Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
PLoS Pathog ; 18(4): e1009925, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35443018

RESUMO

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable but low levels of antiviral antibodies after infusion. In comparison to the control animals, CCP-treated animals had similar levels of viral RNA in upper and lower respiratory tract secretions, similar detection of viral RNA in lung tissues by in situ hybridization, but lower amounts of infectious virus in the lungs. CCP-treated animals had a moderate, but statistically significant reduction in interstitial pneumonia, as measured by comprehensive lung histology. Thus overall, therapeutic benefits of CCP were marginal and inferior to results obtained earlier with monoclonal antibodies in this animal model. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , Antivirais , COVID-19/terapia , Humanos , Imunização Passiva , Macaca mulatta , RNA Viral , Soroterapia para COVID-19
2.
bioRxiv ; 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34494025

RESUMO

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT 50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable levels of antiviral antibodies after infusion. In comparison to the control animals, they had similar levels of virus replication in the upper and lower respiratory tract, but had significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses. AUTHOR SUMMARY: The results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day, were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP did not result in a detectable reduction in virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of CCP-based therapies.

3.
PLoS Pathog ; 17(7): e1009688, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34228761

RESUMO

There is an urgent need for effective therapeutic interventions against SARS-CoV-2, including new variants that continue to arise. Neutralizing monoclonal antibodies have shown promise in clinical studies. We investigated the therapeutic efficacy of a combination of two potent monoclonal antibodies, C135-LS and C144-LS that carry half-life extension mutations, in the rhesus macaque model of COVID-19. Twelve young adult macaques (three groups of four animals) were inoculated intranasally and intra-tracheally with a high dose of SARS-CoV-2 and 24 hours later, treated intravenously with a high (40 mg/kg) or low (12 mg/kg) dose of the C135-LS and C144-LS antibody combination, or a control monoclonal antibody. Animals were monitored for 7 days. Compared to the control animals, animals treated with either dose of the anti-SARS-CoV-2 antibodies showed similarly improved clinical scores, lower levels of virus replication in upper and lower respiratory tract, and significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. In conclusion, this study provides proof-of-concept in support of further clinical development of these monoclonal antibodies against COVID-19 during early infection.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , COVID-19/terapia , Pulmão/patologia , SARS-CoV-2/imunologia , Replicação Viral , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/patologia , COVID-19/virologia , Modelos Animais de Doenças , Feminino , Pulmão/diagnóstico por imagem , Macaca mulatta , Masculino , Análise Multivariada , Radiografia , Sistema Respiratório/virologia , SARS-CoV-2/fisiologia , Fatores de Tempo , Resultado do Tratamento , Replicação Viral/imunologia
4.
J Med Primatol ; 49(6): 322-331, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32621339

RESUMO

BACKGROUND: The emergence of SARS-CoV-2 and the ensuing COVID-19 pandemic prompted the need for a surveillance program to determine the viral status of the California National Primate Research Center non-human primate breeding colony, both for reasons of maintaining colony health and minimizing the risk of interference in COVID-19 and other research studies. METHODS: We collected biological samples from 10% of the rhesus macaque population for systematic testing to detect SARS-CoV-2 virus by RT-PCR and host antibody response by ELISA. Testing required the development and validation of new assays and an algorithm using in laboratory-developed and commercially available reagents and protocols. RESULTS AND CONCLUSIONS: No SARS-CoV-2 RNA or antibody was detected in this study; therefore, we have proposed a modified testing algorithm for sentinel surveillance to monitor for any future transmissions. As additional reagents and controls become available, assay development and validation will continue, leading to the enhanced sensitivity, specificity, accuracy, and efficiency of testing.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/veterinária , Macaca mulatta/virologia , Doenças dos Macacos/virologia , Pandemias/veterinária , Pneumonia Viral/veterinária , Animais , Anticorpos Antivirais/sangue , Betacoronavirus/genética , Betacoronavirus/imunologia , COVID-19 , Infecções por Coronavirus/virologia , Fezes/virologia , Humanos , Pneumonia Viral/virologia , RNA Viral/isolamento & purificação , SARS-CoV-2 , Vigilância de Evento Sentinela/veterinária
5.
DNA Repair (Amst) ; 9(7): 737-44, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20434408

