IL-12-Overexpressed Nanoparticles Suppress the Proliferation of Melanoma Through Inducing ICD and Activating DC, CD8+ T, and CD4+ T Cells.

Purpose: The drug resistance and low response rates of immunotherapy limit its application. This study aimed to construct a new nanoparticle (CaCO3-polydopamine-polyethylenimine, CPP) to effectively deliver interleukin-12 (IL-12) and suppress cancer progress through immunotherapy. Methods: The size distribution of CPP and its zeta potential were measured using a Malvern Zetasizer Nano-ZS90. The morphology and electrophoresis tentative delay of CPP were analyzed using a JEM-1400 transmission electron microscope and an ultraviolet spectrophotometer, respectively. Cell proliferation was analyzed by MTT assay. Proteins were analyzed by Western blot. IL-12 and HMGB1 levels were estimated by ELISA kits. Live/dead staining assay was performed using a Calcein-AM/PI kit. ATP production was detected using an ATP assay kit. The xenografts in vivo were estimated in C57BL/6 mice. The levels of CD80+/CD86+, CD3+/CD4+ and CD3+/CD8+ were analyzed by flow cytometry. Results: CPP could effectively express EGFP or IL-12 and increase ROS levels. Laser treatment promoted CPP-IL-12 induced the number of dead or apoptotic cell. CPP-IL-12 and laser could further enhance CALR levels and extracellular HMGB1 levels and decrease intracellular HMGB1 and ATP levels, indicating that it may induce immunogenic cell death (ICD). The tumors and weights of xenografts in CPP-IL-12 or laser-treated mice were significantly reduced than in controls. The IL-12 expression, the CD80+/CD86+ expression of DC from lymph glands, and the number of CD3+/CD8+T or CD3+/CD4+T cells from the spleen increased in CPP-IL-12-treated or laser-treated xenografts compared with controls. The levels of granzyme B, IFN-γ, and TNF-α in the serum of CPP-IL-12-treated mice increased. Interestingly, CPP-IL-12 treatment in local xenografts in the back of mice could effectively inhibit the growth of the distant untreated tumor. Conclusion: The novel CPP-IL-12 could overexpress IL-12 in melanoma cells and achieve immunotherapy to melanoma through inducing ICD, activating CD4+ T cell, and enhancing the function of tumor-reactive CD8+ T cells.

m6A-methylated KCTD21-AS1 regulates macrophage phagocytosis through CD47 and cell autophagy through TIPR.

Blocking immune checkpoint CD47/SIRPα is a useful strategy to engineer macrophages for cancer immunotherapy. However, the roles of CD47-related noncoding RNA in regulating macrophage phagocytosis for lung cancer therapy remain unclear. This study aims to investigate the effects of long noncoding RNA (lncRNA) on the phagocytosis of macrophage via CD47 and the proliferation of non-small cell lung cancer (NSCLC) via TIPRL. Our results demonstrate that lncRNA KCTD21-AS1 increases in NSCLC tissues and is associated with poor survival of patients. KCTD21-AS1 and its m6A modification by Mettl14 promote NSCLC cell proliferation. miR-519d-5p gain suppresses the proliferation and metastasis of NSCLC cells by regulating CD47 and TIPRL. Through ceRNA with miR-519d-5p, KCTD21-AS1 regulates the expression of CD47 and TIPRL, which further regulates macrophage phagocytosis and cancer cell autophagy. Low miR-519d-5p in patients with NSCLC corresponds with poor survival. High TIPRL or CD47 levels in patients with NSCLC corresponds with poor survival. In conclusion, we demonstrate that KCTD21-AS1 and its m6A modification promote NSCLC cell proliferation, whereas miR-519d-5p inhibits this process by regulating CD47 and TIPRL expression, which further affects macrophage phagocytosis and cell autophagy. This study provides a strategy through miR-519-5p gain or KCTD21-AS1 depletion for NSCLC therapy by regulating CD47 and TIPRL.

High-Valence Metal Doping Induced Lattice Expansion for M-FeNi LDH toward Enhanced Urea Oxidation Electrocatalytic Activities.

