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1.
Vaccine ; 36(27): 3917-3925, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29843999

RESUMO

Newcastle disease (ND), caused by virulent class II avian paramyxovirus 1 (Newcastle disease virus, NDV), occurs sporadically in poultry despite their having been immunized with commercial vaccines. These vaccines were all derived from NDV strains isolated around 70 years ago. Since then, class II NDV strains have evolved into 18 genotypes. Whether the vaccination failure results from genotype mismatches between the currently used vaccine strains and field-circulating velogenic strains or from an impaired immune response in the vaccination remains unclear. To test the first hypothesis, we performed a heterologous genotype II vaccine/genotype XI challenge in one-day old specific pathogen free (SPF) chicks and reproduced viral shedding. We then produced two attenuated strains of genotype II and XI by reverse genetics and used them to immunize two-week old SPF chickens that were subsequently challenged with velogenic strains of genotypes II, VII and XI. We found that both vaccines could induce antibodies with hemagglutination inhibition titers higher than 6.5 log2. Vaccination also completely prevented disease, viral shedding in swabs, and blocked viral replication in tissues from different genotypes in contrast to unvaccinated chickens that died shortly after challenge. Taken together, our results support the hypothesis that, in immunocompetent poultry, genotype mismatch is not the main reason for vaccination failure.


Assuntos
Anticorpos Antivirais/imunologia , Pareamento Incorreto de Bases/imunologia , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/genética , Doenças das Aves Domésticas/prevenção & controle , Vacinas Virais/genética , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Galinhas/imunologia , Cricetinae , Ensaio de Imunoadsorção Enzimática/veterinária , Genótipo , Doença de Newcastle/terapia , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/terapia , Vacinação/veterinária , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Eliminação de Partículas Virais
2.
Int J Cancer ; 126(11): 2635-43, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19856313

RESUMO

Regulatory T cells (T(reg)) inhibit the generation of host-versus-tumor immunity via suppression of tumor-specific effector T-cell responses and development of immune tolerance to neoplastic cells. The transcription factor forkhead box P3 (FOXP3) is an intracellular key molecule for T(reg) development and function and is considered to represent the most specific T(reg) cell marker. The aim of this study was to analyze the frequency and prognostic impact of tumor-infiltrating FOXP3(+) T(reg) in colorectal cancer (CRC) stratified by mismatch-repair (MMR) status. Using the tissue microarray technique, 1,420 tumor samples were immunohistochemically stained for FOXP3 and stratified into 1,197 MMR-proficient and 223 MMR-deficient CRCs. Additionally, the 1,197 MMR-proficient CRCs were randomized into 2 subgroups (Test Groups 1 and 2; n = 613 and 584, respectively). In both MMR-proficient CRC subgroups high frequency tumor-infiltrating FOXP3(+) T(reg) was associated with early T stage (p = 0.001 and <0.001), tumor location (p = 0.01 and 0.045) and increased 5-year survival rate (p = 0.004 and <0.001), whereas in MMR-deficient CRCs an association between FOXP3(+) T(reg) and absence of lymph node involvement (p = 0.023), absence of vascular invasion (p = 0.023) and improved 5-year survival rate (p = 0.029) could be detected. In a multivariable analysis including age, gender, T stage, N stage, tumor grade, vascular invasion, and tumor border configuration, a high FOXP3(+) T(reg) frequency was an independent prognostic factor in both MMR-proficient CRC subsets (p = 0.019 and p = 0.007), but not in the MMR-deficient CRCs (p = 0.13). Therefore, high frequency of tumor-infiltrating FOXP3(+) T(reg) is associated with early T stage and independently predicts improved disease-specific survival in MMR-proficient CRC patients.


Assuntos
Pareamento Incorreto de Bases/imunologia , Neoplasias Colorretais/imunologia , Fatores de Transcrição Forkhead/análise , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Pareamento Incorreto de Bases/genética , Neoplasias Colorretais/patologia , Feminino , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Taxa de Sobrevida
3.
J Immunol Methods ; 324(1-2): 26-37, 2007 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-17553518