RESUMO

The repair of DNA double-strand breaks (DSBs) by homologous recombinational repair (HRR) underlies the high radioresistance and low mutability observed in S-phase mammalian cells. To evaluate the contributions of HRR and non-homologous end-joining (NHEJ) to overall DSB repair capacity throughout the cell cycle after gamma-irradiation, we compared HRR-deficient RAD51D-knockout 51D1 to CgRAD51D-complemented 51D1 (51D1.3) CHO cells for survival and chromosomal aberrations (CAs). Asynchronous cultures were irradiated with 150 or 300cGy and separated by cell size using centrifugal elutriation. Cell survival of each synchronous fraction ( approximately 20 fractions total from early G1 to late G2/M) was measured by colony formation. 51D1.3 cells were most resistant in S, while 51D1 cells were most resistant in early G1 (with survival and chromosome-type CA levels similar to 51D1.3) and became progressively more sensitive throughout S and G2. Both cell lines experienced significantly reduced survival from late S into G2. Metaphases were collected from every third elutriation fraction at the first post-irradiation mitosis and scored for CAs. 51D1 cells irradiated in S and G2 had approximately 2-fold higher chromatid-type CAs and a remarkable approximately 25-fold higher level of complex chromatid-type exchanges compared to 51D1.3 cells. Complex exchanges in 51D1.3 cells were only observed in G2. These results show an essential role for HRR in preventing gross chromosomal rearrangements in proliferating cells and, with our previous report of reduced survival of G2-phase NHEJ-deficient prkdc CHO cells [Hinz et al., DNA Repair 4, 782-792, 2005], imply reduced activity/efficiency of both HRR and NHEJ as cells transition from S to G2.


Assuntos
Aberrações Cromossômicas , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Raios gama , Recombinação Genética/fisiologia , Animais , Células CHO , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , Rad51 Recombinase/genética
6.
Mutat Res ; 683(1-2): 91-7, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-19896956

RESUMO

DNA double-strand breaks (DSB) are generally considered the most critical lesion induced by ionizing radiation (IR) and may initiate carcinogenesis and other disease. Using an immunofluorescence assay to simultaneously detect nuclear foci of the phosphorylated forms of histone H2AX and ATM kinase at sites of DSBs, we examined the response of 25 apparently normal and 10 DNA repair-deficient (ATM, ATR, NBN, LIG1, LIG4, and FANCG) primary fibroblast strains irradiated with low doses of (137)Cs gamma-rays. Quiescent G(0)/G(1)-phase cultures were exposed to 5, 10, and 25 cGy and allowed to repair for 24h. The maximum level of IR-induced foci (0.15 foci per cGy, at 10 or 30 min) in the normal strains showed much less inter-individual variation (CV approximately 0.2) than the level of spontaneous foci, which ranged from 0.2-2.6 foci/cell (CV approximately 0.6; mean+/-SD of 1.00+/-0.57). Significantly slower focus formation post-irradiation was observed in seven normal strains, similar to most mutant strains examined. There was variation in repair efficiency measured by the fraction of IR-induced foci remaining 24h post-irradiation, curiously with the strains having slower focus formation showing more efficient repair after 25 cGy. Interestingly, the ranges of spontaneous and residual induced foci levels at 24h in the normal strains were as least as large as those observed for the repair-defective mutant strains. The inter-individual variation in DSB foci parameters observed in cells exposed to low doses of ionizing radiation in this small survey of apparently normal people suggests that hypomorphic genetic variants in genomic maintenance and/or DNA damage signaling and repair genes may contribute to differential susceptibility to cancer induced by environmental mutagens.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA , Fibroblastos/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Imunofluorescência , Histonas/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Radiação Ionizante , Proteínas Supressoras de Tumor/metabolismo
7.
Nucleic Acids Res ; 35(11): 3733-40, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17517774

RESUMO

Fanconi anemia (FA) is a chromosomal instability disorder in which DNA-damage processing defects are reported for translesion synthesis (TLS), non-homologous end joining (NHEJ) and homologous recombination (HR; both increased and decreased). To reconcile these diverse findings, we compared spontaneous mutagenesis in FA and HR mutants of hamster CHO cells. In the fancg mutant we find a reduced mutation rate accompanied by an increased proportion of deletions within the hprt gene. Moreover, in fancg cells gene amplification at the CAD and dhfr loci is elevated, another manifestation of inappropriate processing of damage during DNA replication. In contrast, the rad51d HR mutant has a greatly elevated rate of hprt mutations, >85% of which are deletions. Our analysis supports the concept that HR faithfully restores broken replication forks, whereas the FA pathway acts more globally to ensure chromosome stability by promoting efficient end joining of replication-derived breaks, as well as TLS and HR.


