RESUMEN
Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.
Asunto(s)
Linfocitos B , ADN Glicosilasas , Reparación de la Incompatibilidad de ADN , Cambio de Clase de Inmunoglobulina , Proteínas de la Membrana , Mutación , Proteínas de Neoplasias , Hipermutación Somática de Inmunoglobulina , Animales , Femenino , Humanos , Ratones , Linfocitos B/metabolismo , Sistemas CRISPR-Cas , ADN Glicosilasas/antagonistas & inhibidores , ADN Glicosilasas/metabolismo , Epistasis Genética , Células HEK293 , Cambio de Clase de Inmunoglobulina/genética , Región de Cambio de la Inmunoglobulina/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Hipermutación Somática de Inmunoglobulina/genéticaRESUMEN
Epstein-Barr virus (EBV), a member of the γ-herpesvirus family, is one of the most prevalent and persistent human viruses, infecting up to 90% of the adult population globally. EBV's life cycle includes primary infection, latency, and lytic reactivation, with the virus primarily infecting B cells and epithelial cells. This virus has evolved sophisticated strategies to evade both innate and adaptive immune responses, thereby maintaining a lifelong presence within the host. This persistence is facilitated by the expression of latent genes such as EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), which play crucial roles in viral latency and oncogenesis. In addition to their well-known roles in several types of cancer, including nasopharyngeal carcinoma and B-cell lymphomas, recent studies have identified the pathogenic roles of EBV in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. This review highlights the intricate interactions between EBV and the host immune system, underscoring the need for further research to develop effective therapeutic and preventive strategies against EBV-associated diseases.
Asunto(s)
Enfermedades Autoinmunes , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Evasión Inmune , Humanos , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/patogenicidad , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/virología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/virología , Animales , Latencia del Virus/inmunologíaRESUMEN
The SARS-CoV-2 virus, causing the devastating COVID-19 pandemic, has been reported to affect platelets and cause increased thrombotic events, hinting at the possible bidirectional interactions between platelets and the virus. In this review, we discuss the potential mechanisms underlying the increased thrombotic events as well as altered platelet count and activity in COVID-19. Inspired by existing knowledge on platelet-pathogen interactions, we propose several potential antiviral strategies that platelets might undertake to combat SARS-CoV-2, including their abilities to internalize the virus, release bioactive molecules to interfere with viral infection, and modulate the functions of immune cells. Moreover, we discuss current and potential platelet-targeted therapeutic strategies in controlling COVID-19, including antiplatelet drugs, anticoagulants, and inflammation-targeting treatments. These strategies have shown promise in clinical settings to alleviate the severity of thrombo-inflammatory complications and reduce the mortality rate among COVID-19 patients. In conclusion, an in-depth understanding of platelet-SARS-CoV-2 interactions may uncover novel mechanisms underlying severe COVID-19 complications and could provide new therapeutic avenues for managing this disease.
RESUMEN
The newly identified shieldin complex, composed of SHLD1, SHLD2, SHLD3, and REV7, lies downstream of 53BP1 and acts to inhibit DNA resection and promote NHEJ. Here, we show that Shld2-/- mice have defective class switch recombination (CSR) and that loss of SHLD2 can suppress the embryonic lethality of a Brca1Δ11 mutation, highlighting its role as a key effector of 53BP1. Lymphocyte development and RAG1/2-mediated recombination were unaffected by SHLD2 deficiency. Interestingly, a significant fraction of Shld2-/- primary B-cells and 53BP1- and shieldin-deficient CH12F3-2 B-cells permanently lose expression of immunoglobulin upon induction of CSR; this population of Ig-negative cells is also seen in other NHEJ-deficient cells and to a much lesser extent in WT cells. This loss of Ig is due to recombination coupled with overactive resection and loss of coding exons in the downstream acceptor constant region. Collectively, these data show that SHLD2 is the key effector of 53BP1 and critical for CSR in vivo by suppressing large deletions within the Igh locus.