The precise design of low-cost, efficient, and definite electrocatalysts is the key to sustainable renewable energy. The urea oxidation reaction (UOR) offers a promising alternative to the oxygen evolution reaction for energy-saving hydrogen generation. In this study, by tuning the lattice expansion, a series of M-FeNi layered double hydroxides (M-FeNi LDHs, M: Mo, Mn, V) with excellent UOR performance are synthesized. The hydrolytic transformation of Fe-MIL-88A is assisted by urea, Ni2+ and high-valence metals, to form a hollow M-FeNi LDH. Owing to the large atomic radius of the high-valence metal, lattice expansion is induced, and the electronic structure of the FeNi-LDH is regulated. Doping with high-valence metal is more favorable for the formation of the high-valence active species, NiOOH, for the UOR. Moreover, the hollow spindle structure promoted mass transport. Thus, the optimal Mo-FeNi LDH showed outstanding UOR electrocatalytic activity, with 1.32 V at 10 mA cm-2 . Remarkably, the Pt/C||Mo-FeNi LDH catalyst required a cell voltage of 1.38 V at 10 mA·cm-2 in urea-assisted water electrolysis. This study suggests a new direction for constructing nanostructures and modulating electronic structures, which is expected to ultimately lead to the development of a class of auxiliary electrocatalysts.

[Correlation of Treg and IL-15 expression in the peripheral blood of patients with oral lichen planus].

PURPOSE: To investigate the expression of Treg and IL-15 in the peripheral blood of patients with oral lichen planus (OLP). METHODS: The peripheral blood was obtained from 36 OLP patients and 20 healthy controls. Flow cytometry was used to evaluate the proportion of Treg cells, and the level of IL-15 was measured by ELISA. The data were analyzed by SPSS 16.0 software package. RESULTS: In the peripheral blood of OLP patients, the proportion of CD4+CD25+Foxp3+ Treg cells and IL-15 content were significantly higher than those of the healthy controls (P<0.05), but there were no significantly difference between two clinical subtypes of OLP(P>0.05). The proportion of CD4+CD25+Foxp3+Treg cells was positively correlated with the level of IL-15(r=0.70,P<0.05). CONCLUSIONS: The high expression of Treg cells and IL-15 in the perpheral blood of OLP patients may be responsible for the pathogenesis of OLP.

[The up-regulated expression of LAIR-1 in tumor patients PBMC].

AIM: To investigate the expression of LAIR-1 in patients with tumors and the function of LAIR-1 in anticancer immunity. METHODS: A sandwich ELISA was employed to detect the serum sLAIR-1 from tumor patients and healthy individuals. The expression of LAIR-1 in CD4+ T cells, CD8+ T cells, NK cells and B cells isolated from the peripheral blood of cancer patients was examined by fluorescence staining and flow cytometry. RESULTS: The level of serum sLAIR-1 in tumor patients was higher than that in healthy individuals [4.6+/-3.2 microg/L vs 3.9+/-3.0 microg/L, P<0.05]. NK cells, CD4+ T cells and CD8+ T cells expressed the up-regulated LAIR-1. The ratio of CD4/CD8 in lung cancer patients decreased significantly while the percentage of B cells in cancer patients increased greatly compared with healthy individuals. CONCLUSION: The expression of LAIR-1 is up-regulated in tumor patients, which may contribute to cancer immune escape.

[Identification of cell lines expressing the putative ligand(s) for LAIR-1(CD305) and LAIR-2(CD306)].

AIM: To identify cell lines expressing the putative ligand(s) for LAIR-1 and LAIR-2. METHODS: CHO cell lines secreting LAIR-1-Fc or LAIR-2-Fc fusion protein were prepared and the supernatants from the CHO cell lines were collected and purified by protein A affinity chromatography column. Several cell lines were stained with the purified LAIR-1-Fc and LAIR-2-Fc fusion proteins and then analyzed by flow cytometry for detecting the expression of their putative ligand(s). To exclude non-specific binding between the cells and the fusion proteins, the cell lines expressing their putative ligand(s) were pre-incubated with the mAbs against LAIR-1 or LAIR-2, stained with the fusion proteins, and analyzed by flow cytometry. RESULTS: Both LAIR-1 and LAIR-2 fusion proteins bound strongly with human amnion-derived epithelial cell line WISH, and moderately with human melanoma cell line C32, human embryo kidney epithelial cell line 293T, and human umbilical vein endothelial cell line ECV304. Furthermore, this binding could be blocked by the mAb FMU-LAIR-2.2 (recognizing both LAIR-1 and LAIR-2) or mAb FMU-LAIR-2.1(recognizing LAIR-2). CONCLUSION: The putative ligand(s) for LAIR-1 and LAIR-2 were expressed on WISH, C32, 293T and ECV304 cell lines. LAIR-1 and LAIR-2 may share the same ligand(s). These findings lay the foundation for the molecular cloning of the putative ligand(s) for LAIR-1 and LAIR-2.

