RESUMO
In the past decade, remarkable progress has been made in immunotherapy against cancer. Specifically, the introduction of immune checkpoint inhibitors has revolutionized the field. However, many patients are unable to benefit significantly from this treatment option. One of the major reasons for this is most likely the absence of an adequate tumor-specific T cell response in these patients. A way to circumvent this problem might be to combine immune checkpoint inhibitor treatment with new strategies to activate tumor-specific T cells. One such strategy could be to activate and mature dendritic cells in situ. Dendritic cells carry an array of external and internal pattern recognition receptors that induce cell activation and maturation when interacting with their corresponding damage-associated or pathogen-associated molecular patterns (DAMPs or PAMPs). Targeting such molecular patterns directly to dendritic cells might be a way to evoke stronger immune responses. Here, we review our recent findings using antibody-targeted DNA. We summarize the results from our experiments showing that dendritic cells can be actively targeted in vivo through the αXß2 integrin subunit CD11c, and that DNA delivered through this receptor in vitro leads to maturation of dendritic cells via the cytosolic cGAS/STING DNA-sensing pathway.
Assuntos
DNA/imunologia , Células Dendríticas/imunologia , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Vacinas de DNA/imunologia , Animais , Humanos , Imunidade Inata , Vacinas de DNA/administração & dosagemRESUMO
Common variable immunodeficiency (CVID) is a heterogeneous primary immunodeficiency disease, leading to recurrent bacterial airway infections and often also autoimmune complications. To shed light on the regulatory lymphocytes from these patients, we analyzed the levels of regulatory B (pro-B10) cell and regulatory T (Treg) cell subpopulations in PBMCs from twenty-six patients diagnosed with CVID using multi-color flowcytometry. Pro-B10 cells were induced by 48h in vitro stimulation prior to analysis. Suppressor function was measured on a subset of patients with splenomegaly and autoimmune complications. The levels of regulatory B and T cells were correlated to clinical manifestations, including autoimmunity, splenomegaly and CVID EUROclass subgroups. We demonstrate a significant association between elevated levels of pro-B10 cells, decreased levels of Tregs and autoimmune phenomena in CVID patients. The finding of marked abnormalities in regulatory lymphocyte populations contribute to our understanding of the pathogenesis of CVID and potentially be valuable in the clinical management and treatment of patients.
Assuntos
Linfócitos B Reguladores/imunologia , Imunodeficiência de Variável Comum/fisiopatologia , Esplenomegalia/fisiopatologia , Linfócitos T Reguladores/imunologia , Idade de Início , Autoimunidade/imunologia , Linfócitos B Reguladores/citologia , Imunodeficiência de Variável Comum/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/citologiaRESUMO
An issue with many current vaccines is the dependency on broadly inflammatory adjuvants, such as aluminum hydroxide or aluminum salts that affect many immune- and non-immune cells. These adjuvants are not necessarily activating all antigen-presenting cells (APCs) that take up the antigen and most likely they also activate APCs with no antigen uptake, as well as many non-immune cells. Conjugation of antigen and adjuvant would enable the use of smaller amounts of adjuvant and avoid unnecessary tissue damage and activation of bystander cells. It would ensure that all APCs that take up the antigen would also become activated and avoid that immature and non-activated APCs present the antigen to T cells without a co-stimulatory signal, leading to tolerogenesis. We have developed a novel vaccine that co-deliver antigen and a nucleotide adjuvant to the same APC and lead to a strong activation response in dendritic cells and macrophages. The vaccine is constructed as a fusion-protein with an antigen fused to the DNA/RNA-binding domain from the Hc2 protein from Chlamydia trachomatis. We have found that the fusion protein is able to package polyinosinic:polycytidylic acid (poly(I:C)) or dsDNA into small particles. These particles were taken up by macrophages and dendritic cells and led to strong activation and maturation of these cells. Immunization of mice with the fusion protein packaged poly(I:C) led to a stronger antibody response compared to immunization with a combination of poly(I:C) and antigen without the Hc2 DNA/RNA-binding domain.