Asunto(s)
Roturas del ADN de Doble Cadena , Cambio de Clase de Inmunoglobulina , Animales , Cambio de Clase de Inmunoglobulina/genética , RatonesRESUMEN
Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Línea Celular Tumoral , Cristalografía por Rayos X , Reparación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos de 4 o más Anillos/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación/efectos de los fármacos , Modelos Moleculares , Estructura MolecularRESUMEN
Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC, the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda(-/-) GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda(-/-) GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition, we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation, demonstrating the importance of apoptosis in GC homeostasis and clonal selection.
Asunto(s)
Apoptosis/fisiología , Síndrome Linfoproliferativo Autoinmune/inmunología , Subgrupos de Linfocitos B/inmunología , Caspasa 8/fisiología , Selección Clonal Mediada por Antígenos , Citidina Desaminasa/fisiología , Centro Germinal/inmunología , Síndromes de Inmunodeficiencia/patología , Traslado Adoptivo , Animales , Antígenos/inmunología , Subgrupos de Linfocitos B/patología , Caspasa 8/genética , División Celular , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , Roturas del ADN de Doble Cadena , Centro Germinal/patología , Inmunización , Cambio de Clase de Inmunoglobulina , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Células Plasmáticas/patología , Quimera por Radiación , Receptores de Antígenos de Linfocitos B/inmunología , Hipermutación Somática de InmunoglobulinaRESUMEN
Upon activation with T-dependent Ag, B cells enter germinal centers (GC) and upregulate activation-induced deaminase (AID). AID(+) GC B cells then undergo class-switch recombination and somatic hypermutation. Follicular dendritic cells (FDC) are stromal cells that underpin GC and require constitutive signaling through the lymphotoxin (LT) ß receptor to be maintained in a fully mature, differentiated state. Although it was shown that FDC can be dispensable for the generation of affinity-matured Ab, in the absence of FDC it is unclear where AID expression occurs. In a mouse model that lacks mature FDC, as well as other LT-sensitive cells, we show that clusters of AID(+)PNA(+)GL7(+) Ag-specific GC B cells form within the B cell follicles of draining lymph nodes, suggesting that FDC are not strictly required for GC formation. However, later in the primary response, FDC-less GC dissipated prematurely, correlating with impaired affinity maturation. We examined whether GC dissipation was due to a lack of FDC or other LTß receptor-dependent accessory cells and found that, in response to nonreplicating protein Ag, FDC proved to be more critical for long-term GC maintenance. Our study provides a spatial-temporal analysis of Ag-specific B cell activation and AID expression in the context of a peripheral lymph node that lacks FDC-M1(+) CD35(+) FDC and other LT-sensitive cell types, and reveals that FDC are not strictly required for the induction of AID within an organized GC-like environment.
Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/metabolismo , Células Dendríticas Foliculares/metabolismo , Centro Germinal/citología , Animales , Diferenciación Celular , Células Cultivadas , Citidina Desaminasa/biosíntesis , Células Dendríticas Foliculares/citología , Células Dendríticas Foliculares/inmunología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Receptor beta de Linfotoxina/inmunología , Receptor beta de Linfotoxina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Complemento 3b/metabolismoRESUMEN
Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder in which maternal antibodies cross the placenta and destroy fetal/neonatal platelets. It has been demonstrated that the neonatal Fc receptor (FcRn) regulates immunoglobulin G (IgG) homeostasis and plays an important role in transplacental IgG transport. However, the role of FcRn in the pathogenesis and therapy of FNIT has not been studied. Here, we developed an animal model of FNIT using combined ß3 integrin-deficient and FcRn-deficient (ß3(-/-)FcRn(-/-)) mice. We found that ß3(-/-)FcRn(-/-) mice are immunoresponsive to ß3(+/+)FcRn(-/-) platelets. The generated antibodies were ß3 integrin specific and were maintained at levels that efficiently induced thrombocytopenia in adult ß3(+/+)FcRn(-/-) mice. FNIT was observed when immunized ß3(-/-)FcRn(+/+) females were bred with ß3(+/+)FcRn(+/+) males, while no FNIT occurred in ß3(-/-)FcRn(-/-) females bred with ß3(+/+)FcRn(-/-) males, suggesting that FcRn is indispensable for the induction of FNIT. We further demonstrated that fetal FcRn was responsible for the transplacental transport of various IgG isotypes. We found that anti-FcRn antibody and intravenous IgG prevented FNIT, and that intravenous IgG ameliorated FNIT through both FcRn-dependent and -independent pathways. Our data suggest that targeting FcRn may be a potential therapy for human FNIT as well as other maternal pathogenic antibody-mediated diseases.
