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1.
J Med Virol ; 93(3): 1732-1738, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32965677

RESUMO

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) had led to a serious public health crisis, and no specific treatments or vaccines are available yet. A nucleocapsid protein (NP)-based enzyme-linked immunosorbent assay (ELISA) detection method is not only important in disease diagnosis, but is required for the evaluation of vaccine efficacy during the development of an inactivated SARS-CoV-2 vaccine. In this study, we expressed both the NP and N-terminally truncated NP (ΔN-NP) of SARS-CoV-2 in an Escherichia coli expression system and described the purification of the soluble recombinant NP and ΔN-NP in details. The identities of the NP and ΔN-NP were confirmed with mass spectrometry. We then used immunoglobulin G detection ELISAs to compare the sensitivity of NP and ΔN-NP in detecting anti-SARS-CoV-2 antibodies. ΔN-NP showed greater sensitivity than NP in the analysis of serially diluted sera from mice and rabbits vaccinated with inactive SARS-CoV-2 and in human sera diluted 1:400. ΔN-NP showed a positive detection rate similar to that of the SARS-CoV-2 S protein in human sera. We conclude that ΔN-NP is a better serological marker than NP for evaluating the immunogenicity of inactivated SARS-CoV-2.


Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Produtos Inativados/imunologia , Animais , COVID-19/prevenção & controle , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Coelhos , SARS-CoV-2/genética , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Glicoproteína da Espícula de Coronavírus/genética
2.
Arthritis Rheumatol ; 71(12): 2005-2015, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31259485

RESUMO

OBJECTIVE: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) plays a crucial role in innate and adaptive immune signaling by modulating the threshold for activation of immune cells, including Treg cells. Therefore, MALT-1 is regarded to be an interesting therapeutic target in several immune-mediated diseases. The goal of this study was to examine the role of MALT-1 in experimental animal models of rheumatoid arthritis (RA). METHODS: MALT-1 activation was assessed by measuring cleavage of the deubiquitinase CYLD in lymphocytes from mice with collagen-induced arthritis (CIA). Furthermore, the impact of MALT-1 deficiency on arthritis was evaluated in Malt1KO mice with CIA or with collagen antibody-induced arthritis (CAIA). T cell-specific MALT-1 deficiency was measured in mice with deletion of T cell-specific MALT-1 (Malt1Tcell KO ), and the time-dependent effects of MALT-1 deficiency were assessed in mice with deletion of tamoxifen-inducible T cell-specific MALT-1 (Malt1iTcell KO ). Bone density was determined in MALT-1-deficient mice using micro-computed tomography and femur-bending tests. Reconstitution of Treg cells was performed using adoptive transfer experiments. RESULTS: MALT-1 activation was observed in the lymphocytes of mice with CIA. T cell-specific MALT-1 deletion in the induction phase of arthritis (incidence of arthritis, 25% in control mice versus 0% in Malt1iTcell KO mice; P < 0.05), but not in the effector phase of arthritis, completely protected mice against the development of CIA. Consistent with this finding, MALT-1 deficiency had no impact on CAIA, an effector phase model of RA. Finally, mice with MALT-1 deficiency showed a spontaneous decrease in bone density (mean ± SEM trabecular thickness, 46.3 ± 0.7 µm in control mice versus 40 ± 1.1 µm in Malt1KO mice; P < 0.001), which was linked to the loss of Treg cells in these mice. CONCLUSION: Overall, these data in murine models of RA highlight MALT-1 as a master regulator of T cell activation, which is relevant to the pathogenesis of autoimmune arthritis. Furthermore, these findings show that MALT-1 deficiency can lead to spontaneous osteoporosis, which is associated with impaired Treg cell numbers.


Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Osteoporose/genética , Deleção de Sequência/imunologia , Animais , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Ativação Linfocitária/genética , Camundongos , Osteoporose/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia
3.
BMC Res Notes ; 12(1): 340, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208438

