Xiao-Lian Zhang

The Ubiquitin Ligase RNF5 Regulates Antiviral Responses by Mediating Degradation of the Adaptor Protein MITA

Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

Cancer Stem Cell Vaccination Confers Significant Antitumor Immunity

Most studies of cancer stem cells (CSC) involve the inoculation of cells from human tumors into immunosuppressed mice, preventing an assessment on the immunologic interactions and effects of CSCs. In this study, we examined the vaccination effects produced by CSC-enriched populations from histologically distinct murine tumors after their inoculation into different syngeneic immunocompetent hosts. Enriched CSCs were immunogenic and more effective as an antigen source than unselected tumor cells in inducing protective antitumor immunity. Immune sera from CSC-vaccinated hosts contained high levels of IgG which bound to CSCs, resulting in CSC lysis in the presence of complement. CTLs generated from peripheral blood mononuclear cells or splenocytes harvested from CSC-vaccinated hosts were capable of killing CSCs in vitro. Mechanistic investigations established that CSC-primed antibodies and T cells were capable of selective targeting CSCs and conferring antitumor immunity. Together, these proof-of-concept results provide a rationale for a new type of cancer immunotherapy based on the development of CSC vaccines that can specifically target CSCs.

Aptamers That Preferentially Bind Type IVB Pili and Inhibit Human Monocytic-Cell Invasion by Salmonella enterica Serovar Typhi

ABSTRACT Salmonella enterica serovar Typhi is an important pathogen exclusively for humans and causes typhoid or enteric fever. It has been shown that type IVB pili, encoded by the S. enterica serovar Typhi pil operon located in Salmonella pathogenicity island 7, are important in the pathogenic process. In this study, by using both an adhesion-invasion assay and fluorescence quantitative PCR analysis, we demonstrated that the entry of type IVB piliated S. enterica serovar Typhi A21-6 (pil+ Kmr) into human THP-1 monocytic cells was greater than that of a nonpiliated S. enterica serovar Typhi pilS::Kmr (pil mutant) strain. We have applied a systematic evolution of ligands by exponential enrichment approach to select oligonucleotides (aptamers) as ligands that specifically bind to type IVB pili. Using this approach, we identified a high-affinity single-stranded RNA aptamer (S-PS8.4) as a type IVB pilus-specific ligand and further found that the selected aptamer (S-PS8.4) could significantly inhibit the entry of the piliated strain (but not that of the nonpiliated strain) into human THP-1 cells. The binding affinities between aptamers and pre-PilS (structural protein of type IVB pili) were determined by nitrocellulose filter-binding assays, and the Kd value was determined to be 8.56 nM for the S-PS8.4 aptamer alone. As an example of an aptamer against type IVB pili of S. enterica serovar Typhi, the aptamer S-PS8.4 can serve as a tool for analysis of bacterial type IVB pilus-host cell interactions and may yield information for the development of putative new drugs against S. enterica serovar Typhi bacterial infections, useful both in prevention of infection and in therapeutic treatment.

CS-SELEX Generates High-Affinity ssDNA Aptamers as Molecular Probes for Hepatitis C Virus Envelope Glycoprotein E2

Currently, the development of effective diagnostic reagents as well as treatments against Hepatitis C virus (HCV) remains a high priority. In this study, we have described the development of an alive cell surface -Systematic Evolution of Ligands by Exponential Enrichment (CS-SELEX) technique and screened the functional ssDNA aptamers that specifically bound to HCV envelope surface glycoprotein E2. Through 13 rounds of selection, the CS-SELEX generated high-affinity ssDNA aptamers, and the selected ssDNA aptamer ZE2 demonstrated the highest specificity and affinity to E2-positive cells. HCV particles could be specifically captured and diagnosed using the aptamer ZE2. A good correlation was observed in HCV patients between HCV E2 antigen-aptamer assay and assays for HCV RNA quantities or HCV antibody detection. Moreover, the selected aptamers, especially ZE2, could competitively inhibit E2 protein binding to CD81, an important HCV receptor, and significantly block HCV cell culture (HCVcc) infection of human hepatocytes (Huh7.5.1) in vitro. Our data demonstrate that the newly selected ssDNA aptamers, especially aptamer ZE2, hold great promise for developing new molecular probes, as an early diagnostic reagent for HCV surface antigen, or a therapeutic drug specifically for HCV.