Asunto(s)
Feto/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Integrina beta3/inmunología , Receptores Fc/inmunología , Trombocitopenia Neonatal Aloinmune/inmunología , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos/metabolismo , Plaquetas/inmunología , Regulación hacia Abajo , Femenino , Feto/fisiopatología , Eliminación de Gen , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inmunoglobulinas Intravenosas/uso terapéutico , Integrina beta3/genética , Masculino , Intercambio Materno-Fetal , Ratones , Modelos Animales , Placenta/metabolismo , Embarazo , Transporte de Proteínas , Receptores Fc/genética , Trombocitopenia Neonatal Aloinmune/tratamiento farmacológicoRESUMEN
Immune thrombocytopenia (ITP) is characterized by platelet clearance mediated primarily by autoantibodies against the platelet GPIIbIIIa and/or GPIbα. Steroid therapy is a first-line treatment for ITP. However, some patients are refractory to this therapy and currently no method can predict which patients will respond. To evaluate whether steroids are equally efficacious in treating patients with ITP caused by anti-GPIIbIIIa versus anti-GPIbα antibodies, we performed a retrospective study on 176 newly diagnosed patients with acute ITP who had severe bleeding symptoms and were admitted as resident patients to the hospital. The patients were treated first with intravenous administration of high-dose dexamethasone (DXM), followed by oral administration of prednisone. Response to therapy was observed in a majority of patients with antibodies specific for GPIIbIIIa (31/43) or without detectable antibodies against either GPIIbIIIa or GPIbα (36/45). In contrast, the steroid response was significantly lower in patients with anti-GPIbα antibodies (9/34) or with antibodies against both GPIbα and GPIIbIIIa (16/54). The preliminary findings of this study suggest that in future prospective clinical trials including corticosteroids, the anti-GPIbα, and -GPIIbIIIa status should be assessed in order to test its potential relevance in deciding future treatments.
Asunto(s)
Autoanticuerpos/inmunología , Dexametasona/uso terapéutico , Glucocorticoides/uso terapéutico , Glicoproteínas de Membrana/inmunología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Prednisona/uso terapéutico , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Plaquetas/patología , Dexametasona/farmacología , Esquema de Medicación , Monitoreo de Drogas , Femenino , Glucocorticoides/farmacología , Humanos , Masculino , Persona de Mediana Edad , Complejo GPIb-IX de Glicoproteína Plaquetaria , Prednisona/farmacología , Púrpura Trombocitopénica Idiopática/inmunología , Púrpura Trombocitopénica Idiopática/patología , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Here, we developed a new synthetic method for the production of a new class of polymeric inorganic hybrid biomaterial that has potential for dental implant applications and, in general, other orthopedic applications owing to its excellent mechanical properties and biomechanical compatibility. The new hybrid biomaterial is a composite consisting of polyetherketoneketone (PEKK) and hydroxyapatite (HA). This hybrid material boasts several unique features, including its high HA loading (up to 50 wt%), which is close to that of natural human bone; the homogeneous HA distribution in the PEKK matrix without phase separation; and the fact that the addition of HA has no effect on the molecular weight of PEKK. Nanoindentation analysis was used to investigate the mechanical properties of the composite, and its nano/microstructure variations were investigated through a structural model developed here. Through nanoindentation technology, the newly developed PEKK/HA hybrid biomaterial has an indentation modulus of 12.1 ± 2.5 GPa and a hardness of 0.42 ± 0.09 GPa, which are comparable with those of human bone. Overall, the new PEKK/HA biomaterial exhibits excellent biomechanical compatibility and shows great promise for application to dental and orthopedic devices.