RESUMO

OBJECTIVE: As part of a larger study to understand how Envelope N-glycosylation influences HIV-1 pathogenesis, we selected a participant infected with a single Subtype C variant and determined whether deletion of specific potential N-glycan sites (PNGs) impacted Envelope function longitudinally. RESULTS: We deleted five PNGs previously linked to HIV-1 transmission of two matched Envelope clones representing variants at 5 and 173 weeks post-infection. The transmitted founder (TF) had significantly better pseudovirus entry efficiency than the chronic infection (CI) variant. Deletion of all PNGs significantly reduced TF entry efficiency, binding to dendritic cell-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) receptor and trans-infection. However, mutational analysis did not affect the phenotype of the CI Envelope to the same extent. Notably, deletion of the PNGs at N241 and N448 had no effect on CI Envelope function, suggesting that some PNGs might only be important during acute infection. Therefore, vaccines that elicit antibodies against N-glycans important for TF Envelope function could drive the loss of PNGs during immune escape, abrogating viral replication. Conversely, changes in N-glycosylation might have no effect on some variants, reducing vaccine efficacy. This finding highlights the need for further investigation into the role of Envelope N-glycosylation in HIV-1 pathogenesis.


Assuntos
Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Sequência de Aminoácidos , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Progressão da Doença , Feminino , Glicosilação , Células HEK293 , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Deleção de Sequência/imunologia , Replicação Viral/genética , Replicação Viral/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
4.
J Gen Virol ; 100(4): 642-655, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30230443

RESUMO

Koi herpesvirus (KHV, Cyprinidherpesvirus 3) causes a fatal disease of koi and common carp. To obtain safe and efficacious live vaccines, we generated deletion mutants of KHV lacking the nonessential genes encoding two enzymes of nucleotide metabolism, thymidine kinase (TK, ORF55) and deoxyuridine-triphosphatase (DUT, ORF123). Since single-deletion mutants based on a KHV isolate from Israel (KHV-I) only exhibited partial attenuation (Fuchs W, Fichtner D, Bergmann SM, Mettenleiter TC. Arch Virol 2011;156 : 1059-1063), a corresponding double mutant was generated and tested in vivo, and shown to be almost avirulent but still protective. To overcome the low in vitro virus titres of KHV-I (≤105 p.f.u. ml-1), single and double TK and DUT deletions were also introduced into a cell culture-adapted KHV strain from Taiwan (KHV-T). The deletions did not affect in vitro virus replication, and all KHV-T mutants exhibited wild-type-like plaque sizes and titres exceeding 107 p.f.u. ml-1, as a prerequisite for economic vaccine production. Compared to wild-type and revertant viruses, the single-deletion mutants of KHV-T were significantly attenuated in vivo, and immersion of juvenile carp in water containing high doses of the double mutant caused almost no fatalities. Nevertheless, the deletion mutants induced similar levels of KHV-specific serum antibodies to the parental wild-type virus, and conferred solid protection against disease after challenge with wild-type KHV. For the convenient differentiation of DNA samples prepared from gill swabs of carp infected with wild-type and TK-deleted KHV we developed a triplex real-time PCR. Thus, KHV-TΔDUT/TK might be suitable as a genetic DIVA vaccine in the field.


Assuntos
Herpesviridae/genética , Herpesviridae/imunologia , Pirofosfatases/genética , Pirofosfatases/imunologia , Timidina Quinase/genética , Timidina Quinase/imunologia , Animais , Carpas/imunologia , Carpas/virologia , Células Cultivadas , DNA Viral/genética , DNA Viral/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Israel , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
5.
Cell Immunol ; 332: 129-133, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30093071

RESUMO

GARP is a transmembrane protein that presents latent TGF-ß1 on the surface of regulatory T cells (Tregs). Neutralizing anti-GARP monoclonal antibodies that prevent the release of active TGF-ß1, inhibit the immunosuppressive activity of human Tregs in vivo. In this study, we investigated the contribution of GARP on mouse Tregs to immunosuppression in experimental tumors. Unexpectedly, Foxp3 conditional garp knockout (KO) mice challenged orthotopically with GL261 tumor cells or subcutaneously with MC38 colon carcinoma cells did not show prolonged survival or delayed tumor growth. Also, the suppressive function of KO Tregs was similar to that of wild type Tregs in the T cell transfer model in allogeneic, immunodeficient mice. In conclusion, garp deletion in mouse Tregs is not sufficient to impair their immunosuppressive activity in vivo.


