VSIG4 induces the immunosuppressive microenvironment by promoting the infiltration of M2 macrophage and Tregs in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma and has a poor prognosis. Despite the impressive advancements in treating ccRCC using immune checkpoint (IC) blockade, such as PD-1/PD-L1 inhibitors, a considerable number of ccRCC patients experience adaptive resistance. Therefore, exploring new targetable ICs will provide additional treatment options for ccRCC patients. We comprehensively analyzed multi-omics data and performed functional experiments, such as pathologic review, bulk transcriptome data, single-cell sequencing data, Western blotting, immunohistochemistry and in vitro/in vivo experiments, to explore novel immunotherapeutic targets in ccRCC. It was found that immune-related genes VSIG4, SAA1, CD7, FOXP3, IL21, TNFSF13B, BATF, CD72, MZB1, LTB, CCL25 and KLRK1 were significantly upregulated in ccRCC (Student's t test and p-value < 0.05; 36 normal and 267 ccRCC tissues in raining cohort; 36 normal and 266 ccRCC tissues in validation cohort) and correlated with the poor prognosis of ccRCC patients (Wald test and p-value < 0.05 in univariate cox analysis; log-rank test and p-value < 0.05 in Kaplan-Meier method; 267 patients in training cohort and 266 in validation cohort). In particular, we found the novel IC target VSIG4 was specifically expressed in inhibitory immune cells M2-biased tumor-associated macrophages (TAMs), conventional dendritic cell 2 (cDC2) cells, and cycling myeloid cells in ccRCC microenvironment. Moreover, VSIG4 showed a closely relation with resistance of Ipilimumab/Nivolumab immunotherapy in ccRCC. Furthermore, VSIG4 promoted the infiltration of M2 macrophages, Tregs, and cDC2 in ccRCC tissues. VSIG4+ TAMs and VSIG4+ cDC2s may be a kind of immune cell subtypes related to immunosuppression. VSIG4 may play similar roles with other IC ligands, as it is highly expressed on the surface of antigen-presenting cells and ccRCC cells to inhibit T cells activity and facilitate immune escape. Targeting IC gene VSIG4 may provide a novel immunotherapeutic strategy to ccRCC patients with resistance to existing targeted therapy options.

SARS-CoV-2 ORF10 impairs cilia by enhancing CUL2ZYG11B activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal pathogen of the ongoing global pandemic of coronavirus disease 2019 (COVID-19). Loss of smell and taste are symptoms of COVID-19, and may be related to cilia dysfunction. Here, we found that the SARS-CoV-2 ORF10 increases the overall E3 ligase activity of the CUL2ZYG11B complex by interacting with ZYG11B. Enhanced CUL2ZYG11B activity by ORF10 causes increased ubiquitination and subsequent proteasome-mediated degradation of an intraflagellar transport (IFT) complex B protein, IFT46, thereby impairing both cilia biogenesis and maintenance. Further, we show that exposure of the respiratory tract of hACE2 mice to SARS-CoV-2 or SARS-CoV-2 ORF10 alone results in cilia-dysfunction-related phenotypes, and the ORF10 expression in primary human nasal epithelial cells (HNECs) also caused a rapid loss of the ciliary layer. Our study demonstrates how SARS-CoV-2 ORF10 hijacks CUL2ZYG11B to eliminate IFT46 and leads to cilia dysfunction, thereby offering a powerful etiopathological explanation for how SARS-CoV-2 causes multiple cilia-dysfunction-related symptoms specific to COVID-19.

A High Throughput Cell-Based Screen Assay for LINE-1 ORF1p Expression Inhibitors Using the In-Cell Western Technique.

Long interspersed nuclear element 1 (LINE-1) is a dominant autonomous retrotransposon in human genomes which plays a role in affecting the structure and function of somatic genomes, resulting in human disorders including genetic disease and cancer. LINE-1 encoded ORF1p protein which possesses RNA-binding and nucleic acid chaperone activity, and interacts with LINE-1 RNA to form a ribonucleoprotein particle (RNP). ORF1p can be detected in many kinds of tumors and its overexpression has been regarded as a hallmark of histologically aggressive cancers. In this study, we developed an In-Cell Western (ICW) assay in T47D cells to screen the compounds which can decrease the expression of ORF1p. Using this assay, we screened 1,947 compounds from the natural products library of Target Mol and Selleckchem, among which three compounds, Hydroxyprogesterone, 2,2':5',2″-Terthiophene and Ethynyl estradiol displayed potency in diminishing LINE-1 ORF1p expression level. Further mechanistic studies indicated the compounds act by affecting LINE-1 RNA transcription. Notably, we demonstrated that the compounds have an inhibitory effect on the proliferation of several lung and breast cancer cell lines. Taken together, we established a high throughput screening system for ORF1p expression inhibitors and the identified compounds provide some clues to the development of a novel anti-tumor therapeutic strategy by targeting ORF1p.

