A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses.

Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17ß-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyer's patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-ß, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.

B Cell Endosomal RAB7 Promotes TRAF6 K63 Polyubiquitination and NF-κB Activation for Antibody Class-Switching.

Upon activation by CD40 or TLR signaling, B lymphocytes activate NF-κB to induce activation-induced cytidine deaminase and, therefore, Ig class switch DNA recombination, as central to the maturation of the Ab and autoantibody responses. In this study, we show that NF-κB activation is boosted by colocalization of engaged immune receptors, such as CD40, with RAB7 small GTPase on mature endosomes, in addition to signals emanating from the receptors localized on the plasma membrane, in mouse B cells. In mature endosomes, RAB7 directly interacts with TRAF6 E3 ubiquitin ligase, which catalyzes K63 polyubiquitination for NF-κB activation. RAB7 overexpression in Cd19+/creRosa26fl-STOP-fl-Rab7 mouse B cells upregulates K63 polyubiquitination activity of TRAF6, enhances NF-κB activation and activation-induced cytidine deaminase induction, and boosts IgG Ab and autoantibody levels. This, together with the extensive intracellular localization of CD40 and the strong correlation of RAB7 expression with NF-κB activation in mouse lupus B cells, shows that RAB7 is an integral component of the B cell NF-κB activation machinery, likely through interaction with TRAF6 for the assembly of "intracellular membrane signalosomes."

HoxC4 binds to the promoter of the cytidine deaminase AID gene to induce AID expression, class-switch DNA recombination and somatic hypermutation.

The cytidine deaminase AID (encoded by Aicda in mice and AICDA in humans) is critical for immunoglobulin class-switch recombination (CSR) and somatic hypermutation (SHM). Here we show that AID expression was induced by the HoxC4 homeodomain transcription factor, which bound to a highly conserved HoxC4-Oct site in the Aicda or AICDA promoter. This site functioned in synergy with a conserved binding site for the transcription factors Sp1, Sp3 and NF-kappaB. HoxC4 was 'preferentially' expressed in germinal center B cells and was upregulated by engagement of CD40 by CD154, as well as by lipopolysaccharide and interleukin 4. HoxC4 deficiency resulted in impaired CSR and SHM because of lower AID expression and not some other putative HoxC4-dependent activity. Enforced expression of AID in Hoxc4(-/-) B cells fully restored CSR. Thus, HoxC4 directly activates the Aicda promoter, thereby inducing AID expression, CSR and SHM.

Small Molecule Inhibition of Rab7 Impairs B Cell Class Switching and Plasma Cell Survival To Dampen the Autoantibody Response in Murine Lupus.

IgG autoantibodies mediate pathology in systemic lupus patients and lupus-prone mice. In this study, we showed that the class-switched IgG autoantibody response in MRL/Faslpr/lpr and C57/Sle1Sle2Sle2 mice was blocked by the CID 1067700 compound, which specifically targeted Ras-related in brain 7 (Rab7), an endosome-localized small GTPase that was upregulated in activated human and mouse lupus B cells, leading to prevention of disease development and extension of lifespan. These were associated with decreased IgG-expressing B cells and plasma cells, but unchanged numbers and functions of myeloid cells and T cells. The Rab7 inhibitor suppressed T cell-dependent and T cell-independent Ab responses, but it did not affect T cell-mediated clearance of Chlamydia infection, consistent with a B cell-specific role of Rab7. Indeed, B cells and plasma cells were inherently sensitive to Rab7 gene knockout or Rab7 activity inhibition in class switching and survival, respectively, whereas proliferation/survival of B cells and generation of plasma cells were not affected. Impairment of NF-κB activation upon Rab7 inhibition, together with the rescue of B cell class switching and plasma cell survival by enforced NF-κB activation, indicated that Rab7 mediates these processes by promoting NF-κB activation, likely through signal transduction on intracellular membrane structures. Thus, a single Rab7-inhibiting small molecule can target two stages of B cell differentiation to dampen the pathogenic autoantibody response in lupus.

Epigenetics of the antibody response.

Epigenetic marks, such as DNA methylation, histone post-translational modifications and miRNAs, are induced in B cells by the same stimuli that drive the antibody response. They play major roles in regulating somatic hypermutation (SHM), class switch DNA recombination (CSR), and differentiation to plasma cells or long-lived memory B cells. Histone modifications target the CSR and, possibly, SHM machinery to the immunoglobulin locus; they together with DNA methylation and miRNAs modulate the expression of critical elements of that machinery, such as activation-induced cytidine deaminase (AID), as well as factors central to plasma cell differentiation, such as B lymphocyte-induced maturation protein-1 (Blimp-1). These inducible B cell-intrinsic epigenetic marks instruct the maturation of antibody responses. Their dysregulation plays an important role in aberrant antibody responses to foreign antigens, such as those of microbial pathogens, and self-antigens, such as those targeted in autoimmunity, and B cell neoplasia.

Histone deacetylase inhibitors upregulate B cell microRNAs that silence AID and Blimp-1 expression for epigenetic modulation of antibody and autoantibody responses.

Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require activation-induced cytidine deaminase (AID), and plasma cell differentiation, which requires B lymphocyte-induced maturation protein-1 (Blimp-1), are critical for the generation of class-switched and hypermutated (mature) Ab and autoantibody responses. We show that histone deacetylase inhibitors valproic acid and butyrate dampened AICDA/Aicda (AID) and PRDM1/Prdm1 (Blimp-1) mRNAs by upregulating miR-155, miR-181b, and miR-361 to silence AICDA/Aicda, and miR-23b, miR-30a, and miR-125b to silence PRDM1/Prdm1, in human and mouse B cells. This led to downregulation of AID, Blimp-1, and X-box binding protein 1, thereby inhibiting CSR, SHM, and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of histone deacetylase inhibitor-mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important inducers/modulators of AICDA/Aicda), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2, or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a, and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell-intrinsic epigenetic mechanisms, valproic acid blunted class-switched and hypermutated T-dependent and T-independent Ab responses in C57BL/6 mice. In addition, it decreased class-switched and hypermutated autoantibodies, ameliorated disease, and extended survival in lupus MRL/Fas(lpr/lpr) mice. Our findings outline epigenetic mechanisms that modulate expression of an enzyme (AID) and transcription factors (Blimp-1 and X-box binding protein 1) that are critical to the B cell differentiation processes that underpin Ab and autoantibody responses. They also provide therapeutic proof-of-principle in autoantibody-mediated autoimmunity.

Expression of Derlin-1 and its effect on expression of autophagy marker genes under endoplasmic reticulum stress in lung cancer cells.

BACKGROUND: Recent findings indicated that Derlin-1 has an important function in tumour progression. In this study, we aimed to determine whether Derlin-1 has an oncogene function as a cross-talk molecule with autophagy. METHODS: Cancer cells were treated with tunicamycin (TM) for 8 and 24 h. The expression of Derlin-1 and autophagy-related genes was determined by western blot. Autophagy was analysed by fluorescence microscopy after staining the cancer cells with monodansylcadaverine. The interaction between Derlin-1 and other proteins was identified using co-immunoprecipitation assay. RESULTS: Our study demonstrated high Derlin-1 expression levels in most non-small lung cancer cell lines. Derlin-1 expression was enhanced under endoplasmic reticulum (ER) stress. Previous studies revealed that TM triggers the initiation of autophagy by activating Beclin 1, converting LC3I to LC3II and degrading p62. Knockdown of Derlin-1 did not affect Beclin 1 and LC3II expression but disrupted the degradation of p62 under ER stress, which resulted in the blockage of autophagy flux. Furthermore, Derlin-1 and p62 were observed to interact under ER stress. CONCLUSION: This study is the first report about the interaction between Derlin-1 and p62. Derlin-1 may function in tumour progression partially by interacting with p62.