Assuntos
Proteínas de Membrana/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/imunologia , Imunossupressores/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Knockout , Deleção de Sequência/imunologia , Fator de Crescimento Transformador beta1/imunologia
6.
Radiat Res ; 186(6): 568-576, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27869554

RESUMO

Until the end of the 20th century, mouse germ cell data on induced mutation rates, which were collected using classical genetic methods at preselected specific loci, provided the principal basis for estimates of genetic risks from radiation in humans. The work reported on here is an extension of earlier efforts in this area using molecular methods. It focuses on validating the use of array comparative genomic hybridization (array CGH) methods for identifying radiation-induced copy number variants (CNVs) and specifically for DNA deletions. The emphasis on deletions stems from the view that it constitutes the predominant type of radiation-induced genetic damage, which is relevant for estimating genetic risks in humans. In the current study, deletion mutations were screened in the genomes of F1 mice born to unirradiated or 4 Gy irradiated sires at the spermatogonia stage (100 offspring each). The array CGH analysis was performed using a "2M array" with over 2 million probes with a mean interprobe distance of approximately 1 kb. The results provide evidence of five molecularly-confirmed paternally-derived deletions in the irradiated group (5/100) and one in the controls (1/100). These data support a calculation, which estimates that the mutation rate is 1 × 10-2/Gy per genome for induced deletions; this is much lower than would be expected if one assumes that the specific locus rate of 1 × 10-5/locus per Gy (at 34 loci) is applicable to other genes in the genome. The low observed rate of induced deletions suggests that the effective number of genes/genomic regions at which recoverable deletions could be induced would be only approximately 1,000. This estimate is far lower than expected from the size of the mouse genome (>20,000 genes). Such a discrepancy between observation and expectation can occur if the genome contains numerous genes that are far less sensitive to radiation-induced deletions, if many deletion-bearing offspring are not viable or if the current method is substandard for detecting small deletions.


Assuntos
Hibridização Genômica Comparativa , Genômica , Mutagênese/efeitos da radiação , Análise de Sequência com Séries de Oligonucleotídeos , Deleção de Sequência/imunologia , Espermatogônias/metabolismo , Espermatogônias/efeitos da radiação , Animais , Feminino , Masculino , Camundongos , Deleção de Sequência/efeitos da radiação
7.
Integr Biol (Camb) ; 6(11): 1088-99, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25212181

RESUMO

The bacterial two-component system (TCS) regulates genes that are crucial for virulence in several pathogens. One of such TCS, the PhoPR system, consisting of a transmembrane sensory histidine kinase protein (PhoR) and an intracellular response regulator protein (PhoP), has been reported to have a major role in mycobacterial pathogenesis. We knocked out the phoP in C. pseudotuberculosis, the causal organism of caseous lymphadenitis (CLA), and using a combination of in vitro and in vivo mouse system, we showed for the first time, that the PhoP of C. pseudotuberculosis plays an important role in the virulence and pathogenicity of this bacterium. Furthermore, we modeled the PhoP of C. pseudotuberculosis and our docking results showed that several natural compounds including Rhein, an anthraquinone from Rheum undulatum, and some drug-like molecules may target PhoP to inhibit the TCS of C. pseudotuberculosis, and therefore may facilitate a remarkable attenuation of bacterial pathogenicity being the CLA. Experiments are currently underway to validate these in silico docking results.


Assuntos
Proteínas de Bactérias/imunologia , Infecções por Corynebacterium/imunologia , Corynebacterium pseudotuberculosis/patogenicidade , Transdução de Sinais/imunologia , Animais , Antraquinonas/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Bioensaio , Linhagem Celular , Sobrevivência Celular/imunologia , Infecções por Corynebacterium/genética , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/imunologia , DNA Bacteriano/química , DNA Bacteriano/genética , Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Reação em Cadeia da Polimerase , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Virulência
8.
J Immunol ; 193(1): 252-7, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24879790

RESUMO

The DNA deaminase activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) by deaminating cytidines to uridines at V region (V) genes and switch (S) regions. The mechanism by which AID is recruited to V genes and S region DNA is poorly understood. In this study, we used the CH12 B lymphoma line to demonstrate that, although S regions can efficiently recruit AID and undergo mutations and deletions, AID neither binds to nor mutates the V gene, thus clearly demonstrating intraimmunoglobulin locus specificity. Depletion of the RNA-binding protein polypyrimidine tract binding protein-2, previously shown to promote recruitment of AID to S regions, enables stable association of AID with the V gene. Surprisingly, AID binding to the V gene does not induce SHM. These results unmask a striking lack of correlation between AID binding and its mutator activity, providing evidence for the presence of factors required downstream of AID binding to effect SHM. Furthermore, our findings suggest that S regions are preferred targets for AID and, aided by polypyrimidine tract binding protein-2, act as "sinks" to sequester AID activity from other genomic regions.


