Discovery of RORγ Allosteric Fluorescent Probes and Their Application: Fluorescence Polarization, Screening, and Bioimaging.

Retinoic acid receptor-related orphan receptor γ (RORγ) acts as a crucial transcription factor in Th17 cells and is involved in diverse autoimmune disorders. RORγ allosteric inhibitors have gained significant research focus as a novel strategy to inhibit RORγ transcriptional activity. Leveraging the high affinity and selectivity of RORγ allosteric inhibitor MRL-871 (1), this study presents the design, synthesis, and characterization of 11 allosteric fluorescent probes. Utilizing the preferred probe 12h, we established an efficient and cost-effective fluorescence polarization-based affinity assay for screening RORγ allosteric binders. By employing virtual screening in conjunction with this assay, 10 novel RORγ allosteric inhibitors were identified. The initial SAR studies focusing on the hit compound G381-0087 are also presented. The encouraging outcomes indicate that probe 12h possesses the potential to function as a powerful tool in facilitating the exploration of RORγ allosteric inhibitors and furthering understanding of RORγ function.

The role of autophagy-related proteins in the pathogenesis of neuromyelitis optica spectrum disorders.

This study aimed to investigate the expression of autophagy-related proteins in a mouse model of neuromyelitis optica (NMO). Mice were assigned to one of four groups: an animal experimental model group (NMO-EAE group, given with exogenous IL-17A), Interleukin-17 monoclonal antibody intervention group (NMO-EAE_0IL17inb), No exogenous interleukin-17 enhanced immune intervention group (NMO-EAE_0IL17), and a control group. Behavioral scores were assessed in each group, and the protein expressions of sequestosome 1 (P62), Beclin-1, the mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K-I), and LC3II/LC3I were detected using Western blotting. In the NMO-EAE_0IL17 group, the expression of Beclin-1 decreased, the LC3II/LC3I ratio was lower, and the expressions of P62, mTOR, and PI3K-I increased; after administration of IL-17A inhibitor into the brain tissue, however, the expression of Beclin-1 increased significantly, along with the LC3II/LC3I ratio, while the expressions of P62, mTOR and PI3K-I protein decreased significantly. In terms of behavioral scores, the scores of optic neuritis and myelitis were more serious, onset occurred earlier and the progress was faster, after the administration of IL-17A. In the mechanism of NMO animal model, IL-17A may regulate autophagy and affect the disease process through the activation of the PI3K-mTOR signaling pathway.

Senescent mesenchymal stem/stromal cells and restoring their cellular functions.

Mesenchymal stem/stromal cells (MSCs) have various properties that make them promising candidates for stem cell-based therapies in clinical settings. These include self-renewal, multilineage differentiation, and immunoregulation. However, recent studies have confirmed that aging is a vital factor that limits their function and therapeutic properties as standardized clinical products. Understanding the features of senescence and exploration of cell rejuvenation methods are necessary to develop effective strategies that can overcome the shortage and instability of MSCs. This review will summarize the current knowledge on characteristics and functional changes of aged MSCs. Additionally, it will highlight cell rejuvenation strategies such as molecular regulation, non-coding RNA modifications, and microenvironment controls that may enhance the therapeutic potential of MSCs in clinical settings.

[Recurrent skin blisters for more than 7 months in a girl aged 15 months].

A girl, aged 15 months, attended the hospital due to recurrent skin erythema, blisters, and desquamation for more than 7 months. Giemsa staining and immunohistochemical staining showed mast cell infiltration and degranulation. Hematoxylin staining showed spinous layer edema and blister formation under the epidermis, with a large amount of serous fluid and a small number of inflammatory cells in the blister. Marked edema was observed in the dermis, with diffused mononuclear cell infiltration. The girl was diagnosed with mastocytosis. Mastocytosis should be considered for children with recurrent skin erythema and blisters.

Prognostic value of soluble MICA levels in the serum of patients with advanced hepatocellular carcinoma.

Serum levels of soluble MHC class I-related chain A (sMICA) are related with the prognosis of various types of cancer; however, few studies on the prognostic value of sMICA in hepatocellular carcinoma (HCC) have been reported. In this study, we retrospectively investigated the relationship between sMICA levels and clinical features of advanced HCC, and we assessed the prognostic value of sMICA in advanced HCC. Furthermore, the relationship of serum sMICA levels and natural killer group 2, member D (NKG2D) expression on natural killer (NK) cells was also evaluated. We detected sMICA levels in the serum of 60 advanced HCC patients using enzyme-linked immunosorbent assay (ELISA) and measured expression levels of NKG2D on NK cells using flow cytometry. We found that serum sMICA levels in HCC patients were in the range of 0.10-6.21 ng/mL. Chi-square analyses showed that sMICA level was significantly related with only tumor size. Survival analysis showed that a high sMICA level was significantly related with poor prognosis among HCC patients. Multivariate analyses indicated that sMICA was an independent prognostic factor. In addition, the levels of CD56+NKG2D+ NK cells were within the range of 11.2%-55.4%, and correlation analyses indicated that sMICA level was negatively correlated with the level of NKG2D+ NK cells. Our results suggest that serum sMICA levels may be an independent prognostic factor for advanced HCC.