RESUMEN
OBJECTIVE: This study aims to evaluate the effectiveness and safety of hepatic arterial infusion chemotherapy (HAIC) following the simultaneous placement of self-expandable metallic stent (SEMS) and iodine-125 (125I) seed strands for the management of advanced cholangiocarcinoma (CCA) patients presenting with malignant obstructive jaundice (MOJ). METHODS: Data from 74 patients with MOJ caused by advanced CCA treated with stent placement with 125I seed strands with or without HAIC between November 2015 and October 2020 were analysed retrospectively. Eighteen patients received 5 sessions of HAIC after SEMS placement with 125I seed strands (HAIC group), and 56 patients only underwent SEMS placement with 125I seed strands and served as controls (control group). HAIC consisted of infusions of gemcitabine (600-1000 mg/m2 given over 30 min) followed by oxaliplatin (60-100 mg/m2 given over 2 h), with an interval of 4 weeks. Propensity score matching (PSM) analysis was used to adjust for differences in the baseline characteristics of the groups (including age, total bilirubin, and serum alanine aminotransferase level). Overall survival (OS), stent patency, and adverse events were compared between the two groups. RESULTS: OS and stent patency were significantly better in patients in the HAIC group than in those in the control group (median survival time: before PSM, 362 vs. 185 days, p = 0.005; after PSM, 357 vs. 183 days, p = 0.012; median duration of stent patency: before PSM, 294 vs. 156 days, p = 0.001; after PSM, 287 vs. 183 days, p = 0.039). All adverse reactions were controllable by temporary symptomatic treatment. Serious complications and treatment-related deaths were not observed. CONCLUSION: Our preliminary study showed that HAIC following SEMS placement with 125I seed strands is effective and safe for the management of advanced CCA patients presenting with MOJ and could improve stent patency and patient survival.
Asunto(s)
Neoplasias de los Conductos Biliares , Colangiocarcinoma , Ictericia Obstructiva , Stents Metálicos Autoexpandibles , Neoplasias de los Conductos Biliares/complicaciones , Neoplasias de los Conductos Biliares/terapia , Conductos Biliares Intrahepáticos , Colangiocarcinoma/complicaciones , Colangiocarcinoma/terapia , Humanos , Radioisótopos de Yodo , Ictericia Obstructiva/etiología , Ictericia Obstructiva/terapia , Puntaje de Propensión , Estudios Retrospectivos , Stents Metálicos Autoexpandibles/efectos adversos , Resultado del TratamientoRESUMEN
Although eosinophils are important contributors to mucosal immune responses, mechanisms that regulate their accumulation in mucosal-associated lymphoid tissues remain ill-defined. Combining bone marrow chimeras and pharmacological inhibition approaches, here we find that lymphotoxin-beta receptor (LTßR) signaling during the neonatal period is required for the accumulation of eosinophils in the mesenteric lymph nodes (MLN) during an enteric viral infection in adult male and female mice. We demonstrate that MLN stromal cells express genes that are important for eosinophil migration and survival, such as Ccl-11 (eotaxin-1), Ccl7, Ccl9, and Cxcl2, and that expression of most of these genes is downregulated as a consequence of neonatal LTßR blockade. We also find that neonatal LTßR signaling is required for the generation of a rotavirus-specific IgA antibody response in the adult MLN, but eosinophils are dispensable for this response. Collectively, our studies reveal a role for neonatal LTßR signaling in regulating eosinophil numbers in the adult MLN.