[Expression and biology identification of the human epididymis-specific gene ESC42 in E. coli].

OBJECTIVE: To provide materials for the study of the function of ESC42 protein specifically expressed in the human epididymis. METHODS: The ESC42 gene was amplified from the human epididymis cDNA library by PCR and then cloned into prokaryotic expression vector pGEX-4T-1, expressed and purified by recombinant DNA techniques. The specificity of ESC42 protein was identified by Western blot and MALDI-TOF-MS. The database was searched by Ms-Fit. RESULTS: The recombinant plasmid expressed a Mr 38 x 10(3) fusion protein in E. coli at a level of 30% of the total protein, and the purity was as high as 99%. The ESC42 protein was identified by ESC42 monoclonal antibody and its molecular weight was 11 978.12, tested by MALDI-TOF-MS. The peptide mass fingerprint analysis showed that the coverage rate of the sequence reached 48% with 100% matching. The motif scan in Prosite database reveal that ESC42 belonged to the beta-defensin family and had antibacterial activity. CONCLUSION: Obtaining high purity of rhESC42 protein may lay a foundation for the study of its functions.

[Cloning, prokaryotic expression of LAIR and identification of their immunological reactivities].

AIM: To clone and express the leukocyte-associated immunoglobulin-like receptor (LAIR) and to identify the immune reactivity of LAIR-2 to anti-LAIR-1 specific monoclonal antibodies(mAb). METHODS: Genes encoding LAIR-1 and LAIR-2 were cloned by RT-PCR from human peripheral blood mononuclear cells(PBMC) and two leukemia cell lines Jurkat and HL-60. The extracellular region gene of LAIR1 and LAIR-2 were inserted into vector pGEX-4T-3 expressing GST fusion protein, expressed on IPTG induction and purified through glutathione-sepharose 4B column. The immunological reactivity of expressed LAIR-2 to anti-LAIR-1 mAb was identified by indirect ELISA. RESULTS: LAIR-1 and LAIR-2 cDNAs had been cloned and expressed. Five new LAIR-1 cDNA isoforms were cloned. Among them, two isoforms from HL-60 included LAIR-1 open reading frames (ORF) and three isoforms from Jurkat were LAIR-1 cDNA segments. The LAIR-1 and LAIR-2 showed different immunological reactivities. CONCLUSION: The transcription, processing after transcription and expression of LAIRs may be related to disparities in individuals and disease status. The difference in immunological reactivity may be involved in their structure.

[Preparation, characterization and application of monoclonal antibodies to Fc fragment of Ig fusion protein].

AIM: To prepare and characterize mAb to Fc fragment of Ig fusion protein, and to establish sandwich ELISA for detecting Ig fusion proteins and affinity chromatography method for Ig fusion protein purification. METHODS: hBCMA-Ig was used as antigen to immune BALB/c mice. The lymphocyte hybridoma technique was used to establish hybridoma cell lines stably secreting anti-Fc mAb. ELISA was employed to detect the isotype, epitopes, and species specificity of mAbs. Western blot was used to assess the reactivity between anti-Fc mAbs and denatured Ig fusion protein. LAIR1-Ig fusion protein was purified through affinity column anti-Fc mAb cross-linked to Sepharose 4B. RESULTS: Seven hybridoma cell lines(FMUFc1-FMUFc7) were acquired. A sandwich ELISA was successfully established using FMUFc4 as coating mAb and FMUFc5 as enzyme-labeled mAb. FMUFc6 could be used for Western blot. LAIR1-Ig fusion protein was effectively purified through FMUFc5-Sepharose affinity chromatography column. CONCLUSION: mAb against Fc fragment of Ig fusion protein has been prepared successfully, and methods to detect and purify Ig fusion protein are established. These mAbs provide useful tool for further application of Ig fusion protein.