Assuntos
Sequência de Bases , Citidina Desaminase/imunologia , DNA/imunologia , Proteínas do Tecido Nervoso/imunologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/imunologia , Deleção de Sequência/imunologia , Animais , Linhagem Celular Tumoral , Citidina Desaminase/genética , DNA/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Ligação Proteica
9.
Integr Biol (Camb) ; 6(4): 438-49, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24599496

RESUMO

Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involved in modulating migration towards a chemical signal are limited because they do not employ stable, precisely controlled chemical gradients. Here, we develop a positive selection microfluidic-based genetic screen that allows us to identify Salmonella virulence factors that manipulate DC migration within stable, linear chemokine gradients. Our screen identified 7 Salmonella effectors (SseF, SifA, SspH2, SlrP, PipB2, SpiC and SseI) that inhibit DC chemotaxis toward CCL19. This method is widely applicable for identifying novel microbial factors that influence normal host cell chemotaxis as well as revealing new mammalian genes involved in directed cell migration.


Assuntos
Movimento Celular/imunologia , Quimiocina CCL19/imunologia , Células Dendríticas/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Fatores de Virulência/imunologia , Animais , Células Dendríticas/microbiologia , Interações Hospedeiro-Patógeno , Camundongos , Camundongos da Linhagem 129 , Microfluídica , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Salmonella typhimurium/genética , Deleção de Sequência/imunologia , Estatísticas não Paramétricas , Fatores de Virulência/genética
10.
AAPS J ; 15(4): 1160-7, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23990502

RESUMO

Immunogenicity assessment of fully human monoclonal antibody-based biotherapeutics requires sensitive and specific ligand binding assays. One of the components of specificity is the depletion of signal by a relevant biotherapeutic that is commonly based on an arbitrary depletion criterion of inhibition of the original response or reduction of the signal below the screening assay cut point (ACP). Hence, there is a need to develop a statistically derived physiologically relevant specificity criterion. We illustrate an optimization approach to determine the concentration of biotherapeutic required for the specificity evaluation. Naïve donor sample sets with and without circulating drug and antitherapeutic/drug antibody (ADA) were prepared. Next, a depletion cut point (DCP) using naïve and ADA-containing donor sets with the optimized biotherapeutic concentration was evaluated. A statistically derived design of experiment was used to establish a validated DCP. A reliable DCP requires naïve (no ADA) donors treated only with an optimized concentration of biotherapeutic. The additional DCPs generated using two distinct concentrations of ADA-spiked sample sets led to a physiologically irrelevant criterion that was not necessarily representative of real-time samples. This increased the risk of false positives or negatives. In this study, well-defined bioanalytical and statistical methods were employed to validate a DCP to confirm the presence of biotherapeutic specific ADA in human serum samples. A physiologically relevant and effective strategy to confirm specificity in immune reactive samples, especially those that are close to the ACP, is proposed through this study.


Assuntos
Anticorpos Monoclonais/sangue , Fenômenos Imunogenéticos/fisiologia , Imunoglobulina G/sangue , Análise Serial de Proteínas/normas , Deleção de Sequência/imunologia , Terapia Biológica/normas , Feminino , Humanos , Fenômenos Imunogenéticos/efeitos dos fármacos , Masculino , Análise Serial de Proteínas/estatística & dados numéricos , Deleção de Sequência/genética
11.
Infect Immun ; 81(9): 3128-38, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23774595

