assuntos
Divisão Celular/efeitos dos fármacos , Fragmentos de Peptídeos/biossíntese , Proteínas de Plantas/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida , Hidrólise , Camundongos , Fragmentos de Peptídeos/farmacologia , Proteínas de Plantas/químicaRESUMO
To create a tool for eukaryotic surface display, this approach is aimed at demonstrating a direct modification of the native envelope protein gp64 of Autographa californica NPV without disturbing viral infectivity. Short affinity-tag peptides, the biotin mimic streptagII, and the gp41 amino-acid motif ELDKWA of HIV-1, specific for the human monoclonal antibody 2F5, were engineered into the baculovirus major coat protein gp64 and presented on the viral surface. Two different streptag peptides were inserted at the naturally occurring NotI site at amino-acid 278 of gp64. Additionally, the ten-amino-acid peptide GG-ELDKWA-GG, containing the epitope of mAb 2F5, was introduced into gp64 envelope protein at the same position. In all cases we were able to propagate viable virus-achieving infectious titers in the range of wild-type AcMNPV. Streptag and ELDKWA-epitope surface localization on purified virus particles was demonstrated by flow cytometry and Western blot analysis. We could also show selective retention of mutant viruses by specific interaction between chimeric virions and their target counterparts, recognizing the epitope or the streptag peptide in the viral envelope. These data provide evidence that altering the surface properties of the baculovirus virion could be of value in improving baculovirus display technology and developing new applications.
Assuntos
Nucleopoliedrovírus/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Western Blotting , Epitopos , Dados de Sequência Molecular , Spodoptera , Proteínas do Envelope Viral/imunologia , Vírion/isolamento & purificação , Replicação ViralRESUMO
Regulation of cellular processes that eventually lead to a state of growth arrest is an important manifestation of in vitro cellular senescence caused and accompanied by variations of the gene expression pattern. Whereas these changes at the mRNA level have been studied mainly in fibroblast cultures, we concentrated on endothelial cells that represent an accepted model for vascular systems and may be involved in the pathogenesis of diseases related to aging. To isolate differentially expressed genes, we created a subtractive cDNA library using mRNA from senescent (35 passages) and young (five passages) human umbilical vein endothelial cells (HUVECs). Candidate clones were isolated from the cDNA library, differential expression was confirmed by Northern blot analyses and sequences were compared with a genbank data base. Because many mRNAs were below the detection limit of Northern blot analysis, we were forced to establish a more sensitive PCR based method (ATAC-PCR) to quantify and confirm altered levels of gene expression. Several mRNAs were found to be upregulated in senescent HUVECs including two components of the extracellular matrix (ECM): plasminogen activator inhibitor and fibronectin. Elevated expression of both has already been described in senescent cells. The mRNAs of TGF-beta-inducible gene H3 (beta-IG-H3; ECM protein), insulin-like growth factor binding protein (IGFBP-3), p53-inducible gene (PIG3) a protein involved in vesicular transport (SEC13R) and ribosomal protein L28 have likewise been shown to be preferentially expressed in senescent cells. Because studies support the involvement of ECM components, TGF-beta and p53 in tumor suppressing mechanisms, our data supports the hypothesis that cellular senescence and upregulation of ECM proteins may be associated with tumor preventive functions.