Proteomic analysis of underlying apoptosis mechanisms of human retinal pigment epithelial ARPE-19 cells in response to mechanical stretch.

Our previous study demonstrated mechanical stretch (MS) could induce the apoptosis of retinal pigment epithelial (RPE) cells, but the related mechanisms remained unclear. This study was to characterize the protein expression profile in RPE cell line ARPE-19 exposed to MS, cytochalasin D (CD; an inhibitor of actin polymerization) or CD + MS at 2-time points (6, 24 hr; n = 3, at each time point) by using proteomics technique. Our data highlighted that compared with control, ECE1 was continuously downregulated in ARPE-19 cells treated by MS or CD + MS from 6 to 24 hr. Function and protein-protein interaction network analyses showed ATAD2 was downregulated in all three treatment groups compared with control, but successive upregulation of RPS13 and RPL7 and downregulation of AHSG were specifically induced by MS. ATAD2 was enriched in cell cycle; AHSG was associated with membrane organization; RPS13 and RPL7 participated in ribosome biogenesis. Furthermore, transcription factor CREB1 that was upregulated in MS group at 24 hr after treatment, may negatively regulate ATAD2. The expressions of all crucial proteins in ARPE-19 cells were confirmed by western blot analysis. Overexpression of ATAD2 and AHSG were also shown to reverse the apoptosis of ARPE-19 cells induced by MS or CD + MS, with significantly decreased apoptotic rates and caspase-3 activities. Accordingly, our findings suggest downregulation of ATAD2 and AHSG may be potential contributors to the apoptosis of RPE cells induced by MS. Overexpression of them may represent underlying preventive and therapeutic strategies for MS-induced retinal disorders.

Expression of SASP, DNA Damage Response, and Cell Proliferation Factors in Early Gastric Neoplastic Lesions: Correlations and Clinical Significance.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated senescence-associated secretory phenotype (SASP) pathway has recently been identified in the suppression and promotion of cancers. However, its practical role in carcinogenesis remains to be comprehensively elucidated. Here, we describe an investigation analysing SASP activity and its correlations with DNA damage response (DDR), genomic mutations, and cell proliferation in gastric carcinogenesis among 30 cases with available endoscopic submucosal dissection (ESD) specimens of early neoplastic lesions (including low-grade dysplasia [LGD], high-grade dysplasia [HGD], and intramucosal carcinoma). The positive cells of senescence-associated ß-galactosidase staining and cGAS, STING, interferon-regulatory factor 3 (IRF3), and signal transducer and activator of transcription 6 (STAT6) expression levels using immunostaining were elevated in HGD and in cancers. Similarly, increased expression of the Fanconi anemia group D2 (FANCD2) protein, tumour suppressor p53 binding protein 1 (TP53BP1), and replication protein A (RPA2) (i.e., primary DDR factors) was detected in HGD and in cancers; these increased expression levels were closely correlated with high expression of Ki67 and minichromosome maintenance complex component 7 (MCM7) proteins. Moreover, genomic mutations in TP53 gene were detected in 56.67% of the evaluated cases (17/30) using next-generation sequencing, and positive staining was verified in HGD and in cancers. Statistical analysis revealed that cell proliferation closely correlated with the expression of DDR factors, of which TP53BP1 was positively associated with SASP factors and IRF3 was positively correlated with cell proliferation. In addition, an analysis evaluating clinical features demonstrated that STAT6-positive cases showed a longer progression-free survival time than STAT6-negative cases. Our evaluation, conducted using a limited number of specimens, suggests SASP may be prevalent in early gastric neoplastic lesions and could be activated by accelerated cell proliferation-induced DDR. The clinical significance of SASP still needs to be determined.

Autophagy-Mediated Clearance of Free Genomic DNA in the Cytoplasm Protects the Growth and Survival of Cancer Cells.