Assuntos
Formação de Anticorpos , Vacinas , Animais , Camundongos , Nucleotídeos/metabolismo , Células Dendríticas , Antígenos , Poli I-C , Adjuvantes Imunológicos , DNARESUMO
The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.
Assuntos
Proteínas de Membrana , Neoplasias , Humanos , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Interferons/metabolismo , Nucleotidiltransferases/metabolismo , Neoplasias/terapia , Neoplasias/metabolismo , Retículo Endoplasmático/metabolismo , Microambiente TumoralRESUMO
The immune system targets virus-infected cells by different means. One of the essential antiviral mechanisms is apoptosis induced by ligation of tumor necrosis factor receptor 1 (TNFR1). This receptor can be activated by tumor necrosis factor alpha (TNF-α), which upon binding to TNFR1 induces the assembly of first an inflammatory and later a proapoptotic signaling complex. Here, we report that infection by human herpesvirus 6B (HHV-6B) inhibited poly(ADP-ribose) polymerase (PARP) cleavage, caspase 3 and 8 activation, and IκBα Ser-32 phosphorylation downstream of TNFR1, indicating inhibition of both the inflammatory and apoptotic signaling pathways. We identified a hitherto uncharacterized viral protein, U20, as sufficient for mediating this inhibition. U20 was shown to locate to the cell membrane, and overexpression inhibited PARP cleavage, caspase 3 and 8 activation, IκBα Ser-32 phosphorylation, and NF-κB transcriptional activity. Moreover, small interfering RNA (siRNA) knockdown of U20 demonstrated that the protein is necessary for HHV-6B-mediated inhibition of TNFR signaling during infection. These results suggest an important novel function of U20 as a viral immune evasion protein during HHV-6B infection.
Assuntos
Apoptose , Herpesvirus Humano 6/patogenicidade , Evasão da Resposta Imune , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Transdução de Sinais , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/virologia , Herpesvirus Humano 6/imunologia , HumanosRESUMO
Osteopontin (OPN) is a cytokine with multiple functions, including immune defense mechanisms against invading microorganisms. OPN-deficient mice are impaired in clearing intracellular pathogens, suggesting an important role of OPN during phagocytosis, but it remains to be defined how OPN may enhance this innate immune process. Here, we demonstrate that OPN binds to monocytes, but not resting T cells, NK cells, or B cells, and mediates chemoattraction of IL-1-activated human monocytes. Moreover, OPN binds in a specific manner to all known serotypes of the two bacterial species Streptococcus agalactiae and Staphylococcus aureus and opsonizes these bacteria for phagocytosis. We identify the integrin alpha(X)beta(2) (CD11c/CD18), which is highly expressed on the cell surface of monocytes, as a novel OPN receptor. To eliminate the contribution from other molecular interactions between the bacteria and the phagocyte, we show that OPN-coated synthetic beads are phagocytosed in an alpha(X)beta(2) integrin-dependent manner. The ligand recognition does not involve the RGD motif previously reported to support binding of OPN to integrins. Taken together, these data identify the alpha(X)beta(2) integrin as a novel OPN receptor that is required for OPN-mediated phagocytosis, thereby elucidating an important mechanism of an innate immune function of OPN.
Assuntos
Integrina alfaXbeta2/metabolismo , Osteopontina/imunologia , Fagocitose , Animais , Sítios de Ligação , Imunidade Inata , Camundongos , Camundongos Knockout , Monócitos/metabolismo , Osteopontina/metabolismo , Ligação Proteica , Staphylococcus aureus/imunologia , Streptococcus agalactiae/imunologiaRESUMO
The pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) involves an abnormal chemokine regulation. The chemokine receptor CCR4 is necessary for T cell migration to the skin. We, therefore, studied if CCR4 and its ligand macrophage-derived chemokine (MDC/CCL22) could participate in spreading the disease between skin and joints by examining RA, PsA and osteoarthritis (OA) patients. In synovial fluid from RA and PsA patients we observed a significantly higher MDC/CCL22 level compared to OA patients. Additionally, the MDC/CCL22 protein was found to be elevated in RA and PsA plasma compared to OA and healthy volunteers. Flow cytometry revealed that most CD4(+)CCR4(+) lymphocytes also co-expressed CD45RO. Neither the MDC/CCL22 level nor the expression of CCR4 correlated to CRP. Immunohistochemistry of the RA and OA synovial membrane demonstrated CCR4 to be expressed by mononuclear cells and endothelial cells. Our results show that MDC/CCL22 is present within the synovial membrane of RA and OA patients and in high amount in the synovial fluid of patients with RA and PsA. This will enable migration of CCR4 expressing memory cells supporting that MDC/CCR4 could play a role in attracting skin specific memory T cells to the joints.
Assuntos
Artrite Psoriásica/imunologia , Artrite Reumatoide/imunologia , Quimiocina CCL22/metabolismo , Osteoartrite/imunologia , Receptores CCR4/metabolismo , Idoso , Animais , Artrite Psoriásica/patologia , Artrite Reumatoide/patologia , Quimiocina CCL22/genética , Feminino , Humanos , Memória Imunológica/imunologia , Pessoa de Meia-Idade , Osteoartrite/patologia , Receptores CCR4/genética , Pele/imunologia , Líquido Sinovial/imunologia , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologiaRESUMO
Immunotherapeutic activation of tumor-specific T cells has proven to be an interesting approach in anticancer treatment. Particularly, anti-CTLA-4 and anti-PD-1/PD-L1 treatment looks promising, and conceivably, even better clinical results might be obtained if such treatment could be combined with boosting the existing tumor-specific T-cell response. One way to achieve this could be by increasing the level of maturation of dendritic cells locally and in the draining lymph nodes. When exposed to cancer cells, dendritic cells may spontaneously mature because of danger-associated molecular patterns derived from the tumor cells. Double-stranded DNA play a particularly important role in the activation of the dendritic cells, through engagement of intracellular DNA-sensors, and signaling through the adaptor protein STING. In the present study, we have investigated the maturational response of human monocyte-derived dendritic cells (moDC) and human monocytic THP-1 cells to targeted and untargeted DNA. We used an anti-CD11c antibody conjugated with double-stranded DNA to analyze the maturation status of human moDCs, as well as maturation using a cGAS KO and STING KO THP-1 cell maturation model. We found that dendritic cells can mature after exposure to cytoplasmic double-stranded DNA delivered through CD11c-mediated endocytosis. Moreover, we show that THP-1 cells matured using IL-4, GM-CSF, and ionomycin upregulate DC-maturation markers after CD11c-targeted delivery of double-stranded DNA. This upregulation is completely abrogated in cGAS KO and STING KO cells.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Células Dendríticas/fisiologia , Imunoterapia/métodos , Proteínas de Membrana/metabolismo , Neoplasias/terapia , Nucleotidiltransferases/metabolismo , Antígeno B7-H1/imunologia , Antígeno CD11c/metabolismo , Antígeno CTLA-4/imunologia , Diferenciação Celular , Citocinas/metabolismo , DNA/imunologia , Endocitose , Humanos , Proteínas de Membrana/genética , Neoplasias/imunologia , Nucleotidiltransferases/genética , Receptor de Morte Celular Programada 1/imunologia , RNA Interferente Pequeno/genética , Células THP-1RESUMO
BACKGROUND: Various forms of cellular stress can activate the tumour suppressor protein p53, an important regulator of cell cycle arrest, apoptosis, and cellular senescence. Cells infected by human herpesvirus 6B (HHV-6B) accumulate aberrant amounts of p53. OBJECTIVES: The aim of this study was to investigate the role of p53 accumulation in the HHV-6B-induced cell cycle arrest. STUDY DESIGN: The role of p53 was studied using the p53 inhibitor pifithrin-a, and cells genetically deficient in functional p53 by homologous recombination. RESULTS: In response to HHV-6B infection, epithelial cells were arrested in the G1/S phase of the cell cycle concomitant with an aberrant accumulation of p53. However, the known p53-induced mediator of cell cycle arrest, p21, was not upregulated. Approximately 90% of the cells expressed HHV-6B p41, indicative of viral infection. The presence of pifithrin-a, a p53 inhibitor, did not reverse the HHV-6B-induced cell cycle block. In support of this, HHV-6B infection of p53(-/-) cells induced a cell cycle block before S-phase with kinetics similar to or faster than that observed by infection in wt cells. CONCLUSIONS: HHV-6B infection inhibited host cell proliferation concomitantly with p53 accumulation, but importantly the block in cell cycle occurred by a pathway independent of p53.
Assuntos
Proliferação de Células , Herpesvirus Humano 6/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Benzotiazóis/farmacologia , Ciclo Celular , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Proteínas de Ligação a DNA/biossíntese , Humanos , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/biossíntese , Proteínas Virais/biossíntese , Replicação ViralRESUMO
During recent years, inborn errors of human IL-17 immunity have been demonstrated to underlie primary immunodeficiencies with chronic mucocutaneous candidiasis (CMC). Various defects in receptors responsible for sensing of Candida albicans or downstream signalling to IL-17 may lead to susceptibility to Candida infection. While CMC is common in patients with profound T cell immunodeficiencies, CMC is also recognised as part of other immunodeficiencies in syndromic CMC, or as relatively isolated CMC disease. We describe a 40-year-old woman with a clinical picture involving cutaneous bacterial abscesses, chronic oral candidiasis and extensive dermatophytic infection of the feet. By whole exome sequencing, we identified a STAT1-gain-of-function mutation. Moreover, the patient's peripheral blood mononuclear cells displayed severely impaired Th17 responses. The patient was treated with antifungals and prophylactic antibiotics, which led to resolution of the infection. We discuss the current knowledge within the field of Th17 deficiency and the pathogenesis and treatment of CMC.
Assuntos
Candidíase Mucocutânea Crônica/genética , Dermatoses do Pé/genética , Interleucina-17/deficiência , Ceratodermia Palmar e Plantar Epidermolítica/genética , Fator de Transcrição STAT1/genética , Tinha/genética , Abscesso/diagnóstico , Abscesso/tratamento farmacológico , Abscesso/genética , Adulto , Antibacterianos/uso terapêutico , Antifúngicos/uso terapêutico , Candidíase Mucocutânea Crônica/diagnóstico , Candidíase Mucocutânea Crônica/tratamento farmacológico , Feminino , Dermatoses do Pé/diagnóstico , Dermatoses do Pé/tratamento farmacológico , Humanos , Ceratodermia Palmar e Plantar Epidermolítica/diagnóstico , Ceratodermia Palmar e Plantar Epidermolítica/tratamento farmacológico , Mutação , Dermatopatias Infecciosas/diagnóstico , Dermatopatias Infecciosas/tratamento farmacológico , Dermatopatias Infecciosas/genética , Tinha/diagnóstico , Tinha/tratamento farmacológicoRESUMO
Herpes simplex encephalitis (HSE) in children has previously been linked to defects in type I interferon (IFN) production downstream of Toll-like receptor 3. Here, we describe a novel genetic etiology of HSE by identifying a heterozygous loss-of-function mutation in the IFN regulatory factor 3 (IRF3) gene, leading to autosomal dominant (AD) IRF3 deficiency by haploinsufficiency, in an adolescent female patient with HSE. IRF3 is activated by most pattern recognition receptors recognizing viral infections and plays an essential role in induction of type I IFN. The identified IRF3 R285Q amino acid substitution results in impaired IFN responses to HSV-1 infection and particularly impairs signaling through the TLR3-TRIF pathway. In addition, the R285Q mutant of IRF3 fails to become phosphorylated at S386 and undergo dimerization, and thus has impaired ability to activate transcription. Finally, transduction with WT IRF3 rescues the ability of patient fibroblasts to express IFN in response to HSV-1 infection. The identification of IRF3 deficiency in HSE provides the first description of a defect in an IFN-regulating transcription factor conferring increased susceptibility to a viral infection in the CNS in humans.