Discovery of Potent Antiallergic Agents Based on an o-Aminopyridinyl Alkynyl Scaffold.

Effective therapeutic agents are highly desired for immune-mediated allergic diseases. Herein, we report the design, synthesis, and structure-activity relationship of an o-aminopyridinyl alkyne series as novel orally bioavailable antiallergic agents, which was identified through phenotypic screening. Compound optimization yielded a highly potent compound 36, which effectively suppressed mast cell degranulation in a dose-dependent manner (IC50, 2.54 nM for RBL-2H3 cells; 48.28 nM for peritoneal mast cells (PMCs)) with a good therapeutic index. It also regulated the activation of FcεRI-mediated downstream signaling proteins in IgE/Ag-stimulated RBL-2H3 cells. In addition, 36 exhibited excellent in vivo pharmacokinetic properties and antiallergic efficacy in both passive systemic anaphylaxis (PSA) and house dust mite (HDM)-induced murine models of pulmonary allergic inflammation. Furthermore, preliminary analysis of the kinases profile identified Src-family kinases as potential targets for 36. Compound 36 may serve as a new valuable lead compound for future antiallergic drug discovery.

Efficacy and safety of bipolar versus monopolar transurethral resection of bladder tumors: A meta-analysis of randomized controlled trials.

CONTEXT: In the management of bladder tumors bipolarenergy has been used as a common alternative to the conventional monopolar transurethral resection of the bladder (M-TURB). AIM: This study aims to examine the clinical efficacy and safety of bipolar versus monopolar TURB tumors (TURBTs). SUBJECTS AND METHODS: We conducted a systematic literature search in the PubMed, Cochrane Library, and Embase databases for the identification of prospective randomized controlled trials (RCTs) that compared the outcomes between the two procedures. THE STATISTICAL TOOL: Meta-analysis was performed using the software Review Manager 5.3. RESULTS: We identified nine RCTs involving 1193 patients. In terms of the surgical outcomes, there was no significant difference between the bipolar and monopolar TURBT. However, there was significantly reduced bladder perforation (risk ratio [RR] = 0.48; 95% confidence interval [CI] = 0.30-0.77; P = 0.002) and shorter hospital stay (mean difference = 0.43; 95% CI = 0.83-0.03, P = 0.01) in the bipolar TURBT group. There was also a lower incidence of thermal damage, which causes histopathological artifacts for patients treated via bipolar TURBT relative to those treated via monopolar TURBT (RR = 0.66; 95% CI = 0.55-0.78; P < 0.00001). P < 0.05 was considered to be statistically significant. However, after bipolar and monopolar TURBT, we had no sufficient evidence regarding the recurrence rate. CONCLUSION: This meta-analysis suggests that the use of bipolar technology, which is associated with less bladder perforation and lower thermal artifacts in TURBT is safer and more effective.

A novel IgG1 monoclonal antibody against xanthine oxidase alleviates inflammation induced by potassium oxonate in mice.

Xanthine oxidase (XOD) is a key enzyme that catalyzes xanthine to uric acid. Most of the urate-lowering medicines targeting XOD have a limited effect on alleviating inflammation in spite of significant effects on decreasing serum uric acid level. In this study, we produced and characterized a novel monoclonal antibody (Anti-XOD mAb) using hybridoma technology based on a novel peptide OI5P-1(O-IA2(5)-P2-1),which containing a B-cell epitope of XOD and a novel Th2 built-in adjuvant I5P-1(IA2(5)-P2-1). Results of western blotting and cross-reactivity assay indicated that the mAb binds specifically to XOD and the affinity was 2.523×1010L/mol. The mAb reduced serum uric acid level and hepatic xanthine oxidase activity in potassium oxonate induced mice. A decreased methane dicarboxylic aldehyde level and an improved superoxide dismutase level in mAb treated mice indicated anti-lipid peroxidation effects of the mAb. Moreover, the mAb showed a significant immunomodulatory effect which could shift Th1/Th2 balance to Th2-dominant immunity. The mAb treatment alleviates inflammation induced by potassium oxonate, superior to the small molecule allopurinol treatment. For the first time, these results showed that the anti-XOD mAb may serve as a promising therapeutic approach for inflammatory response related to uric acid.