The cGAS (GMP-AMP synthase)-mediated senescence-associated secretory phenotype (SASP) and DNA-induced autophagy (DNA autophagy) have been extensively investigated in recent years. However, cGAS-mediated autophagy has not been elucidated in cancer cells. The described investigation revealed that active DNA autophagy but not SASP activity could be detected in the BT-549 breast cancer cell line with high micronucleus (MN) formation. DNA autophagy was identified as selective autophagy of free genomic DNA in the cytoplasm but not nucleophagy. The process of DNA autophagy in the cytosol could be initiate by cGAS and usually cooperates with SQSTM1-mediated autophagy of ubiquitinated histones. Cytoplasmic DNA, together with nuclear proteins such as histones, could be derived from DNA replication-induced nuclear damage and MN collapse. The inhibition of autophagy through chemical inhibitors as well as the genomic silencing of cGAS or SQSTM1 could suppress the growth and survival of cancer cells, and induced DNA damage could increase the sensitivity to these inhibitors. Furthermore, expanded observations of several other kinds of human cancer cells indicated that high relative DNA autophagy or enhancement of DNA damage could also increase or sensitize these cells to inhibition of DNA autophagy.

Allogeneic dendritic cells induce potent antitumor immunity by activating KLRG1+CD8 T cells.

The graft-versus-leukemia effect reminds us to observe the allogeneic cell elicited anti-tumor immune responses. Here we immunized recipient B6 mice with different types of allogenic leukocytes and found that vaccination with allogenic dendritic cells (alloDC) elicited the most efficient protection against broad-spectrum tumors. The recipient lymphocytes were analyzed and the data showed that CD8 T cells increased significantly after immunization and expressed effector memory T cell marker KLRG1. Functional evaluation demonstrated that these KLRG1+CD8 T cells could kill tumor cells in vitro and in vivo in Granzyme B- and Fas/FasL-dependent manners with no tumor antigen specificity, and tend to migrate into tumor sites by high expression of heparanase. Adoptive transfer of these cells could provide antitumor protection against tumors. AlloDC could also treat mice with residual tumors and combination of anti-PD1 antibody could enhance this effects. Together, our study showed that alloDC-immunization could induce potent antitumor effect through the expansion of KLRG1+CD8 T cells, which can work as both preventive and therapeutic tumor vaccines.

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells.

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators' ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

CD8(+)NKT-like cells regulate the immune response by killing antigen-bearing DCs.

CD1d-dependent NKT cells have been extensively studied; however, the function of CD8(+)NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8(+)NKT-like cells, which express both T cell markers (TCRß and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8(+)NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8(+)NKT-like cell development is normal in CD1d(-/-) mice, which suggests that CD8(+)NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8(+)NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8(+)NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8(+)NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens.

Blunted IL17/IL22 and pro-inflammatory cytokine responses in the genital tract and blood of HIV-exposed, seronegative female sex workers in Kenya.

BACKGROUND: Identifying the immune correlates of reduced susceptibility to HIV remains a key goal for the HIV vaccine field, and individuals who are HIV-exposed, seronegative (HESN) may offer important clues. Reduced systemic immune activation has been described in HESN individuals. Conversely, pro-inflammatory T cell subsets, particularly CD4+ T cells producing the cytokine IL17 (Th17 cells), may represent a highly susceptible target for HIV infection after sexual exposure. Therefore, we characterized the cellular pro-inflammatory and IL17/IL22 cytokine immune milieu in the genital mucosa and blood of HESN female sex workers (FSWs). METHODS AND RESULTS: Blinded lab personnel characterized basal and mitogen-induced gene and cytokine immune responses in the cervix and blood of HESN FSWs (n = 116) and non-FSW controls (n = 17) using qPCR and ELISA. IL17 and IL22 production was significantly reduced in both the cervix and blood of HESNs, both in resting cells and after mitogen stimulation. In addition, HESN participants demonstrated blunted production of both pro-inflammatory cytokines and ß-chemokines. DISCUSSION AND CONCLUSIONS: We conclude that HIV exposure without infection was associated with blunted IL17/IL22 and pro-inflammatory responses, both systemically and at the site of mucosal HIV exposure. It will be important for further studies to examine the causal nature of the association and to define the cell subsets responsible for these differences.

Evaluation of a quantitative real-time PCR assay to measure HIV-specific mucosal CD8+ T cell responses in the cervix.

Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure these responses. ß-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFNγ, IL-2 and TNFα occurred after 3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required assay input cell numbers; qPCR retained sensitivity with input of at least 2.5×10(4) PBMCs. This optimized qPCR assay was then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals. SEB induced IFNγ mRNA transcription was detected in CMCs and correlated positively with IFNγ protein production. However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless, this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract.