2,3,5,4'- tetrahydroxystilbene-2-O-ß-D- glucopyranoside (TSG)-Driven immune response in the hepatotoxicity of Polygonum multiflorum.

ETHNOPHARMACOLOGICAL RELEVANCE: 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside (TSG) as the primary constituent of Polygonum multiflorum Thumb. (PM) possesses anti-oxidative, antihypercholesterolemic, anti-tumor and many more biological activities. The root of PM has been used as a tonic medicine for thousands of years. However, cases of PM-induced liver injury are occasionally reported, and considered to be related to the host immune status. AIM OF THE STUDY: The primary toxic elements and specific mechanisms PM causing liver damage are still not thoroughly clear. Our study aimed to investigate the influences of TSG on the immune response in idiosyncratic hepatotoxicity of PM. MATERIALS AND METHODS: The male C57BL/6 mice were treated with different doses of TSG and the alterations in liver histology, serum liver enzyme levels, proportions of T cells and cytokines secretion were evaluated by hematoxylin and eosin (HE), RNA sequencing, quantitative real time polymerase chain reaction (qRT-PCR), Flow cytometry (FCM), and enzyme-linked immunosorbent assay (ELISA), respectively. Then, primary spleen cells from drug-naive mice were isolated and cultured with TSG in vitro. T cell subsets proliferation and cytokines secretion after treated with TSG were assessed by CCK8, FCM and ELISA. In addition, mice were pre-treated with anti-CD25 for depleting regulatory T cells (Tregs), and then administered with TSG. Liver functions and immunological alterations were analyzed to evaluate liver injury. RESULTS: Data showed that TSG induced liver damage, and immune cells infiltration in the liver tissues. FCM results showed that TSG could activate CD4+T and CD8+T in the liver. Results further confirmed that TSG notably up-regulated the levels of inflammatory cytokines including TNF-α, IFN-γ, IL-18, perforin and granzyme B in the liver tissues. Furthermore, based on transcriptomics profiles, some immune system-related pathways including leukocyte activation involved in inflammatory response, leukocyte cell-cell adhesion, regulation of interleukin-1 beta production, mononuclear cell migration, antigen processing and presentation were altered in TSG treated mice. CD8+T/CD4+T cells were also stimulated by TSG in vitro. Interestingly, increased proportion of Tregs was observed after TSG treatment in vitro and in vivo. Foxp3 and TGF-ß1 mRNA expressions were up-regulated in the liver tissues. Depletion of Tregs moderately enhanced TSG induced the secretion of inflammatory cytokines in serum. CONCLUSIONS: Our findings showed that TSG could trigger CD4+T and CD8+T cells proliferation, promote cytokines secretion, which revealed that adaptive immune response associated with the mild liver injury cause by TSG administration. Regulatory T cells (Tregs) mainly sustain immunological tolerance, and in this study, the progression of TSG induced liver injury was limited by Tregs. The results of our investigations allow us to preliminarily understand the mechanisms of PM related idiosyncratic hepatotoxicity.

[Leptin promotes the proliferation and migration of MDA-MB-231 breast cancer cells by up regulating MMP14].

Objective To investigate the effect of matrix metalloproteinase 14 (MMP14) on the proliferation and migration of MDA-MB-231 human breast cancer cells treated with leptin. Methods MDA-MB-231 breast cancer cells were randomly divided into control group and (50, 100, 200, 400) ng/mL leptin treated groups. Real-time fluorescence quantitative PCR and Western blot were used to detect the expressions of MMP14 mRNA and protein in cancer cells. The MMP14 of MDA-MB-231 cells and leptin receptor genes were silenced and the silenced cells were stimulated with different concentrations of leptin, then cell proliferation was detected by MTT assay, cell migration was detected by scratch assay, and MMP14 protein expression was detected by Western blot. Results Compared with those in the control group, the mRNA and protein expressions of MMP14 increased in a dose-dependent manner in leptin treated groups. After knockdown of MMP14 and leptin receptor genes, the promoting effect of leptin on the proliferation and migration of MDA-MB-231 cells and the expression of MMP14 protein were weakened. Conclusion Leptin up-regulates the expression of MMP14 in MDA-MB-231 cells and promotes cell proliferation and migration.

THH Relieves CIA Inflammation by Reducing Inflammatory-related Cytokines.

Tripterygium hypoglaucum hutch (THH) is a plant of the genus tripterygium, which is also known as colquhounia, Gelsemiun elegan, and so on. It is mainly distributed in Yunnan, Guizhou, and Sichuan regions and other places in China. To study the immune mechanism of THH on related inflammatory cytokines in collagen II-induced arthritis (CIA) mice, healthy male C57BL/6 mice were used to model CIA mice. Mice received THH 420 mg/kg/day or the same amount of normal saline (NS) by gavage for 20 days. The thickness of the ankle joint in mice was observed, and the arthritis index was calculated. Related inflammatory cytokines were detected by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that after treatment with THH, the CIA mice had less swelling and destruction of the joints as well as decreased foot size and arthritis index. The mRNA and protein levels of TNF-α, IFN-γ, and IL-17A were lower in the THH-treated group than in the NS group (P < 0.05). In summary, THH has great significance in the treatment of CIA mice, including reduced related inflammatory cytokines expression level in both joint tissue and serum. The mechanism of THH in the treatment of CIA may be through the inhibition of the NF-kB-STAT3-IL-17 pathway, which also requires further experimental investigation.

Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen.

Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR) is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv) specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy.

A fully human chimeric immune receptor for retargeting T-cells to CEA-expressing tumor cells.

BACKGROUND: Recombinant chimeric immune receptors (CIRs) with anti-CEA specificity can retarget grafted T-cells to CEA-expressing tumors in an HLA-independent manner. To reduce the immunogenicity of conventional CIR in humans, an attempt was made to generate a CIR encoded by all human genes. MATERIALS AND METHODS: A single-chain variable fragmented (scFv) antibody gene was prepared from variable region genes of the C2-45 human mAb clone specific for CEA. The scFv gene was connected to a gene construct comprised of the cDNAs for the human CD8a hinge region, the human CD28 transmembrane and cytoplasmic domains, and the human CD3zeta intracellular domain. The resulting human CIR gene, designated L45scFv-CIR, was inserted into the pcDNA3.1 expression vector and transfected into human primary T-cells. RESULTS: Flow cytometric analysis using allophycocyanin-labeled CEA demonstrated the expression of the L45scFv-CIR protein on the T-cells and its specific antigen binding activity. CONCLUSION: This L45scFv-CIR gene, consisting of four human genes, may be a useful tool for eradication of CEA-expressing but HLA-downregulated tumor cells.

Possible applications of antibodies or their genes in cancer therapy.

In this review article the possible applications of anti-tumor-associated antigen (TAA) antibodies in the therapy of cancer have been summarized. First, recombinant monoclonal antibodies (MAbs) are increasingly being used as therapeutic agents, especially in combination with anti-cancer drugs. Second, conjugation of antibody therapy with toxins or radioisotopes offers more therapeutic approaches. Third, development of cytotoxic T-lymphocyte (CTL) or natural killer (NK)-cell populations with anti-TAA antibody activity may be important for the success of cancer immunotherapy because the downregulated HLA class I molecules and the non-ubiquitous expression of NK receptor ligands in tumor tissues constitute the major tumor escape mechanism facing tumor-specific CTL- and/or NK-cell-mediated responses. Finally, in cancer gene therapy, the strategies to target viral vectors carrying therapeutic genes to tumor tissues by modifying the tropisms with MAbs or their genes against TAAs are also very promising.

IgG isotype conversion of a novel human anti-carcinoembryonic antigen antibody to increase its biological activity.

BACKGROUND: The IgG isotype of antibodies is very important for their biological functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). To increase the biological activity of a novel human monoclonal antibody (C2-45) against carcinoembryonic antigen (CEA), we tried to genetically convert its isotype from IgG4 to IgG1. MATERIALS AND METHODS: VH and VL genes were cloned from the parental antibody C2-45 (IgG4) and inserted into the pAc-kappa-CH3 expression vector which contained the constant region gene of human IgG1. The recombinant gene was transfected into Sf9 insect cells to produce recombinant protein. The resulting recombinant protein, designated C2-45 (cIgG1), in the culture medium was purified by affinity chromatography and characterized for its CEA binding activity and biological activity. RESULTS: The converted C2-45 (cIgG1) retained the original antigen-binding activity and showed significantly higher CDC and ADCC activities against CEA-expressing tumor cells than did the original C2-45 (IgG4). CONCLUSION: C2-45 (cIgG1) may be useful for antibody-based immunotherapy of human CEA-expressing tumors.

Lipopolysaccharide regulates biosynthesis of cystathionine γ-lyase and hydrogen sulfide through Toll-like receptor-4/p38 and Toll-like receptor-4/NF-κB pathways in macrophages.

Hydrogen sulfide (H2S), formed mainly by the enzyme cystathionine γ-lyase (CSE) in macrophages, is emerging as a novel regulator in inflammation. Although elevated production of H2S has been shown in inflammatory processes, the underlying molecular mechanism remains to be further elucidated. In this study, we compared parallel TLR4 knockout (TLR4(-/-)) mice with their wild-type counterparts following lipopolysaccharide (LPS) treatment. It showed that LPS increased the expressions of CSE and biosynthesis of H2S in C57BL/6 mice both in vivo and in vitro. However, the effects of LPS were not present in TLR4(-/-) mice, indicating the crucial role of TLR4 in LPS-induced expression of CSE and biosynthesis of H2S. We subsequently used JNK inhibitor, P38 inhibitor, and ERK inhibitor to block the downstream MAPK pathways of TLR4 in macrophages, and found that LPS-induced CSE expression and H2S synthesizing activity were inhibited by pretreatment with the p38 inhibitor. Similarly, the NF-κB inhibitor (BAY 11-7082) reversed the effects of LPS. These results suggest that LPS increases the biosynthesis of CSE and H2S in macrophages mainly in a TLR4-p38-dependent and TLR-4-NF-κB-dependent manner. These findings expand our knowledge of H2S biosynthesis during inflammation and provide a foundation for the development of novel H2S-based therapies.