Construction of a novel vaccine by conjugating a B-cell epitope of DPP4 to peptide IA2(5)-P2-1 to significantly control type 1 diabetes in NOD mice.

Type 1 diabetes is a chronic organ-specific autoimmune disease in which selective destruction of insulin-producing ß cells leads to impaired glucose metabolism and its attendant complications. IA2(5)P2-1, a potent immunogenic carrier which designed by our laboratory, can induce high titer specific antibodies when carry a B cell epitope, such as B cell epitopes of DPP4, xanthine oxidase, and Urate transporter protein. In this report, we describe a novel multi-epitope vaccine composing a peptide of DPP4, an anti-diabetic B epitope of Insulinoma antigen-2(IA-2) and a Th2 epitope (P2:IPALDSLTPANED) of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in non-obese diabetic (NOD) mice successfully induced specific anti-DPP4 antibody, inhibited plasma DPP4 activity, and increased serum GLP-1 level. Moreover, this antibody titer was correlated with the dose of immunization (20µg, 100µg). Inoculation of this vaccine in NOD mice significantly control blood glucose level, improved glucose excursion and increased insulin level in vivo. Consistent with a lower diabetic and insulitis incidence, a induced splenic T cells proliferation and tolerance were observed. IFN-γ secretion reduced and IL-10 increased significantly in the D41-IA2(5)-P2-1 treated mice compared to P277 and control group due to the potential immunomodulatory effect of the epitope in the vaccine. Immunohistochemical analysis and cytometry showed a rebalance of Th1/Th2 in NOD mice. Our results demonstrate that this multi-epitope vaccine may serve as a promising therapeutic approach for type 1 diabetes.

A Novel Multi-Epitope Vaccine Based on Urate Transporter 1 Alleviates Streptozotocin-Induced Diabetes by Producing Anti-URAT1 Antibody and an Immunomodulatory Effect in C57BL/6J Mice.

Hyperuricemia (HUA) is related to diabetes. Uric acid-induced inflammation and oxidative stress are risk factors for diabetes and its complications. Human urate transporter 1 (URAT1) regulates the renal tubular reabsorption of uric acid. IA-2(5)-P2-1, a potent immunogenic carrier designed by our laboratory, can induce high-titer specific antibodies when it carries a B cell epitope, such as B cell epitopes of DPP4 (Dipeptidyl peptidase-4), xanthine oxidase. In this report, we describe a novel multi-epitope vaccine composing a peptide of URAT1, an anti-diabetic B epitope of insulinoma antigen-2(IA-2) and a Th2 epitope (P2:IPALDSLTPANED) of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in streptozotocin-induced diabetes C57BL/6J mice successfully induced specific anti-URAT1 antibody, which inhibited URAT1 action and uric acid reabsorption, and increased pancreatic insulin level with a lower insulitis incidence. Vaccination with U-IA-2(5)-P2-1 (UIP-1) significantly reduced blood glucose and uric acid level, increased Th2 cytokines interleukin (IL)-10 and IL-4, and regulated immune reactions through a balanced Th1/Th2 ratio. These results demonstrate that the URAT1-based multi-epitope peptide vaccine may be a suitable therapeutic approach for diabetes and its complications.

A novel multi-epitope vaccine based on Dipeptidyl Peptidase 4 prevents streptozotocin-induced diabetes by producing anti-DPP4 antibody and immunomodulatory effect in C57BL/6J mice.