RESUMO

Human pathogen group A streptococcus (GAS) has developed mechanisms to subvert innate immunity. We recently reported that the secreted esterase produced by serotype M1 GAS (SsE(M1)) reduces neutrophil recruitment by targeting platelet-activating factor (PAF). SsE(M1) and SsE produced by serotype M28 GAS (SsE(M28)) have a 37% sequence difference. This study aims at determining whether SsE(M28) is also a PAF acetylhydrolase and participates in innate immune evasion. We also examined whether SsE evolved to target PAF by characterizing the PAF acetylhydrolase (PAF-AH) activity and substrate specificity of SsE(M1), SsE(M28), SeE, the SsE homologue in Streptococcus equi, and human plasma PAF-AH (hpPAF-AH). PAF incubated with SsE(M28) or SeE was converted into lyso-PAF. SsE(M1) and SsE(M28) had kcat values of 373 s(-1) and 467 s(-1), respectively, that were ≥ 30-fold greater than that of hpPAF-AH (12 s(-1)). The comparison of SsE(M1), SsE(M28), and hpPAF-AH in kcat and Km in hydrolyzing triglycerides, acetyl esters, and PAF indicates that the SsE proteins are more potent hydrolases against PAF and have high affinity for PAF. SsE(M28) possesses much lower esterase activities against triglycerides and other esters than SsE(M1) but have similar potency with SsE(M1) in PAF hydrolysis. Deletion of sse(M28) in a covS deletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas in trans expression of SsE(M28) in GAS reduced neutrophil infiltration and increased skin invasion in subcutaneous infection of mice. These results suggest that the SsE proteins evolved to target PAF for enhancing innate immune evasion and skin invasion.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Streptococcus/imunologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Animais , Esterases/genética , Esterases/imunologia , Esterases/metabolismo , Feminino , Humanos , Hidrólise , Evasão da Resposta Imune/genética , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Fator de Ativação de Plaquetas/genética , Fator de Ativação de Plaquetas/imunologia , Fator de Ativação de Plaquetas/metabolismo , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Infecções Cutâneas Estafilocócicas/genética , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/metabolismo , Infecções Cutâneas Estafilocócicas/microbiologia , Streptococcus/genética , Streptococcus/metabolismo , Especificidade por Substrato/genética , Especificidade por Substrato/imunologia , Triglicerídeos/genética , Triglicerídeos/imunologia , Triglicerídeos/metabolismo
12.
Infect Immun ; 81(9): 3148-62, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23774599

RESUMO

Colanic acid (CA) is a common exopolysaccharide produced by many genera in the Enterobacteriaceae. It is critical for biofilm formation on HEp-2 cells and on chicken intestinal tissue by Salmonella. In this study, we generated different CA synthesis gene mutants and evaluated the immune responses induced by these mutants. One of these mutations, Δ(wza-wcaM)8, which deleted the whole operon for CA synthesis, was introduced into two Salmonella vaccine strains attenuated by auxotrophic traits or by the regulated delayed attenuation strategy (RDAS). The mice immunized with the auxotrophic Salmonella vaccine strain with the deletion mutation Δ(wza-wcaM)8 developed higher vaginal IgA titers against the heterologous protective antigen and higher levels of antigen-specific IgA secretion cells in lungs. In Salmonella vaccine strains with RDAS, the strain with the Δ(wza-wcaM)8 mutation resulted in higher levels of protective antigen production during in vitro growth. Mice immunized with this strain developed higher serum IgG and mucosal IgA antibody responses at 2 weeks. This strain also resulted in better gamma interferon (IFN-γ) responses than the strain without this deletion at doses of 10(8) and 10(9) CFU. Thus, the mutation Δ(wza-wcaM)8 will be included in various recombinant attenuated Salmonella vaccine (RASV) strains with RDAS derived from Salmonella enterica serovar Paratyphi A and Salmonella enterica serovar Typhi to induce protective immunity against bacterial pathogens.


Assuntos
Formação de Anticorpos/imunologia , Óperon/genética , Polissacarídeos/genética , Vacinas contra Salmonella/imunologia , Salmonella/imunologia , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Formação de Anticorpos/genética , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Feminino , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Óperon/imunologia , Polissacarídeos/imunologia , Salmonella/genética , Vacinas contra Salmonella/genética , Deleção de Sequência/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia
13.
J Immunol ; 190(4): 1819-26, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23296705

RESUMO

The processes of Ig gene locus contraction and looping during V(D)J-recombination are essential for creating a diverse Ab repertoire. However, no cis-acting sequence that plays a major role in specifying locus contraction has been uncovered within the Igκ gene locus. In this article, we demonstrate that a 650-bp sequence corresponding to DNase I hypersensitive sites HS1-2 within the mouse Igκ gene V-J intervening region binds CCCTC-binding factor and specifies locus contraction and long-range Vκ gene usage spanning 3.2 Mb in pre-B cells. We call this novel element Cer (for "contracting element for recombination"). Targeted deletion of Cer caused markedly increased proximal and greatly diminished upstream Vκ gene usage, higher allele usage, more splenic Igκ(+) B cells, and nonlineage-specific Igκ rearrangement in T cells. Relative to wild-type mice, Cer-deletion mice exhibited similar levels of Vκ gene germline transcription and H3K4me3 epigenetic marks but displayed a dramatic decrease in locus contraction in pre-B cells. Thus, our studies demonstrate that DNase I hypersensitive sites HS1-2 within the Vκ-Jκ intervening region are essential for controlling locus contraction and creating a diverse Ab repertoire.