Assuntos
Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Mutagênese , Rad51 Recombinase/genética , Recombinação Genética , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Amplificação de Genes , Deleção de Genes , Hipoxantina Fosforribosiltransferase/genética , Modelos Genéticos , Deleção de Sequência
8.
Mutat Res ; 602(1-2): 34-42, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17010390

RESUMO

Fanconi anemia (FA) is a rare cancer predisposition disease caused by mutations in at least 12 genes encoding proteins that cooperate to maintain genomic integrity. Variants of FA genes, including FANCG, have been identified in human population screening, but their potential reduction in protein function and role in cancer susceptibility is unclear. To test for possible dysfunction, we constructed plasmids containing four FANCG polymorphisms found in the human population and introduced them in the Fancg-deficient (fancg) KO40 line derived from AA8 hamster CHO cells. Expression of wild-type human FANCG provided fancg cells with complete phenotypic correction as assessed by resistance to the DNA crosslinking agent mitomycin C (MMC), thus providing a sensitive test for detecting the degree of complementation activity for the FANCG variants. We found that all four variants conferred levels of mitomycin C resistance as well as restoration of monoubiquitination of Fancd2, a key indicator of a functional FA protein pathway, similar to those observed in wild-type transfectants. Under the same conditions, the L71P amino acid substitution mutant, identified in an FA patient, gave no complementation. Using this novel system for determining FANCG functionality, we detect no decrement in function of the human FANCG polymorphic variants examined.


Assuntos
Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Polimorfismo Genético , Animais , Células CHO , Células Cultivadas , Cricetinae , Relação Dose-Resposta a Droga , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/efeitos da radiação , Proteína do Grupo de Complementação G da Anemia de Fanconi/fisiologia , Frequência do Gene , Teste de Complementação Genética , Humanos , Metanossulfonato de Metila/farmacologia , Mitomicina/toxicidade , Ubiquitina/metabolismo
9.
DNA Repair (Amst) ; 5(8): 875-84, 2006 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-16815103

RESUMO

Fanconi anemia (FA) is a developmental and cancer predisposition disorder in which key, yet unknown, physiological events promoting chromosome stability are compromised. FA cells exhibit excess metaphase chromatid breaks and are universally hypersensitive to DNA interstrand crosslinking agents. Published mutagenesis data from single-gene mutation assays show both increased and decreased mutation frequencies in FA cells. In this review we discuss the data from the literature and from our isogenic fancg knockout hamster CHO cells, and interpret these data within the framework of a molecular model that accommodates these seemingly divergent observations. In FA cells, reduced rates of recovery of viable X-linked hypoxanthine phosphoribosyltransferase (hprt) mutants are characteristically observed for diverse mutagenic agents, but also in untreated cultures, indicating the relevance of the FA pathway for processing assorted DNA lesions. We ascribe these reductions to: (1) impaired mutagenic translesion synthesis within hprt during DNA replication and (2) lethality of mutant cells following replication fork breakage on the X chromosome, caused by unrepaired double-strand breaks or large deletions/translocations encompassing essential genes flanking hprt. These findings, along with studies showing increased spontaneous mutability of FA cells at two autosomal loci, support a model in which FA proteins promote both translesion synthesis at replication-blocking lesions and repair of broken replication forks by homologous recombination and DNA end joining. The essence of this model is that the FANC protein pathway serves to restrict the severity of mutational outcome by favoring base substitutions and small deletions over larger deletions and chromosomal rearrangements.


Assuntos
Instabilidade Cromossômica/genética , Dano ao DNA , Reparo do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Modelos Genéticos , Mutagênese/genética , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Hipoxantina Fosforribosiltransferase/genética , Mutagênese/efeitos dos fármacos , Mutagênicos/toxicidade
10.
Nucleic Acids Res ; 34(9): 2833-43, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16717288

RESUMO

In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks (ICLs). Ectopic expression of wild-type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Ligação a DNA/química , Recombinação Genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Mutação , Rad51 Recombinase/metabolismo , Técnicas do Sistema de Duplo-Híbrido
11.
Nucleic Acids Res ; 34(5): 1358-68, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16522646

RESUMO

Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR proficiency. We constructed and characterized a knockout of the paralog Rad51D in widely studied CHO cells. The rad51d mutant (clone 51D1) displays sensitivity to a diverse spectrum of induced DNA damage including gamma-rays, ultraviolet (UV)-C radiation, and methyl methanesulfonate (MMS), indicating the broad relevance of HRR to genotoxicity. Spontaneous chromatid breaks/gaps and isochromatid breaks are elevated 3- to 12-fold, but the chromosome number distribution remains unchanged. Most importantly, 51D1 cells exhibit a 12-fold-increased rate of hprt mutation, as well as 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. Xrcc3 irs1SF cells from the same parental CHO line show similarly elevated mutagenesis at these three loci. Collectively, these results confirm the a priori expectation that HRR acts in an error-free manner to repress three classes of genetic alterations (chromosomal aberrations, loss of gene function and increased gene expression), all of which are associated with carcinogenesis.


Assuntos
Mutagênese , Rad51 Recombinase/fisiologia , Recombinação Genética , Animais , Células CHO , Sobrevivência Celular , Aberrações Cromossômicas , Cricetinae , Cricetulus , Dano ao DNA , Raios gama , Amplificação de Genes , Marcação de Genes , Hipoxantina Fosforribosiltransferase/genética , Rad51 Recombinase/análise , Rad51 Recombinase/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...