Assuntos
Encefalite por Herpes Simples/genética , Fibroblastos/metabolismo , Haploinsuficiência , Herpesvirus Humano 1/metabolismo , Fator Regulador 3 de Interferon/deficiência , Mutação de Sentido Incorreto , Adolescente , Substituição de Aminoácidos , Encefalite por Herpes Simples/metabolismo , Encefalite por Herpes Simples/patologia , Feminino , Fibroblastos/patologia , Fibroblastos/virologia , Herpesvirus Humano 1/genética , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/biossíntese , Interferon Tipo I/genética , Fosforilação , Multimerização Proteica/genéticaRESUMO
In order to establish a successful infection, it is of crucial importance for invading viruses to alter the activities of the regulatory protein p53. Beta-herpesviruses stabilize p53 and likely direct its activities towards generation of a replication-friendly environment. We here study the mechanisms behind HHV-6B-induced stabilization and inactivation of p53. Stable transgene expression of the HHV-6B protein U19 was sufficient to achieve upregulation of p53. U19 bound directly to the p53-regulating protein HDM2 in vitro, co-precipitated together with HDM2 in lysates, and co-localized with HDM2 in the nucleus when overexpressed. U19 contained a sequence with a putative p53 BOX I-motif for HDM2 binding. Mutation of the two key amino acids within this motif was sufficient to inhibit all the described U19 functions. Our study provides further insight into p53-modulating strategies used by herpesviruses and elucidates a mechanism used by HHV-6B to circumvent the antiviral response.
Assuntos
Herpesvirus Humano 6/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Infecções por Roseolovirus/metabolismo , Transativadores/química , Transativadores/metabolismo , Proteína Supressora de Tumor p53/química , Proteínas Virais/química , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Herpesvirus Humano 6/química , Herpesvirus Humano 6/genética , Humanos , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/genética , Infecções por Roseolovirus/genética , Infecções por Roseolovirus/virologia , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/genéticaRESUMO
HHV-6B infection inhibits cell proliferation in G2/M, but no protein has so far been recognized to exert this function. Here we identify the protein product of direct repeat 6, DR6, as an inhibitor of G2/M cell-cycle progression. Transfection of DR6 reduced the total number of cells compared with mock-transfected cells. Lentiviral transduction of DR6 inhibited host cell DNA synthesis in a p53-independent manner, and this inhibition was DR6 dose-dependent. A deletion of 66 amino acids from the N-terminal part of DR6 prevented efficient nuclear translocation and the ability to inhibit DNA synthesis. DR6-induced accumulation of cells in G2/M was accompanied by an enhanced expression of cyclin B1 that accumulated predominantly in the cytoplasm. Pull-down of cyclin B1 brought down pCdk1 with the inactivating phosphorylation at Tyr15. Together, DR6 delays cell cycle with an accumulation of cells in G2/M and thus might be involved in HHV-6B-induced cell-cycle arrest.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular , Herpesvirus Humano 6/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular , Infecções por Roseolovirus/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Proliferação de Células , Ciclina B1/genética , Ciclina B1/metabolismo , Herpesvirus Humano 6/química , Herpesvirus Humano 6/genética , Humanos , Infecções por Roseolovirus/genética , Infecções por Roseolovirus/fisiopatologia , Infecções por Roseolovirus/virologia , Proteína Supressora de Tumor p53/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1.
Assuntos
DNA/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Interferon Tipo I/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Apoptose , Transporte Biológico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Citosol/metabolismo , DNA/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interferon Tipo I/metabolismo , Interleucina-2/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transfecção , Replicação Viral/imunologiaRESUMO
B cell subsets in newly diagnosed untreated, relapsing-remitting multiple sclerosis (MS) patients were examined. The fraction of CD20(+) B cells was significantly increased in MS. Among subsets of B cells, MS patients had increased frequency of naïve cells, but reduced frequency of memory and B1 cells. The frequencies of B1 cells were inversely correlated with the time since last attack. B1 cells resembled the phenotype of either lymphocytes (CD11b(-) B1 cells) or monocytes (CD11b(+) B1 cells) and a small fraction of cells was CD3(+)CD20(+) by confocal microscopy.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Regulação para Baixo/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/patologia , Adulto , Antígenos CD20/biossíntese , Subpopulações de Linfócitos B/metabolismo , Complexo CD3/biossíntese , Feminino , Humanos , Imunofenotipagem , Contagem de Linfócitos , Masculino , Esclerose Múltipla Recidivante-Remitente/metabolismo , Regulação para Cima/imunologiaRESUMO
Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein.