Type 1 diabetes is a chronic organ-specific autoimmune disease in which selective destruction of insulin-producing ß-cells leads to impaired glucose metabolism and its attendant complications. A series of Dipeptidyl peptidase 4 (DPP4) inhibitors have been developed and granted approval in the treatment of type 2 diabetes mellitus by inhibiting the enzymatic degradation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). An increasing number of studies have shown the potential benefits of DPP4 inhibitors for type 1 diabetes. In this report, we describe a novel multi-epitope vaccine comprising a B cell epitope of DPP4, an anti-diabetic B cell epitope of Insulinoma antigen-2 (IA-2) and a Th2 epitope of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in streptozotocin (STZ) treated mice successfully induced specific anti-DPP4 antibody and increased serum GLP-1 level. Moreover, this antibody lasted for more than 7 weeks. Inoculation of this vaccine in C57BL/6J mice significantly reduced blood glucose level, improved glucose excursion and increased plasma insulin concentration. Consistent with a lower diabetic and insulitis incidence, induced splenic T cell proliferation and tolerance were observed. IFN-γ and IL-2 secretion reduced, but IL-10 and IL-4 increased significantly in the Dipeptidyl Peptidase 41-Insulinoma antigen-2(5)-P2-1 (D41-IP) treated mice compared to the Insulinoma antigen-2(5)-P2-1 (IA2(5)P2-1) and control group due to the potential immunomodulatory effect of the epitopes in the vaccine. Our results demonstrate that this multi-epitope vaccine may serve as a promising therapeutic approach against type 1 diabetes.

miR-377 functions as a tumor suppressor in human clear cell renal cell carcinoma by targeting ETS1.

Clear cell renal cell carcinoma (ccRCC) is the most common form of neoplasm occurring in the adult kidney. Although significant advances have been made in treatment for ccRCC, a significant percent of patients have no benefit from currently available treatment option. MicroRNAs (miRNAs) have been reported to play central roles in regulating tumor progression, and are being explored as potentially more effective therapies and diagnostic tools for various cancers. The transcription factor E26 transformation specific-1 (ETS1) is believed to be intimately involved in tumor progression, and is frequently upregulated in tumors, including ccRCC. However, few studies have investigated the implications of ETS1 in ccRCC, and no studies have investigated the dysregulated mechanisms responsible for aberrant ETS1 expression in ccRCC. We used databases, clinical samples and target prediction algorithms to investigate aberrant miR-377 expression and potential targets in ccRCC. Our results indicate that miR-377 is downregulated in ccRCC, and that miR-377 can regulate the expression of ETS1. Further, using cell cycle analysis, MTT, luciferase and knockdown experiments, we found evidence to suggest that ETS1 is central in the development of the proliferative, metastatic and invasive phenotype of ccRCC cells. Furthermore, miR-377 was found to directly downregulate the expression of ETS1 and reduce the ability of ccRCC cells to proliferate, migrate and invade. As miR-377 was found to be differentially expressed in ccRCC, and in light of the apparent central role of ETS1 in tumor development, our results indicate that miR-377 could be useful for ccRCC diagnostics, prognostics and therapeutics.

[Expression of E-cadherin and ß-catenin and their significance in non-small cell lung cancer].

BACKGROUND: Because invasion and metastasis of cancer threaten seriously human's life, it is of more important clinical significance to understand and evaluate the biologic behavior of can-(cer) correctly. Abnormal expression of E-cadherin and ß-catenin plays important roles in invasion and metastasis of cancer. The objective of this study is to investigate their expression in non-small cell lung cancer (NSCLC) and to find out their correlation with histological type, cell differentiation, metastasis and prognosis of NSCLC. METHODS: The expression of E-cadherin and ß-catenin was detected in 129 NSCLC tissues by high sensitive S-P immunohistochemical method. RESULTS: The abnormal expression rate of E-cadherin and ß-catenin was 62.0% and 65.1% respectively. The abnormal expression rate of E-cadherin in squamous cell carcinoma was much higher than that in adenocarcinoma (P < 0.05). The abnormal expression rate of E-cadherin and ß-catenin in poorly differentiated cells was significantly higher than that in well and moderately differentiated cells (P < 0.05, P < 0.05). Stage III/IV NSCLC showed markedly higher abnormal expression rate of E-cadherin and ß-catenin than stage I/II NSCLC did (P < 0.01, P < 0.01). The abnormal expression rate of E-cadherin and ß-catenin in patients with lymphatic metastasis was significantly higher than that in those without lymphatic metastasis (P < 0.05, P < 0.01). The mean survival duration and 5-year survival rate in patients with normal E-cadherin and ß-catenin expression were remarkably higher than those in patients with abnormal expression (P < 0.05, P < 0.01). CONCLUSIONS: The abnormal expression of E-cadherin and ß-catenin is closely related to histological type, differentiation and metastasis in NSCLC. Detection of their expression might be helpful to predict prognosis of NSCLC.