Specifically binding of L-ficolin to N-glycans of HCV envelope glycoproteins E1 and E2 leads to complement activation.

L-ficolin, one of lectin families, is a recently identified complement factor that initiates lectin pathway of complement. Little is known about its role in viral hepatitis. In the present study, we found that L-ficolin in serum from 103 patients with hepatitis C virus (HCV), were significantly higher than that in 150 healthy controls. We further found that L-ficolin expressions were significantly increased in vitro study by HCV JFH-1 infected human hepatocyte cell line Huh7.5.1. Investigation of the mechanisms of the L-ficolin action on HCV demonstrated that L-ficolin protein could recognize and bind to envelope glycoproteins E1 and E2 of HCV, activating the lectin complement pathway-mediated cytolytic activity in HCV-infected hepatocyte. This interaction between L-ficolin and HCV E1 and E2 glycoproteins was attributed to the N-glycans of E1 and E2. These findings provide new insights into the biological functions of L-ficolin in clinically important hepatic viral diseases.

L-Ficolin Binds to the Glycoproteins Hemagglutinin and Neuraminidase and Inhibits Influenza A Virus Infection Both in vitro and in vivo

L-ficolin, one of the complement lectins found in human serum, is a novel pattern recognition molecule that can specifically bind to microbial carbohydrates, thereby activating the lectin complement pathway and mounting a protective innate immune response. However, little is known about the role of L-ficolin during viral infections in vivo. In the present study, we used a mouse model of influenza A virus infection to demonstrate that the administration of exogenous L-ficolin or ficolin A (FCNA – an L-ficolin-like molecule in the mouse) is protective against the virus. Furthermore, FCNA-null mice have a greatly increased susceptibility to infection with the influenza A virus. Moreover, we found recombinant human L-ficolin inhibited influenza A virus entry into Madin-Darby canine kidney cells. More importantly, L-ficolin can recognize and bind hemagglutinin (HA) and neuraminidase (NA) glycoproteins and different subtypes of influenza A virus, and these interactions can be competitively inhibited by N-acetyl-D-glucosamine. In addition, the binding of L-ficolin and FCNA may lead to the activation of the lectin complement pathway. To our knowledge, this is the first report demonstrating that L-ficolin can block influenza virus infections both in vitro and in vivo using FCNA-knockout mice, possibly by interacting with the carbohydrates of HA and NA. Therefore, these data may provide new immunotherapeutic strategies based on the innate immune molecule L-ficolin against the influenza A virus.

Concise Review: Targeting Cancer Stem Cells Using Immunologic Approaches

Cancer stem cells (CSCs) represent a small subset of tumor cells which have the ability to self‐renew and generate the diverse cells that comprise the tumor bulk. They are responsible for local tumor recurrence and distant metastasis. However, they are resistant to conventional radiotherapy and chemotherapy. Novel immunotherapeutic strategies that specifically target CSCs may improve the efficacy of cancer therapy. To immunologically target CSC phenotypes, innate immune responses to CSCs have been reported using Natural killer cells and γδ T cells. To target CSC specifically, in vitro CSC‐primed T cells have been successfully generated and shown targeting of CSCs in vivo after adoptive transfer. Recently, CSC‐based dendritic cell vaccine has demonstrated significant induction of anti‐CSC immunity both in vivo in immunocompetent hosts and in vitro as evident by CSC reactivity of CSC vaccine‐primed antibodies and T cells. In addition, identification of specific antigens or genetic alterations in CSCs may provide more specific targets for immunotherapy. ALDH, CD44, CD133, and HER2 have served as markers to isolate CSCs from a number of tumor types in animal models and human tumors. They might serve as useful targets for CSC immunotherapy. Finally, since CSCs are regulated by interactions with the CSC niche, these interactions may serve as additional targets for CSC immunotherapy. Targeting the tumor microenvironment, such as interrupting the immune cell, for example, myeloid‐derived suppressor cells, and cytokines, for example, IL‐6 and IL‐8, as well as the immune checkpoint (PD1/PDL1, etc.) may provide additional novel strategies to enhance the immunological targeting of CSCs. Stem Cells 2015;33:2085–2092

Ficolins: structure, function and associated diseases.