Asunto(s)
Eosinófilos , Ganglios Linfáticos , Animales , Femenino , Inmunidad Mucosa , Inmunoglobulina A , Recuento de Leucocitos , Masculino , RatonesRESUMEN
Platelets, small anucleate cells circulating in the blood, are critical mediators in haemostasis and thrombosis. Interestingly, recent studies demonstrated that platelets contain both pro-inflammatory and anti-inflammatory molecules, equipping platelets with immunoregulatory function in both innate and adaptive immunity. In the context of infectious diseases, platelets are involved in early detection of invading microorganisms and are actively recruited to sites of infection. Platelets exert their effects on microbial pathogens either by direct binding to eliminate or restrict dissemination, or by shaping the subsequent host immune response. Reciprocally, many invading microbial pathogens can directly or indirectly target host platelets, altering platelet count or/and function. In addition, microbial pathogens can impact the host auto- and alloimmune responses to platelet antigens in several immune-mediated diseases, such as immune thrombocytopenia, and fetal and neonatal alloimmune thrombocytopenia. In this review, we discuss the mechanisms that contribute to the bidirectional interactions between platelets and various microbial pathogens, and how these interactions hold relevant implications in the pathogenesis of many infectious diseases. The knowledge obtained from "well-studied" microbes may also help us understand the pathogenesis of emerging microbes, such as SARS-CoV-2 coronavirus.
Asunto(s)
Betacoronavirus , Plaquetas/inmunología , Plaquetas/metabolismo , Infecciones por Coronavirus/inmunología , Interacciones Huésped-Patógeno/inmunología , Neumonía Viral/inmunología , Inmunidad Adaptativa , COVID-19 , Infecciones por Coronavirus/virología , Hemostasis , Humanos , Inmunidad Innata , Inflamación/inmunología , Pandemias , Neumonía Viral/virología , SARS-CoV-2 , Trombosis/metabolismoRESUMEN
Redundant mechanisms support immunoglobulin A (IgA) responses to intestinal antigens. These include multiple priming sites [mesenteric lymph nodes (MLNs), Peyer's patches, and isolated lymphoid follicles] and various cytokines that promote class switch to IgA, even in the absence of T cells. Despite these backup mechanisms, vaccination against enteric pathogens such as rotavirus has limited success in some populations. Genetic and environmental signals experienced during early life are known to influence mucosal immunity, yet the mechanisms for how these exposures operate remain unclear. Here, we used rotavirus infection to follow antigen-specific IgA responses through time and in different gut compartments. Using genetic and pharmacological approaches, we tested the role of the lymphotoxin (LT) pathway-known to support IgA responses-at different developmental stages. We found that LT-ß receptor (LTßR) signaling in early life programs intestinal IgA responses in adulthood by affecting antibody class switch recombination to IgA and subsequent generation of IgA antibody-secreting cells within an intact MLN. In addition, early-life LTßR signaling dictates the phenotype and function of MLN stromal cells to support IgA responses in the adult. Collectively, our studies uncover new mechanistic insights into how early-life LTßR signaling affects mucosal immune responses during adulthood.