Characterization of bridging integrator 1 (BIN1) as a potential tumor suppressor and prognostic marker in hepatocellular carcinoma.

It has been shown that bridging integrator 1 (BIN1) can interact with c-myelocytomatosis (c-Myc) oncoprotein in cancer. However, the role of BIN1 in hepatocellular carcinoma (HCC) is not clear. In the present study, we investigated the expression and prognostic role of BIN1 in primary HCC and evaluated the function of BIN1 in hepatocarcinogenesis. Using real-time polymerase chain reaction and Western blot analysis, we found significantly decreased expression of BIN1 in primary HCC tumor tissues (n = 42) compared with adjacent normal tissues and in HCC cell lines. Immunohistochemistry analysis also found decreased BIN1 expression in HCC tumor tissues (n = 117). In clinicopathological analysis, loss of BIN1 expression correlated significantly (P < 0.05) with differentiation scores and tumor size. Importantly, decreased expression of BIN1 in tumors was found to be closely associated with a poor prognosis, and we conclude that BIN1 was an independent prognostic factor in a multivariate analysis. In mechanistic studies, restoring BIN1 expression in BIN1-null HCC cells significantly inhibited cell proliferation and colony formation and induced apoptosis of HCC cells. Furthermore, we found that BIN1 overexpression could significantly suppress the motility and invasion of HCC cells in vitro. Our results indicate that BIN1 may function as a potential tumor suppressor and serve as a novel prognostic marker in HCC patients. The BIN1 molecule might play an important role in tumor growth, cell motility and invasion. Modulation of BIN1 expression may lead to clinical applications of this critical molecule in the control of hepatocellular carcinoma as well as in early and effective diagnosis of this aggressive tumor.

Induction of DIMBOA accumulation and systemic defense responses as a mechanism of enhanced resistance of mycorrhizal corn (Zea mays L.) to sheath blight.

Arbuscular mycorrhizas are the most important symbioses in terrestrial ecosystems and they enhance the plant defense against numerous soil-borne pathogenic fungi and nematodes. Two corn (Zea mays) varieties, Gaoyou-115 that is susceptible to sheath blight disease caused by Rhizoctonia solani and Yuenong-9 that is resistant, were used for mycorrhizal inoculation in this study. Pre-inoculation of susceptible Gaoyou-115 with arbuscular mycorrhizal fungus (AMF) Glomus mosseae significantly reduced the disease incidence and disease severity of sheath blight of corn. HPLC analysis showed that AMF inoculation led to significant increase in 2,4-dihydroxy-7-methoxy-2 H-1,4-benzoxazin-3(4 H)-one (DIMBOA) accumulation in the roots of both corn varieties and in leaves of resistant Yuenong-9. R. solani inoculation alone did not result in accumulation of DIMBOA in both roots and leaves of the two corn varieties. Our previous study showed that DIMBOA strongly inhibited mycelial growth of R. solani in vitro. Real-time PCR analysis showed that mycorrhizal inoculation itself did not affect the transcripts of most genes tested. However, pre-inoculation with G. mosseae induced strong responses of three defense-related genes PR2a, PAL, and AOS, as well as BX9, one of the key genes in DIMBOA biosynthesis pathway, in the leaves of corn plants of both Yuenong-9 and Gaoyou-115 after the pathogen attack. Induction of defense responses in pre-inoculated plants was much higher and quicker than that in non-inoculated plants upon R. solani infection. These results indicate that induction of accumulation of DIMBOA, an important phytoalexin in corn, and systemic defense responses by AMF, plays a vital role in enhanced disease resistance of mycorrhizal plants of corn against sheath blight. This study also suggests that priming is an important mechanism in mycorrhiza-induced resistance.

Decreased expression of XPO4 is associated with poor prognosis in hepatocellular carcinoma.