Assuntos
DNA Intergênico/genética , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Rearranjo Gênico do Linfócito B/genética , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Animais , Técnicas de Introdução de Genes , Humanos , Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Integrases/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores de Células Precursoras de Linfócitos B/genética , Receptores de Células Precursoras de Linfócitos B/metabolismo , Deleção de Sequência/imunologia
14.
J Gen Virol ; 94(Pt 1): 50-58, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23052391

RESUMO

Highly pathogenic avian influenza (HPAI) H7N1 viruses caused a series of epizootics in Italy between 1999 and 2001. The emergence of these HPAI viruses coincided with the deletion of the six amino acids R(225)VESEV(230) at the C terminus of NS1. In order to assess how the truncation of NS1 affected virus replication, we used reverse genetics to generate a wild-type low-pathogenic avian influenza (LPAI) H7N1 virus with a 230aa NS1 (H7N1(230)) and a mutant virus with a truncated NS1 (H7N1(224)). The 6aa truncation had no impact on virus replication in duck or chicken cells in vitro. The H7N1(230) and H7N1(224) viruses also replicated to similar levels and induced similar immune responses in ducks or chickens. No significant histological lesions were detected in infected ducks, regardless of the virus inoculated. However, in chickens, the H7N1(230) virus induced a more severe interstitial pneumonia than did the H7N1(224) virus. These findings indicate that the C-terminal extremity of NS1, including the PDZ-binding motif ESEV, is dispensable for efficient replication of an LPAI virus in ducks and chickens, even though it may increase virulence in chickens, as revealed by the intensity of the histological lesions.


Assuntos
Galinhas/virologia , Patos/virologia , Vírus da Influenza A Subtipo H7N1/genética , Vírus da Influenza A Subtipo H7N1/metabolismo , Influenza Aviária/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Embrião de Galinha , Galinhas/imunologia , Patos/imunologia , Vírus da Influenza A Subtipo H7N1/imunologia , Influenza Aviária/genética , Influenza Aviária/imunologia , Influenza Aviária/virologia , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Proteínas não Estruturais Virais/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
15.
J Immunol ; 189(6): 2879-89, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22875804

RESUMO

Influenza is a major cause of morbidity and mortality in the United States. Studies have shown that excessive T cell activity can mediate pneumonitis in the setting of influenza infection, and data from the 2009 H1N1 pandemic indicate that critical illness and respiratory failure postinfection were associated with greater infiltration of the lungs with CD8+ T cells. T cell Ig and mucin domain 3 (Tim3) is a negative regulator of Th1/Tc1-type immune responses. Activation of Tim3 on effector T cells has been shown to downregulate proliferation, cell-mediated cytotoxicity, and IFN-γ production, as well as induce apoptosis. In this article, we demonstrate that deletion of the terminal cytoplasmic domain of the Tim3 gene potentiates its ability to downregulate Tc1 inflammation, and that this enhanced Tim3 activity is associated with decreased phosphorylation of the TCR-CD3ζ-chain. We then show that mice with this Tim3 mutation infected with influenza are protected from morbidity and mortality without impairment in viral clearance or functional heterotypic immunity. This protection is associated with decreased CD8+ T cell proliferation and decreased production of inflammatory cytokines, including IFN-γ. Furthermore, the Tim3 mutation was protective against mortality in a CD8+ T cell-specific model of pneumonitis. These data suggest that Tim3 could be targeted to prevent immunopathology during influenza infection and demonstrate a potentially novel signaling mechanism used by Tim3 to downregulate the Tc1 response.


Assuntos
Infecções por Orthomyxoviridae/imunologia , Receptores Virais/metabolismo , Regulação para Cima/imunologia , Animais , Complexo CD3/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Citotoxicidade Imunológica/genética , Modelos Animais de Doenças , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Receptor Celular 2 do Vírus da Hepatite A , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/mortalidade , Fosforilação/genética , Fosforilação/imunologia , Receptores Virais/genética , Receptores Virais/fisiologia , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Análise de Sobrevida , Regulação para Cima/genética
16.
J Immunol ; 188(5): 2235-43, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22291186

RESUMO

CD1d is an MHC class I-like molecule that presents glycolipid Ags to types I and II NKT cells. The YxxI motif in the cytoplasmic tail of CD1d contributes to its intracellular localization to the endolysosomal compartment and is important for Ag presentation to type I NKT cells. In this study, we identified the (327-329)RRR motif in CD1d and showed that it is critical for the control of CD1d intracellular trafficking and Ag presentation. The replacement of the arginines in this motif with alanines resulted in the extensive accumulation of CD1d in lysosomes but did not affect the cell surface expression. The defect in its cellular localization was accompanied by defects in Ag presentation to both type I and type II NKT cells. These results demonstrated that the (327-329)RRR motif of CD1d is required for proper cellular distribution of CD1d and optimal Ag presentation to both type I and type II NKT cells.


Assuntos
Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Antígenos CD1d/genética , Citoplasma/genética , Citoplasma/imunologia , Mutagênese Sítio-Dirigida , Células T Matadoras Naturais/imunologia , Motivos de Aminoácidos/genética , Animais , Antígenos CD1d/biossíntese , Antígenos CD1d/metabolismo , Arginina/genética , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Membrana Celular/genética , Membrana Celular/imunologia , Citoplasma/enzimologia , Líquido Intracelular/enzimologia , Líquido Intracelular/imunologia , Lisossomos/enzimologia , Lisossomos/genética , Lisossomos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células T Matadoras Naturais/classificação , Células T Matadoras Naturais/patologia , Transporte Proteico/genética , Transporte Proteico/imunologia , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Eletricidade Estática
17.
J Immunol ; 188(5): 2285-96, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22291188

RESUMO

The MHC class I (MHC I) molecules play a pivotal role in the regulation of immune responses by presenting antigenic peptides to CTLs and by regulating cytolytic activities of NK cells. In this article, we show that MHC I A in rhesus macaques can be alternatively spliced, generating a novel MHC I A isoform (termed "MHC I A-sv1") devoid of α(3) domain. Despite the absence of ß2-microglobulin (ß2m), the MHC I A-sv1 proteins reached the cell surface of K562-transfected cells as endoglycosidase H-sensitive glycoproteins that could form disulfide-bonded homodimers. Cycloheximide-based protein chase experiments showed that the MHC I A-sv1 proteins were more stable than the full-length MHC I A in transiently or stably transfected cell lines. Of particular interest, our studies demonstrated that MHC I A-sv1 could form ß2m-free heterodimers with its full-length protein in mammalian cells. The formation of heterodimers was accompanied by a reduction in full-length MHC I A ubiquitination and consequent stabilization of the protein. Taken together, these results demonstrated that MHC I A-sv1 and MHC I A can form a novel heterodimeric complex as a result of the displacement of ß2m and illustrated the relevance of regulated MHC I A protein degradation in the ß2m-free heterodimerization-dependent control, which may have some implications for the MHC I A splice variant in the fine tuning of classical MHC I A/TCR and MHC I A/killer cell Ig-like receptor interactions.


Assuntos
Processamento Alternativo/imunologia , Regulação para Baixo/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Isoformas de Proteínas/metabolismo , Ubiquitina/antagonistas & inibidores , Ubiquitina/fisiologia , Microglobulina beta-2/deficiência , Microglobulina beta-2/metabolismo , Processamento Alternativo/genética , Animais , Dissulfetos/metabolismo , Regulação para Baixo/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Células K562 , Macaca mulatta , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Isoformas de Proteínas/genética , Multimerização Proteica , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/imunologia , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Transfecção , Ubiquitina/metabolismo , Microglobulina beta-2/genética
18.
Curr Microbiol ; 63(6): 574-80, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21964939

RESUMO

Actinobacillus pleuropneumoniae is a Gram-negative pathogen that causes porcine pleuropneumonia. The pathogenicity of A. pleuropneumoniae is strongly correlated with the production of active repeat-in-toxin (RTX) proteins such as ApxIVA. We evaluated the contribution of a potential ApxIVA activator, ORF1, to the virulence and immunogenicity of A. pleuropneumoniae in pigs. The orf1 gene in A. pleuropneumoniae SLW03 (serovar 1, ΔapxICΔapxIIC) was deleted, producing strain SLW05 (ΔapxICΔapxIICΔorf1). The virulence of strains SLW03 and SLW05 was compared in pigs. Clinical signs and pulmonary lesions induced by strain SLW05 were slighter than that of strain SLW03 (P < 0.05). The immunogenicity and protective efficacy of strains SLW03 and SLW05 were similar. All pigs immunized with strain SLW03 or SLW05 developed high antibody titers against ApxIA, ApxIIA, and ApxIVA before challenge. Two weeks after a second immunization, pigs were challenged intratracheally with either a fully virulent A. pleuropneumoniae serovar 1 or serovar 3 strain. Vaccination with strains SLW03 or SLW05 provided significantly greater protection compared to the negative control (P < 0.01). Immunized pigs displayed significantly fewer clinical signs and lower lung lesion scores than non-immunized pigs. These results suggested that ORF1 plays an important role in the development of ApxIVA toxicity. Furthermore, strain SLW05 is a highly attenuated strain able to induce protective immunity against A. pleuropneumoniae infection.


Assuntos
Infecções por Actinobacillus/veterinária , Actinobacillus pleuropneumoniae/imunologia , Proteínas de Bactérias/imunologia , Doenças dos Suínos/microbiologia , Infecções por Actinobacillus/imunologia , Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Ensaio de Imunoadsorção Enzimática/veterinária , Histocitoquímica/veterinária , Distribuição Aleatória , Deleção de Sequência/imunologia , Suínos , Doenças dos Suínos/imunologia , Vacinação/veterinária , Virulência/imunologia
19.
J Immunol ; 187(9): 4733-43, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21949022

RESUMO

Class-switch recombination of Ab isotype is mediated by a recombinational DNA deletion event and must be robustly upregulated during Ag-driven differentiation of B cells. The enhancer region 3' of the Cα gene is important for the upregulation of switch recombination. Using a transgene of the entire H chain C region locus, we demonstrate in this study that it is the four 3' enhancer elements themselves (a total of 4.7 kb) that are responsible for the upregulation rather than the 24 kb of DNA in between them. Neither allelic exclusion nor transgenic µ expression is reduced by deletion of the four 3' enhancers. We also test deletions of two or three of the 3' enhancers and show that deletion of more 3' enhancers results in a progressive reduction in both switch recombination and germline transcription of all H chain genes. Nevertheless, we find evidence for special roles for some 3' enhancers; different H chain genes are affected by different 3' enhancer deletions. Thus, we find that the dramatic induction of class-switch recombination during Ag-driven differentiation is the result of an interaction among four separated regulatory elements.


Assuntos
Switching de Imunoglobulina/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Recombinação Genética/imunologia , Deleção de Sequência/imunologia , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Células Cultivadas , Elementos Facilitadores Genéticos/genética , Elementos Facilitadores Genéticos/imunologia , Éxons/genética , Feminino , Imunoglobulina G/biossíntese , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/genética
20.
PLoS One ; 6(8): e22939, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21860662

RESUMO

Dendritic cell (DC)-mediated presentation of MHC class I (MHC-I)/peptide complexes is a crucial first step in the priming of CTL responses, and the cytoplasmic tail of MHC-I plays an important role in modulating this process. Several species express a splice variant of the MHC-I tail that deletes exon 7-encoding amino acids (Δ7), including a conserved serine phosphorylation site. Previously, it has been shown that Δ7 MHC-I molecules demonstrate extended DC surface half-lives, and that mice expressing Δ7-K(b) generate significantly augmented CTL responses to viral challenge. Herein, we show that Δ7-D(b)-expressing DCs stimulated significantly more proliferation and much higher cytokine secretion by melanoma antigen-specific (Pmel-1) T cells. Moreover, in combination with adoptive Pmel-1 T-cell transfer, Δ7-D(b) DCs were superior to WT-D(b) DCs at stimulating anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival. Human DCs engineered to express Δ7-HLA-A*0201 showed similarly enhanced CTL stimulatory capacity. Further studies demonstrated impaired lateral membrane movement and clustering of human Δ7-MHC-I/peptide complexes, resulting in significantly increased bioavailability of MHC-I/peptide complexes for specific CD8+ T cells. Collectively, these data suggest that targeting exon 7-encoded MHC-I cytoplasmic determinants in DC vaccines has the potential to increase CD8+ T-cell stimulatory capacity and substantially improve their clinical efficacy.


Assuntos
Processamento Alternativo/genética , Linfócitos T CD8-Positivos/imunologia , Citoplasma/metabolismo , Células Dendríticas/imunologia , Antígenos HLA/química , Antígenos HLA/genética , Melanoma Experimental/imunologia , Processamento Alternativo/imunologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/citologia , Células Dendríticas/citologia , Epitopos/genética , Epitopos/imunologia , Éxons/genética , Antígenos HLA/imunologia , Humanos , Lentivirus/genética , Melanoma Experimental/genética , Camundongos , Deleção de Sequência/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia
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