Asunto(s)
Inmunoglobulina A/inmunología , Ganglios Linfáticos/inmunología , Receptor beta de Linfotoxina/inmunología , Linfotoxina-alfa/inmunología , Mesenterio/inmunología , Células del Estroma/inmunología , Animales , Heces/microbiología , Femenino , Inmunidad Mucosa , Ganglios Linfáticos/citología , Receptor beta de Linfotoxina/genética , Linfotoxina-alfa/genética , Masculino , Mesenterio/citología , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
OBJECTIVE: To investigate the mechanism of paeoniflorin in preventing hepatic granuloma formation and fibrosis in mice infected with Schistosoma japonicum. METHODS: Model of hepatic granuloma and fibrosis was established by infecting mice with S. japonicum cercariae. The infected mice were randomly divided into 4 groups: group A as model (infected control) group (15 mice), and paeoniflorin being given before, simultaneously and after praziquantel treatment as groups B, C and D. Each of the groups B, C and D was subdivided into 3 subgroups (15 mice each): low dose (paeoniflorin 2 ml, 30 mg/(kg x d) x 30 d), high dose(paeoniflorin 2 ml, 120 mg/(kg x d) x 30 d) and control (2 ml, 0.5% sodium carboxymethylcellulose x 30 d). In group B, paeoniflorin or sodium carboxymethylcellulose was orally administrated on 12 d after infection. In groups C and D, paeoniflorin or sodium carboxymethylcellulose was administrated on 42 d or 72 d after infection. Each of group B, C and D was orally given praziquantel 2 ml (500 mg/(kg x d) x 2 d) on 42 d after infection. On the 102nd day after infection, all animals were sacrificed by cervical dislocation. Serum hyaluronic acid (HA) was detected by radioimmunoassay; area of egg granuloma and degree of hepatic fibrosis were observed via HE and Masson stainings; the expression of transforming growth factor beta1 (TGF-beta1), alpha smooth muscle actin (alpha-SMA and collagen I (Col I) protein were measured by immunohistochemical method. RESULTS: In group B, the level of HA (0.719 +/- 0.239 microg/ml, 0.721 +/- 0.182 microg/ml) in low or high dose subgroups was significantly lower (F = 9.429, P < 0.01) than the control subgroup (1.049 +/- 0.286 microg/ml); the area of granuloma (0.066 +/- 0.005 mm2, 0.064 +/- 0.004 mm2) or the degree of hepatic fibrosis (2.067 +/- 0.458, 1.967 +/- 0.399) in low or high dose subgroups was significantly greater (F = 862.540, F = 29.738, P < 0.01) than the control (0.141 +/- 0.008 mm2, 3.467 +/- 0.834); the expression of alpha-SMA positive cells (2.933 +/- 0.594, 3.000 +/- 0.535) in low or high dose subgroups was significantly lower (F = 12.323, P < 0.01, P < 0.01) than its control (4.800 +/- 1.859); the expression of TGF-beta1 (0.256 +/- 0.057, 0.274 +/- 0.054) in low or high dose subgroups was significantly lower (F = 148.990, P < 0.01) than its control (0.552 +/- 0.047); the content of Col I (0.334 +/- 0.041, 0.339 +/- 0.042) in low or high dose subgroups was significantly lower (F = 180.881, P < 0.01) than its control (0.601 +/- 0.049). In groups C & D, no significant difference was found between the low or high dose subgroups or between the subgroups and their corresponding controls. CONCLUSION: Paeoniflorin can significantly reduce hepatic granuloma formation and fibrosis due to schistosome eggs, and decrease the expression of TGF-beta1, alpha-SMA in mice when it is given before praziquantel administration, which may associate with the activation of hepatic stellate cells and the expression of TGF-beta1 in liver tissue.