BACKGROUND AND AIM: Exportin 4 (XPO4) is a recently-discovered candidate tumor-suppressor gene identified in a liver cancer mouse model. To investigate the role of XPO4 in hepatocellular carcinoma (HCC) pathogenesis, we determined XPO4 expression and its correlation to prognosis in human primary HCC. METHODS: The XPO4 mRNA transcription level in HCC cell lines and tissue samples were detected by real-time quantitative polymerase chain reaction (PCR). XPO4 protein expression in 123 primary HCC clinical surgical specimens were analyzed by immunohistochemical detection. RESULTS: Real-time quantitative PCR showed a decrease in XPO4 expression in HCC cell lines BEL-7402, Hep-G2, and SK-hep1 compared to the normal liver cell line LO2. Decreased XPO4 mRNA was also found in the majority of tumor tissues compared with matched non-tumor liver tissues (P = 0.004). Immunohistochemical detection revealed that XPO4 expression was reduced in 51 of 123 (41.5%) tumor resection samples compared with adjunct non-tumor tissues. We also found XPO4 expression to be significantly correlated with tumor size (P = 0.045) and histopathological classification (P = 0.004). Kaplan-Meier survival curves showed that the downregulation of XPO4 resulted in a significantly poor prognosis (P = 0.008, log-rank test), and multivariate Cox's analysis showed that XPO4 expression was an independent prognostic factor for overall survival of HCC patients (P = 0.013). CONCLUSIONS: Our data suggest that XPO4 could be involved in the progression of human HCC and could serve as a potential target for gene therapy in the treatment of HCC.

Intratumoral expression of IL-17 and its prognostic role in gastric adenocarcinoma patients.

In this study, we characterized the intratumoral expression of IL-17 and CD8(+) TILs in gastric adenocarcinoma patients after resection and determined the correlation between the survival probability of gastric adenocarcinoma patients and the expression of IL-17 in tumor. Expression of IL-17 and CD8 was assessed by immunohistochemistry, and the prognostic effects of intratumoral IL-17 expression and CD8(+) TILs were evaluated by Cox regression and Kaplan-Meier analysis. Immunohistochemical detection revealed the presence of IL-17 and CD8(+) cells in gastric adenocarcinoma tissue samples (90.6%, 174 out of 192 patients and 96.9%, 186 out of 192 patients, respectively). We have also found that intratumoral IL-17 expression was significantly correlated with age (p=0.004) and that the number of CD8(+)TILs was significantly correlated with UICC staging (p=0.012) and the depth of tumor invasion (p=0.022). The five-year overall survival probability among patients intratumorally expressing higher levels of IL-17 was significantly better than those expressing lower levels of IL-17 (p=0.036). Multivariate Cox proportional hazard analyses revealed that intratumoral IL-17 expression (HR: 0.521; 95% CI: 0.329-0.823; p=0.005) was an independent factor affecting the five-year overall survival probability. We conclude that low levels of intratumoral IL-17 expression may indicate poor prognosis in gastric adenocarcinoma patients.

Comparative analysis of cytotoxic T lymphocyte response induced by dendritic cells loaded with hepatocellular carcinoma -derived RNA or cell lysate.

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.

High expression level of EDIL3 in HCC predicts poor prognosis of HCC patients.

AIM: To determine the role of epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3) in pathogenesis of hepatocellular carcinoma (HCC) by investigating the EDIL3 expression in HCC and its prognostic value for HCC. METHODS: EDIL3 expression was detected in 101 HCC surgical tissue samples with immunohistochemistry method, and its relation with clinicopathologic features and prognosis of HCC patients was analyzed. RESULTS: EDIL3 was highly expressed in 48.5% of the HCC patients. Although the EDIL3 expression level did not correlate with any clinicopathological parameters, Kaplan-Meier survival analysis showed that high expression level of EDIL3 resulted in a significantly poor prognosis of HCC patients (log-rank test, P = 0.010). Multivariate Cox's analysis showed that the EDIL3 expression level was a significant and independent prognostic parameter for the overall survival rate of HCC patients (hazard ratio = 1.978, 95% confidence interval = 1.139-3.435, P = 0.015). CONCLUSION: High expression level of EDIL3 predicts poor prognosis of HCC patients. EDIL3 may be a potential target of antiangiogenic therapy for HCC.

Comparative study on anti-tumor immune response of autologous cytokine-induced killer (CIK) cells, dendritic cells-CIK (DC-CIK), and semi-allogeneic DC-CIK.