Intrinsic B cell TLR-BCR linked coengagement induces class-switched, hypermutated, neutralizing antibody responses in absence of T cells.

Maturation of antibody responses entails somatic hypermutation (SHM), class-switch DNA recombination (CSR), plasma cell differentiation, and generation of memory B cells, and it is thought to require T cell help. We showed that B cell Toll-like receptor 4 (TLR4)-B cell receptor (BCR) (receptor for antigen) coengagement by 4-hydroxy-3-nitrophenyl acetyl (NP)-lipopolysaccharide (LPS) (Escherichia coli lipid A polysaccharide O-antigen) or TLR5-BCR coengagement by Salmonella flagellin induces mature antibody responses to NP and flagellin in Tcrß-/-Tcrδ-/- and NSG/B mice. TLR-BCR coengagement required linkage of TLR and BCR ligands, "linked coengagement." This induced B cell CSR/SHM, germinal center-like differentiation, clonal expansion, intraconal diversification, plasma cell differentiation, and an anamnestic antibody response. In Tcrß-/-Tcrδ-/- mice, linked coengagement of TLR4-BCR by LPS or TLR5-BCR by flagellin induced protective antibodies against E. coli or Salmonella Typhimurium. Our findings unveiled a critical role of B cell TLRs in inducing neutralizing antibody responses, including those to microbial pathogens, without T cell help.

Inhibition of autophagy-potentiated chemosensitivity to cisplatin in laryngeal cancer Hep-2 cells.

OBJECTIVE: The purposes of this study were to determine whether autophagy was involved in cisplatin (CDDP) resistance and to investigate the role of the autophagy in the regulation of chemosensitivity to CDDP in laryngeal cancer Hep-2 cells. METHODS: A WST-1 assay was performed to determine cell viability and cell proliferation. Autophagy activation and proapoptotic effects were characterized using monodansylcadaverine labeling and Hoechest staining, respectively. Western blot analysis was used to detect the expression of apoptotic and autophagy-related genes. Flow cytometry was used to assess cell apoptosis ratio. RESULTS: Exposure to CDDP induced the aggregation of autophagosomes in the cytoplasms of Hep-2 cells and up-regulated the expression of Beclin 1 and LC3II. However, CDDP treatment could not lead to obvious inhibition of cell proliferation, which implies that the autophagy may protect CDDP-treated cells from undergoing cell death. Meanwhile, the WST-1 assay indicated that knockdown of the autophagic gene Beclin 1 sensitized Hep-2 cells to CDDP. Furthermore, CDDP-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitor 3-methyladenine or small interfering RNA against Beclin 1. For the definite mechanism of Beclin 1-enhancing chemosensitivity to CDDP, we found that Beclin1 augmented CDDP-induced apoptotic signaling via enhancing caspase-9 and caspase-3 activity but not caspase-8. CONCLUSION: Our results suggest that functional autophagy in response to CDDP may lead to cell survival in Hep-2 cells, whereas defective autophagy may contribute to CDDP-induced apoptosis in Hep-2 cells. Thus, modulators of autophagy may be used beneficially as adjunctive therapeutic agents during the treatment of laryngeal cancer with CDDP therapy.

Rad52 mediates class-switch DNA recombination to IgD.

In B cells, IgD is expressed together with IgM through alternative splicing of primary VHDJH-Cµ-s-m-Cδ-s-m RNAs, and also through IgD class switch DNA recombination (CSR) via double-strand DNA breaks (DSB) and synapse of Sµ with σδ. How such DSBs are resolved is still unknown, despite our previous report showing that Rad52 effects the 'short-range' microhomology-mediated synapsis of intra-Sµ region DSBs. Here we find that induction of IgD CSR downregulates Zfp318, and promotes Rad52 phosphorylation and recruitment to Sµ and σδ, thereby leading to alternative end-joining (A-EJ)-mediated Sµ-σδ recombination with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52-/- B cells aborts IgD CSR in vitro and in vivo and dampens the specific IgD antibody response to OVA. Rad52 knockdown in human B cells also abrogates IgD CSR. Finally, Rad52 phosphorylation is associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in patients with systemic lupus erythematosus and in lupus-prone mice. Our findings thus show that Rad52 mediates IgD CSR through microhomology-mediated A-EJ in concert with Zfp318 downregulation.