Asunto(s)
Benzoatos/uso terapéutico , Hidrocarburos Aromáticos con Puentes/uso terapéutico , Glucósidos/uso terapéutico , Schistosoma japonicum/efectos de los fármacos , Esquistosomiasis Japónica/tratamiento farmacológico , Actinina/biosíntesis , Animales , Benzoatos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Glucósidos/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/parasitología , Masculino , Ratones , Ratones Endogámicos , Monoterpenos , Fitoterapia , Esquistosomiasis Japónica/inmunología , Esquistosomiasis Japónica/patología , Factor de Crecimiento Transformador beta1/biosíntesis , Resultado del TratamientoRESUMEN
OBJECTIVE: To explore the effect of paeoniflorin (PAE) on the production of transforming growth factor beta1 (TGF-beta1) from peritoneal macrophages(PMs) stimulated by soluble egg antigen (SEA) of Schistosoma japonicum. METHODS: SEA was prepared by trituration and added into culture plank, flask and dish containing PMs which were cultured for 24 h. TGF-beta1 secreted from PMs was measured by ELISA. TGF-beta1 mRNA and protein produced from PMs were evaluated by RT-PCR and Western blotting, respectively. SEA (10 mg/L) 5 ml was added into culture flask and dish containing PMs. PMs were cultured for 12 h, and PAE at different concentrations (0, 7.5, 15, 30, 60, 120 mg/L) was added into the culture flask and dish, and PMs were cultured consecutively for another 12 h and 24 h, respectively. TGF-beta1 mRNA and protein from PMs stimulated by SEA were evaluated by RT-PCR and Western blotting, respectively. RESULTS: TGF-beta1 (235.86 +/- 3.43 ng/L) was produced from PMs under stimulation of SEA at 10 mg/L, and the expression of TGF-beta1 mRNA and protein in PMs were depressed significantly by PAE in a concentration-dependent manner (r = -0.827, P < 0.01; r = -0.952, P < 0.01, respectively). CONCLUSION: PAE inhibits the production of TGF-beta1 from PMs stimulated by SEA.
Asunto(s)
Glucósidos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Monoterpenos/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Células Cultivadas , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos , Schistosoma japonicumRESUMEN
Class switch recombination (CSR) has a fundamental function during humoral immune response and involves the induction and subsequent repair of DNA breaks in the immunoglobulin (Ig) switch regions. Here we show the role of Usp22, the SAGA complex deubiquitinase that removes ubiquitin from H2B-K120, in the repair of programmed DNA breaks in vivo. Ablation of Usp22 in primary B cells results in defects in γH2AX and impairs the classical non-homologous end joining (c-NHEJ), affecting both V(D)J recombination and CSR. Surprisingly, Usp22 depletion causes defects in CSR to various Ig isotypes, but not IgA. We further demonstrate that IgG CSR primarily relies on c-NHEJ, whereas CSR to IgA is more reliant on the alternative end joining pathway, indicating that CSR to different isotypes involves distinct DNA repair pathways. Hence, Usp22 is the first deubiquitinase reported to regulate both V(D)J recombination and CSR in vivo by facilitating c-NHEJ.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Enzimas Desubicuitinizantes/metabolismo , Endopeptidasas/metabolismo , Inmunidad Humoral/genética , Cambio de Clase de Inmunoglobulina , Recombinación V(D)J , Animales , Linfocitos B , Enzimas Desubicuitinizantes/genética , Endopeptidasas/genética , Femenino , Histonas/genética , Histonas/metabolismo , Isotipos de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cultivo Primario de Células , Ubiquitina/metabolismo , Ubiquitina TiolesterasaRESUMEN
Immune protection of the body cavities depends on the swift activation of innate and adaptive immune responses in nonclassical secondary lymphoid organs known as fat-associated lymphoid clusters (FALCs). Compared with classical secondary lymphoid organs such as lymph nodes and Peyer's patches, FALCs develop along distinct differentiation trajectories and display a reduced structural complexity. Although it is well established that fibroblastic reticular cells (FRCs) are an integral component of the immune-stimulating infrastructure of classical secondary lymphoid organs, the role of FRCs in FALC-dependent peritoneal immunity remains unclear. Using FRC-specific gene targeting, we found that FRCs play an essential role in FALC-driven immune responses. Specifically, we report that initiation of peritoneal immunity was governed through FRC activation in a myeloid differentiation primary response 88 (MYD88)-dependent manner. FRC-specific ablation of MYD88 blocked recruitment of inflammatory monocytes into FALCs and subsequent CD4+ T cell-dependent B-cell activation and IgG class switching. Moreover, containment of Salmonella infection was compromised in mice lacking MYD88 expression in FRCs, indicating that FRCs in FALCs function as an initial checkpoint in the orchestration of protective immune responses in the peritoneal cavity.