BACKGROUND AND OBJECTIVE: Cytokine-induced killer (CIK) cells and autologous dendritic cells-CIK (DC-CIK) cells co-cultured with autologous dendritic cells (DCs) and CIK cells are commonly used for immunotherapy recently. We compared the anti-tumor immune response of CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells to explore a more effective anti-tumor adoptive immunotherapy approach. METHODS: Peripheral monocytes were isolated from patients with renal carcinoma, lung cancer, or maxillary squamous cell carcinoma and their healthy adult children. Isolated cells were cultured and induced as DCs and CIK cells in vitro. CIK cells from patients were co-cultured with autologous DCs and DCs from their children respectively, generating DC-CIK cells and semi-allogeneic DC-CIK cells. The anti-tumor activities of autologous CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells were measured by LDH assay. Intracellular staining was used to test the secretion of cytokines. Flow cytometry was applied for detecting the phonotype changes of these three types of cells. Cell proliferation and cell apoptosis were detected by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and Annexin V/PI respectively. RESULTS: Compared with autologous CIK cells and DC-CIK cells, semi-allogeneic DC-CIK cells significantly enhanced the anti-tumor activity and IFN-gamma secretion, reduced IL-4 secretion, increased the ratio of CD3(+)CD56(+) cells and CD3(+)CD8(+) cells, decreased the number of CD4(+)CD25(+) cells, promoted cell proliferation, and lessened cell apoptosis. CONCLUSIONS: Semi-allogeneic DC-CIK cells had a stronger anti-tumor effect than did autologous CIK cells and DC-CIK cells. Our results provided experimental evidence for clinical application of DC-CIK cells.

Dendritic cells-mediated CTLs targeting hepatocellular carcinoma stem cells.

Immunotherapy, especially using dendritic cells (DCs)-based vaccine, appears promising in the treatment of hepatocellular carcinoma (HCC) following surgery. However, the therapeutic efficacy of current DC vaccines loaded with HCC antigen is limited in clinical practice. One important reason might be that the DC vaccines for the treatment of HCC were not aimed at targeting the hepatocellular carcinoma cancer stem cells (HCCCSCs). Therefore, establishing an immunotherapy to kill HCC stem cells could be a novel therapeutic strategy. In this study, we have developed an immunotherapy to target CD133(+) HCC cells in the treatment of HCC. This study had three main findings; (1) CD133(+)HCC cells RNA loaded DCs could induce special CD8(+) cytotoxic T lymphocytes (CD133(+)Huh7-CTLs) response against CD133(+) Huh7 cells in vitro. (2) Huh7 cells-induced tumor growth in vivo was effectively inhibited by CD133(+)Huh7-CTLs. (3) the great inhibition potential of CD133(+)Huh7-CTLs to Huh7-induced tumor growth might not be only associated with anti-tumor cytokines such as IFNγ, but also to CD133(+)Huh7-DCs induced specific CTLs. This study shows an experimental proof that CD133(+)HCC cells RNA loaded DC vaccine has potential in treating HCC and may provide a new therapy for clinical post operative adjuvant therapy in future.

Decreased expression of Neurensin-2 correlates with poor prognosis in hepatocellular carcinoma.

AIM: To investigate the expression of Neurensin-2 (NRSN2) in hepatocellular carcinoma (HCC) and its prognostic values in predicting survival. METHODS: The expression and prognostic significance of NRSN2 in HCC was examined by performing immunohistochemical analysis using a total of 110 HCC clinical tissue samples, and Western blotting analysis to further confirm the result. RESULTS: Decreased NRSN2 expression was shown in 70.9% cases. Loss of NRSN2 expression in HCC was significantly related to tumor size (P = 0.006). Larger tumor size was related to negative expression of NRSN2. Patients showing negative NRSN2 expression had a significantly shorter overall survival than those with positive expression (P = 0.008). Multivariate Cox regression analysis indicated that NRSN2 expression level was an independent factor of survival (P = 0.013). Western blotting analysis further confirmed decreased expression of NRSN2 in tumor tissues compared with non-tumorous tissues. CONCLUSION: Our study indicated that NRSN2 could be a tumor suppressor gene for HCC and a candidate biomarker for long-term survival in HCC.

The pivotal role of p38 and NF-kappaB signal pathways in the maturation of human monocyte-derived dendritic cells stimulated by streptococcal agent OK-432.

OK-432, a streptococcal preparation, has been shown as an effective activator to induce human monocyte-derived dendritic cells maturation. During this process, the activation of Toll-like receptor 4 plays an important role. However, the signaling pathway involved in has not been fully understood. In the present study, we investigated the underlying mechanisms, by which OK-432 induced maturation of human monocyte-derived DCs (MoDCs). We observed that exposure of immature MoDCs to OK-432 activated the p38 MAPK and NF-kappaB pathway, accompanied up-regulated the surface expression of maturation markers CD80, CD83, CD86 and HLA-DR, increased secretion of TNF-alpha, IL-12 and chemokine, IP-10. In addition, T cells stimulatory capacity was also enhanced. The maturation of MoDCs stimulated by OK-432 was inhibited by treatment with p38 pathway inhibitor, SB203580, or NF-kappaB pathway inhibitor, BAY-117082. Whereas, blocking of JNK pathway with SP600125 or ERK pathway with PD98059 did not influence OK-432-induced DCs maturation. Taken together, our data indicated that OK-432-induced DCs maturation was due, at least partly to the activation of p38 and NF-kappaB pathway.