RESUMO

The reliable identification of IGHD genes within human immunoglobulin heavy chains is challenging with up to one third of rearrangements having no identifiable IGHD gene. The short, mutated IGHD genes are generally assumed to be indistinguishable from the N-REGIONS of non-template encoded nucleotides that surround them. In this study we have characterised N-REGIONS, demonstrating the importance of nucleotide composition biases in the addition process, including the formation of homopolymer tracts. We then use a simulation approach to determine the likelihood of misidentification of highly mutated IGHD genes among the JUNCTION nucleotides. These likelihoods provide general rules for the identification of mutated D-REGIONs, and suggest that longer D-REGIONs (>25 nucleotides) with as many as ten mutations can be identified with a low risk of error. Shorter D-REGIONs (>16 nucleotides) with as many as four mutations are also identifiable. The reliability of different alignments is dependent upon the junction length (combined N-REGIONs and D-REGION). Data is presented that can guide the alignment of sequences with junction lengths from 5 to 50 nucleotides, including explicit selection between two D-REGION possibilities. The use of such a statistically-based approach to the alignment of IGHD genes will improve the reliability of the partitioning of immunoglobulin sequences, and this in turn will facilitate the study of the many processes that contribute to the diversity of the immunoglobulin repertoire.


Assuntos
Diversidade de Anticorpos/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/genética , Mutação , Pareamento Incorreto de Bases/genética , Pareamento Incorreto de Bases/imunologia , Simulação por Computador , Humanos , Método de Monte Carlo
4.
Mol Cell ; 16(4): 505-8, 2004 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-15546611

RESUMO

The mechanisms underlying somatic hypermutation (SHM) and class switch recombination (CSR) have been the subject of much debate. Recent studies from the Neuberger and Honjo labs have lent insight into these distinct processes, and we discuss a new, comprehensive model for how AID, uracil DNA glycosylase (UNG) and the mismatch repair system function in both SHM and CSR.


Assuntos
Linfócitos B/metabolismo , Pareamento Incorreto de Bases , Switching de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/genética , Animais , Linfócitos B/enzimologia , Linfócitos B/imunologia , Pareamento Incorreto de Bases/imunologia , Citidina Desaminase/metabolismo , DNA Glicosilases/metabolismo , Reparo do DNA , Humanos , Modelos Imunológicos , Edição de RNA , Uracila-DNA Glicosidase
5.
Exp Gerontol ; 39(4): 507-15, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15050284

RESUMO

Many immune functions decline with age and may jeopardize the elderly, as illustrated, for example by the significantly higher mortality rate from influenza in old age. Although innate and humoral immunity are affected by aging, it is the T cell compartment, which manifests most alterations. The mechanisms behind these alterations are still unclear, and several explanations have been offered including thymic involution and Telomere attrition leading to cell senescence. Age related accumulation of mutations has been documented and could serve as an additional mechanism of T cell dysfunction. One effective repair mechanism capable of rectifying errors in DNA replications is the mismatch repair (MMR) system. We previously reported a comparative examination of individual DNA samples from blood cells obtained at 10 year intervals from young and old subjects. We showed significantly higher rates of microsatellite instability (MSI), an indicator of MMR dysfunction in older subjects, compared to young. In the present study we confirm this result, using direct automated sequencing and in addition, we demonstrate that as CD8 lymphocytes from aged individuals, undergo repeated population doublings (PDs) in culture, they develop MSI. CD4 clones that also undergo repeated PDs in culture develop significant MSI as well. Elucidation of this previously unexplored facet of lymphocyte dynamics in relation to aging may help identify novel mechanisms of immunosenescence and pathways that could serve as targets for interventions to restore immune function.


Assuntos
Envelhecimento/genética , Envelhecimento/imunologia , Repetições de Microssatélites/genética , Linfócitos T/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Pareamento Incorreto de Bases/genética , Pareamento Incorreto de Bases/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Senescência Celular/genética , Senescência Celular/imunologia , Metilação de DNA , Reparo do DNA/genética , Reparo do DNA/imunologia , Humanos , Repetições de Microssatélites/imunologia , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética
6.
Exp Gerontol ; 39(4): 499-505, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15050283

RESUMO

Repair of mismatches in mammalian cell DNA is mediated by a complex of proteins that constitute the so-called mismatch repair system (MMR), the main post-replicative pathway for the correction of replication errors. Loss of MMR (as exemplified by germline mutations in some MMR genes, leading to hereditary non-polyposis colorectal cancer) results in increased mutation rates at both coding sequences and in non-coding regions such as microsatellites. In order to evaluate possible functional alterations of this repair system during ageing that could affect immune system efficiency, we studied microsatellite instability at five different loci interspersed in the genome (CD4, VWA31, Tpox, Fes/FPS and p53) in total DNA from T lymphocyte clones derived from hematopoietic stem cells, or peripheral T cells of young or elderly subjects. In addition, these clones had been maintained for different periods in vitro to represent a culture model of ageing. We observed increasing instability accumulating with increasing passages in culture, particularly in CD34+cell-derived clones, but no clear donor age relationship.