Asunto(s)
Fibroblastos/citología , Fibroblastos/inmunología , Grasa Intraabdominal/inmunología , Cavidad Peritoneal/fisiología , Animales , Quimiocina CCL2/inmunología , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVE: To express signaling protein Sj14-3-3 in Pichia pastoris and compare its antigenicity with prokaryotic expression one. METHODS: Sj14-3-3 gene was amplified from pET28a-Sj14-3-3 recombinant plasmid, cloned into vector pMD18-T followed by sequencing. The Sj14-3-3 gene was subcloned into the expression vector pPICZalpha-B and transformed into Pichia pastoris X-33 by electroporation. The transformants were identified by sequencing. Three transformants with high copies were obtained when selected under zeocin, and expression was induced with methanol. The culture supernatant was collected and tested by SDS-PAGE and Western blotting. The specificity and sensitivity of eukaryotic expression rSj14-3-3 in Pichia pastoris were compared with that from prokaryotic expression by detecting sera of patients with schistosomiasis by indirect ELISA. RESULTS: The Sj14-3-3 gene was integrated into Pichia pastoris, and the gene of interest detected by PCR was with 1 300 bp. After induction by methanol, the Sj14-3-3 gene was expressed and secreted into the medium. The molecular weight of the recombinant protein was determined as about Mr 35 000 by SDS-PAGE. Western blotting showed that the protein has a high specificity against mouse-anti-Sjl4-3-3 monoclonal antibody. The recombinant protein had a promising immune reactivity. Indirect ELISA showed that by using eukaryotic expression rSj14-3-3 in Pichia pastoris, the positive rate in 36 cases of acute schistosomiasis was 81%, with no cross-reactivity in 12 cases of Clonorchis sinensis, 9.3% cross-reactivity in 32 cases of normal sera. While using prokaryotic expression rSj14-3-3 in E.coli, the positive rate in 36 cases of acute schistosomiasis was 88.9%, with 16.7% cross-reactivity in 12 cases of Clonorchis sinensis, 12.5% cross-reactivity in 32 cases of normal sera. There was no statistically significant difference of the results (P>0.05). CONCLUSION: The recombinant protein Sj14-3-3 of eukaryotic expression in Pichia pastoris has been successfully harvested and shows a promising immunological potential.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas del Helminto/metabolismo , Schistosoma japonicum/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Humanos , Inmunoglobulina G/sangre , Pichia/genética , Reacción en Cadena de la Polimerasa , Schistosoma japonicum/genética , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica/sangreRESUMEN
The lymphotoxin signaling pathway plays an important role in the homeostasis and function of peripheral and mucosal dendritic cells, and dendritic cell-intrinsic lymphotoxin ß receptor expression is required for optimal responses to opportunistic intestinal bacteria. However, it is unknown whether dendritic cell-intrinsic lymphotoxin ß receptor signaling is required for responses to intestinal viral infections. We explored this question by orally administrating murine rotavirus to chimeric mice that lack lymphotoxin ß receptor signaling in the myeloid compartment but retain lymphoid tissues. We found that although clearance of rotavirus was unimpaired in the lymphotoxin ß receptor(-/-) â wild-type chimeric mice compared with wild-type â wild-type chimeric mice, IFN-γ-producing CD8(+) and CD4(+) T cells were significantly increased in the small intestinal lamina propria of lymphotoxin ß receptor(-/-) â wild-type chimeric mice. In contrast, IL-17-producing CD4(+) T cells were reduced in lymphotoxin ß receptor(-/-) â wild-type chimeric mice in the steady state, and this reduction persisted after rotavirus inoculation. In spite of this altered cytokine profile in the small intestinal lamina propria of lymphotoxin ß receptor(-/-) â wild-type chimeric mice, the local production of rotavirus-specific IgA was unperturbed. Collectively, our results demonstrate that lymphotoxin ß receptor signaling in radio-sensitive myeloid cells regulates the balance of IFN-γ and IL-17 cytokine production within the small intestinal lamina propria; however, these perturbations do not affect mucosal antiviral IgA responses.