Cross-Activation of Hemichannels/Gap Junctions and Immunoglobulin-Like Domains in Innate-Adaptive Immune Responses.

Hemichannels (HCs)/gap junctions (GJs) and immunoglobulin (Ig)-like domain-containing proteins (IGLDCPs) are involved in the innate-adaptive immune response independently. Despite of available evidence demonstrating the importance of HCs/GJs and IGLDCPs in initiating, implementing, and terminating the entire immune response, our understanding of their mutual interactions in immunological function remains rudimentary. IGLDCPs include immune checkpoint molecules of the immunoglobulin family expressed in T and B lymphocytes, most of which are cluster of differentiation (CD) antigens. They also constitute the principal components of the immunological synapse (IS), which is formed on the cell surface, including the phagocytic synapse, T cell synapse, B cell synapse, and astrocytes-neuronal synapse. During the three stages of the immune response, namely innate immunity, innate-adaptive immunity, and adaptive immunity, HCs/GJs and IGLDCPs are cross-activated during the entire process. The present review summarizes the current understanding of HC-released immune signaling factors that influence IGLDCPs in regulating innate-adaptive immunity. ATP-induced "eat me" signals released by HCs, as well as CD31, CD47, and CD46 "don't eat me" signaling molecules, trigger initiation of innate immunity, which serves to regulate phagocytosis. Additionally, HC-mediated trogocytosis promotes antigen presentation and amplification. Importantly, HC-mediated CD4+ T lymphocyte activation is critical in the transition of the innate immune response to adaptive immunity. HCs also mediate non-specific transcytosis of antibodies produced by mature B lymphocytes, for instance, IgA transcytosis in ovarian cancer cells, which triggers innate immunity. Further understanding of the interplay between HCs/GJs and IGLDCPs would aid in identifying therapeutic targets that regulate the HC-Ig-like domain immune response, thereby providing a viable treatment strategy for immunological diseases. The present review delineates the clinical immunology-related applications of HC-Ig-like domain cross-activation, which would greatly benefit medical professionals and immunological researchers alike. HCs/GJs and IGLDCPs mediate phagocytosis via ATP; "eat me and don't eat me" signals trigger innate immunity; HC-mediated trogocytosis promotes antigen presentation and amplification in innate-adaptive immunity; HCs also mediate non-specific transcytosis of antibodies produced by mature B lymphocytes in adaptive immunity.

Estrogen receptors bind to and activate the HOXC4/HoxC4 promoter to potentiate HoxC4-mediated activation-induced cytosine deaminase induction, immunoglobulin class switch DNA recombination, and somatic hypermutation.

Estrogen enhances antibody and autoantibody responses through yet to be defined mechanisms. It has been suggested that estrogen up-regulates the expression of activation-induced cytosine deaminase (AID), which is critical for antibody class switch DNA recombination (CSR) and somatic hypermutation (SHM), through direct activation of this gene. AID, as we have shown, is induced by the HoxC4 homeodomain transcription factor, which binds to a conserved HoxC4/Oct site in the AICDA/Aicda promoter. Here we show that estrogen-estrogen receptor (ER) complexes do not directly activate the AID gene promoter in B cells undergoing CSR. Rather, they bind to three evolutionarily conserved and cooperative estrogen response elements (EREs) we identified in the HOXC4/HoxC4 promoter. By binding to these EREs, ERs synergized with CD154 or LPS and IL-4 signaling to up-regulate HoxC4 expression, thereby inducing AID and CSR without affecting B cell proliferation or plasmacytoid differentiation. Estrogen administration in vivo significantly potentiated CSR and SHM in the specific antibody response to the 4-hydroxy-3-nitrophenylacetyl hapten conjugated with chicken γ-globulin. Ablation of HoxC4 (HoxC4(-/-)) abrogated the estrogen-mediated enhancement of AID gene expression and decreased CSR and SHM. Thus, estrogen enhances AID expression by activating the HOXC4/HoxC4 promoter and inducing the critical AID gene activator, HoxC4.