Assuntos
Envelhecimento/genética , Envelhecimento/imunologia , Repetições de Microssatélites/genética , Linfócitos T/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Pareamento Incorreto de Bases/genética , Pareamento Incorreto de Bases/imunologia , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Senescência Celular/genética , Senescência Celular/imunologia , Células Clonais/patologia , Dano ao DNA/imunologia , Reparo do DNA/genética , Reparo do DNA/imunologia , Genótipo , Células-Tronco Hematopoéticas/patologia , Humanos , Pessoa de Meia-Idade
7.
Eur J Immunol ; 34(2): 504-8, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14768055

RESUMO

Diversification of the primary antibody repertoire in chickens is achieved by a gene conversion process that uses a set of immunoglobulin variable (IgV) pseudogenes as templates. Studies usingthe chicken DT40 B lymphoma cell line have shown that this gene conversion is dependent on activation-induced deaminase, which deaminates deoxycytidine to deoxyuridine in the IgV gene. The mechanism by which the resultant deoxyuridine/deoxyguanosine (dU/dG) mismatch acts to initiate the gene conversion process is unknown but likely involves either (i) recognition of the dU/dG pair by the mismatch repair complex or (ii) recognition of the dU itself by uracil-DNA glycosylase. To discriminate these possibilities, we have investigated the effects on IgV gene conversion of inhibiting uracil-DNA glycosylase. We find that such inhibition diminishes gene conversion, biasing instead towards point mutations. These results demonstrate that IgV gene conversion in DT40 cells is substantially dependent on uracil excision and implies that it proceeds by a pathway involving an abasic site, which could be acted upon by an apyrimidinic endonuclease to generate a DNA strand break facilitating the conversion process.


Assuntos
Galinhas/imunologia , Citidina Desaminase/metabolismo , Desoxiuridina/metabolismo , Conversão Gênica/imunologia , Genes de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Animais , Diversidade de Anticorpos/genética , Diversidade de Anticorpos/imunologia , Pareamento Incorreto de Bases/imunologia , Linhagem Celular Tumoral , Galinhas/genética , DNA Glicosilases/antagonistas & inibidores , DNA Glicosilases/metabolismo , Desaminação , Desoxicitidina/metabolismo , Inibidores Enzimáticos/farmacologia , Região Variável de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Uracila-DNA Glicosidase
8.
J Immunol ; 170(4): 1620-4, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12574322

RESUMO

Affinity maturation of the humoral response is accomplished by somatic hypermutation and class switch recombination (CSR) of Ig genes. Activation-induced cytidine deaminase likely initiates these processes by deamination of cytidines in the V and switch regions of Ig genes. This activity is expected to produce G-U mismatches that can be substrates for MutS homolog 2/MutS homolog 6 heterodimers and for uracil DNA glycosylase. However, G-T and G-U mismatches are also substrates of the methyl-CpG binding domain 4 (Mbd4) glycosylase. To determine whether Mbd4 functions downstream of activation-induced cytidine deaminase activity, we examined somatic hypermutation and CSR in Mbd4(-/-) mice. In this study, we report that CSR, as analyzed by an in vitro switch assay and by in vivo immunizations, is unaffected in Mbd4(-/-) mice. In addition, the hypermutated JH2 to JH4 region in Peyer's patch B cells showed no effects as a result of Mbd4 deficiency. These data indicate that the Mbd4 glycosylase does not significantly contribute to mechanisms of Ab diversification.


Assuntos
Pareamento Incorreto de Bases , Sítios de Ligação de Anticorpos , Ilhas de CpG , Reparo do DNA , Endodesoxirribonucleases/fisiologia , Switching de Imunoglobulina/genética , N-Glicosil Hidrolases/fisiologia , Hipermutação Somática de Imunoglobulina , Animais , Diversidade de Anticorpos/genética , Pareamento Incorreto de Bases/imunologia , Sítios de Ligação de Anticorpos/genética , Ilhas de CpG/imunologia , DNA Glicosilases , Reparo do DNA/imunologia , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Feminino , Guanina , Imunoglobulina G/biossíntese , Imunoglobulina G/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Glicosil Hidrolases/deficiência , N-Glicosil Hidrolases/genética , Estrutura Terciária de Proteína/genética , Uracila
9.
J Immunol ; 170(4): 2214-20, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12574395

RESUMO

Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during DNA replication. Key members of the MMR system include MutSalpha (hMSH2 and hMSH6) and MutSbeta (hMSH2 and hMSH3). To provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer sequences for five key microsatellites. Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis tissue. Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS. When FLS were cultured with S-nitroso-N-acetylpenicillamine, the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6). Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.