Assuntos
Herpesvirus Humano 6/genética , Interações Hospedeiro-Patógeno , Proteína Supressora de Tumor p53/genética , Proteínas Virais/genética , Morte Celular/efeitos da radiação , Dano ao DNA , Raios gama , Regulação da Expressão Gênica/efeitos da radiação , Células HCT116 , Herpesvirus Humano 6/metabolismo , Humanos , Análise em Microsséries , Transdução de Sinais/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/metabolismoRESUMO
Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequency by homologous recombination, and applicability to many cell types, make rAAV an attractive approach for targeted genome editing. Combined with cloning by somatic cell nuclear transfer (SCNT), this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1, and breast cancer. This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination. We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts, which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.
Assuntos
Animais Geneticamente Modificados , Dependovirus/genética , Marcação de Genes , Vetores Genéticos , Suínos/genética , Animais , Técnicas de Transferência de Genes , Humanos , Técnicas de Transferência NuclearRESUMO
An analysis of all known human herpesviruses has not previously been reported on sperm from normal donors. Using an array-based detection method, we determined the cross-sectional frequency of human herpesviruses in semen from 198 Danish sperm donors. Fifty-five of the donors had at least one ejaculate that was positive for one or more human herpesvirus. Of these 27.3% (nâ=â15) had a double herpesvirus infection. If corrected for the presence of multiple ejaculates from some donors, the adjusted frequency of herpesviruses in semen was 27.2% with HSV-1 in 0.4%; HSV-2 in 0.1%; EBV in 6.3%; HCMV in 2.7%; HHV-6A/B in 13.5%; HHV-7 in 4.2%, whereas none of the samples had detectable VZV or HHV-8. Subsequently, we examined longitudinally data on ejaculates from 11 herpesvirus-positive donors. Serial analyses revealed that a donor who tested positive for herpesvirus at one time point did not necessarily remain positive over time. For the most frequently found herpesvirus, HHV-6A/B, we examined its association with sperm. For HHV-6A/B PCR-positive semen samples, HHV-6A/B could be detected on the sperm by flow cytometry. Conversely, PCR-negative semen samples were negative by flow cytometry. HHV-6B was shown to associate with sperm within minutes in a concentration dependent manner. Confocal microscopy demonstrated that HHV-6B associated with the sperm head, but only to sperm with an intact acrosome. Taken together, our data suggest that HHV-6A/B could be transported to the uterus via binding to the sperm acrosome. Moreover, we find a 10 times higher frequency of HHV-7 in semen from healthy individuals than previously detected. Further research is required to determine the potential risk of using herpesvirus-positive donor semen. Longitudinally analyses of ejaculate series indicate that implementation of quarantine for a donor shown to shed a herpesvirus is not a tenable solution.
Assuntos
Acrossomo/virologia , Herpesvirus Humano 6/patogenicidade , Sêmen/virologia , Doadores de Tecidos , Acrossomo/ultraestrutura , Herpesvirus Humano 6/fisiologia , Humanos , Masculino , Infecções por Roseolovirus/epidemiologia , Ligação Viral , Eliminação de Partículas ViraisRESUMO
The SalI-L fragment from human herpesvirus 6A (HHV-6A) encodes a protein DR7 that has been reported to produce fibrosarcomas when injected into nude mice, to transform NIH3T3 cells, and to interact with and inhibit the function of p53. The homologous gene in HHV-6B is dr6. Since p53 is deregulated in both HHV-6A and -6B, we characterized the expression of dr6 mRNA and the localization of the translated protein during HHV-6B infection of HCT116 cells. Expression of mRNA from dr6 was inhibited by cycloheximide and partly by phosphonoacetic acid, a known characteristic of herpesvirus early/late genes. DR6 could be detected as a nuclear protein at 24 hpi and accumulated to high levels at 48 and 72 hpi. DR6 located in dots resembling viral replication compartments. Furthermore, a novel interaction between DR6 and the viral DNA processivity factor, p41, could be detected by confocal microscopy and by co-immunoprecipitation analysis. In contrast, DR6 and p53 were found at distinct subcellular locations. Together, our data imply a novel function of DR6 during HHV-6B replication.