Toll-like receptors and B-cell receptors synergize to induce immunoglobulin class-switch DNA recombination: relevance to microbial antibody responses.

Differentiation of naïve B cells, including immunoglobulin class-switch DNA recombination, is critical for the immune response and depends on the extensive integration of signals from the B-cell receptor (BCR), tumor necrosis factor (TNF) family members, Toll-like receptors (TLRs), and cytokine receptors. TLRs and BCR synergize to induce class-switch DNA recombination in T cell-dependent and T cell-independent antibody responses to microbial pathogens. BCR triggering together with simultaneous endosomal TLR engagement leads to enhanced B-cell differentiation and antibody responses. Te requirement of both BCR and TLR engagement would ensure appropriate antigen-specific activation in an infection. Co-stimulation of TLRs and BCR likely plays a significant role in anti-microbial antibody responses to contain pathogen loads until the T cell-dependent antibody responses peak. Furthermore, the temporal sequence of different signals is also critical for optimal B cell responses, as exemplified by the activation of B cells by initial TLR engagement, leading to the up-regulation of co-stimulatory CD80 and MCH-II receptors, which result in more efficient interactions with T cells, thereby enhancing the germinal center reaction and antibody affinity maturation. Overall, BCR and TLR stimulation and the integration with signals from the pathogen or immune cells and their products determine the ensuing B-cell antibody response.

Clinicopathologic correlations between human papillomavirus 16 infection and Beclin 1 expression in human cervical cancer.

Our earlier study showed that the autophagy gene Beclin 1 could affect cell proliferation in a cervical cancer HeLa cell line. In this study, we examined Beclin 1 protein expression in 81 specimens of cervical squamous carcinoma by immunohistochemistry. Meanwhile, we detected E6 and E7 genes of human papillomavirus 16 in these tissues by polymerase chain reaction. Beclin 1 expression significantly decreased in samples of malignant cervical cancer tissues than in those of normal or cervical intraepithelial neoplasia tissues. The expression of Beclin 1 was associated with pelvic lymph node metastasis and histological grade, but did not correlate with International Federation of Gynecology and Obstetrics stage, age, depth of cervical infiltration, tumor size, and gross type of cervical lesion. The expression of Beclin 1 was not obviously correlated with E6 and E7 genes statistically. Therefore, decreased expression of Beclin 1 may be related to tumorigenesis and the development of cervical cancer, but is not significantly relevant with human papillomavirus 16 infection.

Radical trachelectomy versus radical hysterectomy for the treatment of early cervical cancer: a systematic review.

OBJECTIVE: To assess the efficacy and safety of radical trachelectomy (RT) and radical hysterectomy (RH) for patients with early cervical cancer. DESIGN: Systematic review with meta-analysis. POPULATION: Women who had early cervical cancer. METHODS: Prospective controlled clinical trials comparing RT with RH were identified using a predefined search strategy. Recurrence, five-year recurrence-free survival rate, five-year overall survival rate, postoperative mortality, intraoperative and postoperative complications between the two operations were compared by using the methods provided by the Cochrane Handbook for Systematic Reviews of Interventions. RESULTS: Three controlled clinical trials involving 587 participants were included. Meta-analysis showed that there was no significant difference between the two groups in recurrence rate [1.38; 95% confidence interval (CI) 0.58-3.28, p=0.47], five-year recurrence-free survival rate (1.17; 95% CI 0.54-2.53, p=0.69), five-year overall survival rate (0.86; 95% CI 0.30-2.43, p=0.78), postoperative mortality (1.14; 95% CI 0.42-3.11, p=0.80), intraoperative complications (1.66; 95% CI 0.11-25.28, p=0.72), postoperative complications (0.52; 95% CI 0.11-2.48, p=0.41), blood transfusion (0.29; 95% CI 0.06-1.36, p=0.12) and number of harvested lymph nodes. However, RT, compared with RH, reduced blood loss and shortened duration to normal urine residual volume and postoperative hospital stay. Moreover, RT may achieve to normal conception rates, while RH makes patients sterile. CONCLUSIONS: Radical trachelectomy has similar efficacy and safety to RH as the surgical treatment for early cervical cancer. Moreover, it reduced blood loss and shortened the duration to normal urine residual volumes and postoperative hospital stay. Radical trachelectomy can be used to treat early stage cervical cancer as an alternative operation for patients who wish to preserve fertility.