Assuntos
Artrite Reumatoide/enzimologia , Proteínas de Bactérias , DNA Ligases/biossíntese , Reparo do DNA/imunologia , Proteínas de Ligação a DNA/biossíntese , Repetições de Microssatélites/imunologia , Proteínas Proto-Oncogênicas/biossíntese , Adenosina Trifosfatases/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Pareamento Incorreto de Bases/imunologia , Células Cultivadas , DNA Ligases/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Relação Dose-Resposta Imunológica , Repressão Enzimática/imunologia , Proteínas de Escherichia coli/biossíntese , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Pessoa de Meia-Idade , Proteína MutS de Ligação de DNA com Erro de Pareamento , Proteína 2 Homóloga a MutS , Proteína 3 Homóloga a MutS , Osteoartrite/enzimologia , Osteoartrite/genética , Osteoartrite/patologia , Estresse Oxidativo/genética , Estresse Oxidativo/imunologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Espécies Reativas de Nitrogênio/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Membrana Sinovial/enzimologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia
12.
J Exp Med ; 190(3): 323-30, 1999 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-10430621

RESUMO

Mice deficient in various mismatch repair (MMR) enzymes were examined to determine whether this repair pathway is involved in antibody class switch recombination. Splenic B cells from mice deficient in Msh2, Mlh1, Pms2, or Mlh1 and Pms2 were stimulated in culture with lipopolysaccharide (LPS) to induce immunoglobulin (Ig)G2b and IgG3, LPS and interleukin (IL)-4 to induce IgG1, or LPS, anti-delta-dextran, IL-4, IL-5, and transforming growth factor (TGF)-beta1 to induce IgA. After 4 d in culture, cells were surface stained for IgM and non-IgM isotypes and analyzed by FACS((R)). B cells from MMR-deficient mice show a 35-75% reduction in isotype switching, depending on the isotype and on the particular MMR enzyme missing. IgG2b is the most affected, reduced by 75% in Mlh1-deficient animals. The switching defect is not due to a lack of maturation of the B cells, as purified IgM(+)IgD(+) B cells show the same reduction. MMR deficiency had no effect on cell proliferation, viability, or apoptosis, as detected by [(3)H]thymidine incorporation and by propidium iodide staining. The reduction in isotype switching was demonstrated to be at the level of DNA recombination by digestion-circularization polymerase chain reaction (DC-PCR). A model of the potential role for MMR enzymes in class switch recombination is presented.


Assuntos
Adenosina Trifosfatases , Linfócitos B/enzimologia , Linfócitos B/metabolismo , Pareamento Incorreto de Bases/imunologia , Enzimas Reparadoras do DNA , Reparo do DNA/imunologia , Proteínas de Ligação a DNA , Switching de Imunoglobulina/genética , Isotipos de Imunoglobulinas/biossíntese , Isotipos de Imunoglobulinas/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linfócitos B/citologia , Proteínas de Transporte , Ciclo Celular/genética , Ciclo Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Citometria de Fluxo , Camundongos , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas Nucleares , Proteínas/genética , Proteínas/imunologia , Baço
13.
J Immunol ; 162(6): 3121-4, 1999 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-10092760

RESUMO

During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their V kappa Ox1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1-/- clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum.


Assuntos
Pareamento Incorreto de Bases/imunologia , Reparo do DNA/imunologia , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Mutação/imunologia , Proteínas de Neoplasias/deficiência , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linfócitos B/imunologia , Composição de Bases/imunologia , Proteínas de Transporte , Células Clonais/imunologia , Análise Mutacional de DNA , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Proteína 1 Homóloga a MutL , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares
14.
J Immunol ; 161(11): 6128-32, 1998 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-9834097

RESUMO

Ig somatic hypermutation contributes to the generation of high-affinity Abs that are essential for efficient humoral defense. The presence of multiple point mutations in rearranged Ig V genes and their immediate flanking sequences suggests that the DNA repair system may not be working properly in correcting point mutations introduced to the restricted region of Ig genes. We examined the DNA repair functions of germinal center (GC) centroblasts, which are the cells in which ongoing Ig hypermutation takes place. We found that GC centroblasts express all known components of the human DNA mismatch repair system, and that the system corrects DNA mismatches in a strand-specific manner in vitro. We conclude that general suppression of mismatch repair at the cellular level does not occur during somatic hypermutation.


Assuntos
Pareamento Incorreto de Bases/imunologia , Centro Germinativo/citologia , Centro Germinativo/metabolismo , Linfócitos B/metabolismo , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linfoma de Burkitt , Núcleo Celular/genética , Núcleo Celular/metabolismo , Separação Celular , Células Cultivadas , Células HeLa , Humanos , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo
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