Innate immunity relies upon the ability of a few pattern recognition molecules to sense molecular markers. Ficolins are humoral molecules of the innate immune systems which recognize carbohydrate molecules on pathogens, apoptotic and necrotic cells. Three ficolins have been identified in humans: L-ficolin, H-ficolin and M-ficolin (also referred to as ficolin-2, -3 and -1, respectively). They are soluble oligomeric defence proteins with lectin-like activity and they are structurally similar to the human collectins, mannan-binding lectin (MBL) and surfactant protein A and D. Upon recognition of the infectious agent, the ficolins act through two distinct routes: initiate the lectin pathway of complement activation through attached serine proteases (MASPs), and a primitive opsonophagocytosis thus limiting the infection and concurrently orchestrating the subsequent adaptive clonal immune response. Recently a lot of reports showed that dysfunction or abnormal expressions of ficolins may play crucial roles in the pathogenesis of human diseases including: (1) infectious and inflammatory diseases, e.g., recurrent respiratory infections; (2) apoptosis, and autoimmune disease; (3) systemic lupus erythematosus; (4) IgA nephropathy; (5) clinical syndrome of preeclampsia; (6) other diseases associated factor e.g. C-reactive protein. Precise identification of ficolins functions will provide novel insight in the pathogenesis of these diseases and may provide novel innate immune therapeutic options to treat disease progression. This review discusses the structures, functions, and clinical implications of ficolins and summarizes the reports on the roles of ficolins in human diseases.

Synthesis and antibacterial study of 10, 15, 20-triphenyl-5-(4-hydroxy-3-(trimethylammonium)methyl)phenylporphyrin as models for combination of porphyrin and alkylating agent.

10, 15, 20-Triphenyl-5-(4-hydroxy-3-(trimethylammonium)methyl)phenylporphyrin 4 and its zinc complex 5 have been synthesized and antibacterial activities have been studied for 4 and its derivative. Compound 4 showed stronger inhibition than that of 10, 15, 20-triphenyl-5-(4-hydroxy-3-(dimethylamine)methyl)phenylporphyrin (2) and 10, 15, 20-triphenyl-5-(4-methoxy-3-(trimethylammonium)methyl)phenylporphyrin (6). It is possible that antibacterial activity of compound 4 involved in photoinducing both o-quinone methide intermediate and singlet oxygen formation.

IL‐24 protects against Salmonella typhimurium infection by stimulating early neutrophil Th1 cytokine production, which in turn activates CD8+ T cells

Salmonella are important intracellular pathogens in humans and other animal hosts. IL‐24 is a novel tumour suppressor and can mediate induction of Th1‐type cytokines from PBMC. However, the immunological consequences of this cytokine during intracellular pathogen infection in vivo remain unclear. In the present study, we used a virulent S. typhimurium C5 infected mouse model of typhoid fever to demonstrate that administration of exogenous IL‐24 had a protective effect against the bacteria. The IL‐24 glycosylation site mutant, in contrast, showed a decreased protective effect. Furthermore, the protective effect of IL‐24 was abrogated in IFN‐γ KO mice. More importantly, we demonstrated that IL‐24 predominately stimulated neutrophils to produce IFN‐γ and IL‐12, subsequently activating CD8+ T cells both in vivo and in vitro. In addition, IL‐24 could induce neutrophils to produce NO. These data indicate that the neutrophils activated by IL‐24 may play important roles in host defence against Salmonella infection in vivo. Our findings support the development of a novel cytokine immunotherapy against Salmonella.