[Effect of autophagy gene Beclin 1 on the growth of cervical cancer HeLa cells in vitro and vivo].

OBJECTIVE: To investigate the effects of autophagy gene Beclin 1 on growth of cervical cancer HeLa cells in vitro and vivo. METHODS: The eukaryotic expression vector of Beclin1 was constructed and transfected via lipofectamine into HeLa cells. The experimental cells were classified into 3 groups: pcDNA3.1(+)-Beclin1 group,pcDNA3.1(+) group and HeLa group. Real time-ploymerase chain reaction and Western blot were used for detecting expression of Beclin1 mRNA and protein in the transfected cells. Flow cytometry (FCM) was employed to observe the effect of transfection on the apoptosis of HeLa cells, and proliferation was analyzed by MTT assay. The formation of autophagic vacuoles was measured by MDC staining. HeLa cells transfected with plasmid pcDNA3.1(+)-Beclin1 and pcDNA3.1(+) were inoculated subcutaneously in nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed. RESULTS: Eukaryotic expression vector pcDNA3.1(+)-Beclin1 was constructed successfully. It significantly improved the expression of Beclin1 mRNA and protein in HeLa cells. The proliferation of HeLa cells was inhibited, and the inhibition rate was 58.7%. FCM investigation showed that the apoptotic rate was (28.22 ± 2.34)% of pcDNA3.1(+)-Beclin1 group, significantly higher than the (14.6 ± 4.6)% in the pcDNA3.1(+) group and (11.2 ± 3.0)% in the HeLa group (P < 0.05). The monodansylcadaverin (MDC) staining showed significantly more autophagic vacuoles in the pcDNA3.1(+)-Beclin1 group (10.9%) than that in the pcDNA3.1(+) group (3.1%) and HeLa group (2.5%) (P < 0.05). After transfected with vector pcDNA3.1(+)-Beclin1, the carcinogenic activity of HeLa cells was decreased in nude mice, and the inhibition rate of tumor growth was 52.2%. CONCLUSIONS: Autophagy gene Beclin 1 overexpression can inhibit the proliferation and growth of HeLa cells in vitro and vivo,while promote autophagy and apoptosis of HeLa cells. So it might be one of new gene therapy strategies for cervical carcinoma.

[Effect and mechanism of an autophagy gene beclin 1 on cervical cancer HeLa cells].

OBJECTIVE: To investigate the inhibitory effects and the mechanism of autophagy gene beclin 1 on cervical cancer HeLa cells. METHODS: The eukaryotic expression vector and short hairpin RNA (shRNA) expression vector of beclin 1 were transfected via lipofectamine into HeLa cells. Experimental cells were classified into 5 groups: pcDNA3.1(+)-beclin 1 group, pSUPER-beclin 1 group, pcDNA3.1(+) group, pSUPER group and HeLa group. Real time-PCR and western blot were used for detecting expression of mRNA and protein of beclin 1 and caspase-9 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis, and autophagy of HeLa, while proliferation was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The ultrastructural analysis of autophagic vacuoles was under the electron microscope. Five groups cells were seeded subcutaneously on nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed, and immunohistochemistry was used to detect the protein expression of beclin 1 in tumor tissue. RESULTS: (1) The mRNA expression of beclin 1 and caspase-9: pcDNA3.1(+)-beclin 1 group were 994.72 ± 468.76 and 12.88 ± 2.71, pSUPER-beclin 1 group were 0.18 ± 0.63 and 0.11 ± 0.08, pcDNA3.1(+) group were 0.57 ± 0.12 and 4.28 ± 3.25, pSUPER group were 0.67 ± 0.29 and 2.77 ± 1.27, and HeLa group were 0.74 ± 0.25 and 3.67 ± 3.78, respectively. The eukaryotic expression vector pcDNA3.1(+)-beclin 1 significantly improved the expression of mRNA of beclin 1 and caspase-9 in HeLa cells (P < 0.05), and the shRNA expression vector inhibited the expression of mRNA of beclin 1 and caspase-9(P < 0.05). (2) The cell proliferations: pcDNA3.1(+)-beclin 1 vector significantly inhibited the growth of HeLa cells, while pSUPER-beclin 1 vector significantly improved the growth of HeLa cells (P < 0.05). (3) The rate of apoptosis: pcDNA3.1(+)-beclin 1 group was (28.2 ± 2.3)%, pcDNA3.1(+) group was (14.6 ± 4.6)%, pSUPER-beclin 1 group was (5.7 ± 2.0)%, pSUPER group wa (16.2 ± 3.1)%, and HeLa group was (11.2 ± 3.0)%. The pcDNA3.1(+)-beclin 1 vector significantly increased the apoptosis rate, while the pSUPER-beclin 1 vector significantly decreased the apoptosis rate (P < 0.05). (4) The activity of autophagy: more autophagy cells were identified in pcDNA3.1(+)-beclin 1 group; the rate of autophagy of five group were (10.3 ± 1.5)% in pcDNA3.1(+)-beclin 1 group, (3.6 ± 0.8)% in pcDNA3.1(+) group, (1.2 ± 0.3)% in pSUPER-beclin 1 group, (3.2 ± 1.2)% in pSUPER group and (2.2 ± 1.1)% in HeLa group, there was statistical significances between test groups and control groups (P < 0.05). (5) Carcinogenic activity of HeLa cells in nude mice: the duration of tumorigenesis was the longest in pcDNA3.1(+)-beclin 1 group and the shortest in pSUPER-beclin 1 group among all groups. The tumor size began to grow larger from 7th day after injection in pSUPER-beclin 1 group than in control groups (P < 0.05). The tumor size was smaller from 21st day after injection in pcDNA3.1(+)-beclin 1 group than in control groups (P < 0.05). From 28th day after injection, the tumor weigh was (0.52 ± 0.08) g in pSUPER-beclin 1 group, apparently more than HeLa group (0.37 ± 0.12) g and pSUPER group (0.34 ± 0.24) g (P < 0.05). While in pcDNA3.1(+)-beclin 1 group the tumor weighed (0.18 ± 0.12)g, which was lower than HeLa group and pcDNA3.1(+) group(0.34 ± 0.18) g (P < 0.05). CONCLUSIONS: Autophagy gene beclin 1 overexpression can inhibit proliferation and growth of HeLa cells in vitro and in vivo. Beclin 1 not noly participate in the regulation of autophagy signaling, but also play an important role in the regulation of endogenous apoptosis signaling through caspase-9. So it might be one of the new strategies for gene therapy of cervical carcinoma.

LUBAC Suppresses IL-21-Induced Apoptosis in CD40-Activated Murine B Cells and Promotes Germinal Center B Cell Survival and the T-Dependent Antibody Response.

B cell activation by Tfh cells, i.e., through CD154 engagement of CD40 and IL-21, and survival within GCs are crucial for the T-dependent Ab response. LUBAC, composed of HOIP, SHARPIN, and HOIL-1, catalyzes linear ubiquitination (Linear M1-Ub) to mediate NF-κB activation and cell survival induced by TNF receptor superfamily members, which include CD40. As shown in this study, B cells expressing the Sharpin null mutation cpdm (Sharpincpdm ) could undergo proliferation, CSR, and SHM in response to immunization by a T-dependent Ag, but were defective in survival within GCs, enrichment of a mutation enhancing the BCR affinity, and production of specific Abs. Sharpincpdm B cells stimulated in vitro with CD154 displayed normal proliferation and differentiation, marginally impaired NF-κB activation and survival, but markedly exacerbated death triggered by IL-21. While activating the mitochondria-dependent apoptosis pathway in both Sharpin+/+ and Sharpincpdm B cells, IL-21 induced Sharpincpdm B cells to undergo sustained activation of caspase 9 and caspase 8 of the mitochondria-dependent and independent pathway, respectively, and ultimately caspase 3 in effecting apoptosis. These were associated with loss of the caspase 8 inhibitor cFLIP and reduction in cFLIP Linear M1-Ub, which interferes with cFLIP poly-ubiquitination at Lys48 and degradation. Finally, the viability of Sharpincpdm B cells was rescued by caspase inhibitors but virtually abrogated - together with Linear M1-Ub and cFLIP levels - by a small molecule HOIP inhibitor. Thus, LUBAC controls the cFLIP expression and inhibits the effects of caspase 8 and IL-21-activated caspase 9, thereby suppressing apoptosis of CD40 and IL-21-activated B cells and promoting GC B cell survival.

Epigenetic Modulation of Class-Switch DNA Recombination to IgA by miR-146a Through Downregulation of Smad2, Smad3 and Smad4.

IgA is the predominant antibody isotype at intestinal mucosae, where it plays a critical role in homeostasis and provides a first line of immune protection. Dysregulation of IgA production, however, can contribute to immunopathology, particularly in kidneys in which IgA deposition can cause nephropathy. Class-switch DNA recombination (CSR) to IgA is directed by TGF-ß signaling, which activates Smad2 and Smad3. Activated Smad2/Smad3 dimers are recruited together with Smad4 to the IgH α locus Iα promoter to activate germline Iα-Cα transcription, the first step in the unfolding of CSR to IgA. Epigenetic factors, such as non-coding RNAs, particularly microRNAs, have been shown to regulate T cells, dendritic cells and other immune elements, as well as modulate the antibody response, including CSR, in a B cell-intrinsic fashion. Here we showed that the most abundant miRNA in resting B cells, miR-146a targets Smad2, Smad3 and Smad4 mRNA 3'UTRs and keeps CSR to IgA in check in resting B cells. Indeed, enforced miR-146a expression in B cells aborted induction of IgA CSR by decreasing Smad levels. By contrast, upon induction of CSR to IgA, as directed by TGF-ß, B cells downregulated miR-146a, thereby reversing the silencing of Smad2, Smad3 and Smad4, which, once expressed, led to recruitment of Smad2, Smad3 and Smad4 to the Iα promoter for activation of germline Iα-Cα transcription. Deletion of miR-146a in miR-146a-/- mice significantly increased circulating levels of steady state total IgA, but not IgM, IgG or IgE, and heightened the specific IgA antibody response to OVA. In miR-146a-/- mice, the elevated systemic IgA levels were associated with increased IgA+ B cells in intestinal mucosae, increased amounts of fecal free and bacteria-bound IgA as well as kidney IgA deposition, a hallmark of IgA nephropathy. Increased germline Iα-Cα transcription and CSR to IgA in miR-146a-/- B cells in vitro proved that miR-146a-induced Smad2, Smad3 and Smad4 repression is B cell intrinsic. The B cell-intrinsic role of miR-146a in the modulation of CSR to IgA was formally confirmed in vivo by construction and OVA immunization of mixed bone marrow µMT/miR-146a-/- chimeric mice. Thus, by inhibiting Smad2, Smad3 and Smad4 expression, miR-146a plays an important and B cell intrinsic role in modulation of CSR to IgA and the IgA antibody response.