FASN Inhibition Decreases MHC-I Degradation and Synergizes with PD-L1 Checkpoint Blockade in Hepatocellular Carcinoma.

Immune checkpoint inhibitors (ICI) transformed the treatment landscape of hepatocellular carcinoma (HCC). Unfortunately, patients with attenuated MHC-I expression remain refractory to ICIs, and druggable targets for upregulating MHC-I are limited. Here, we found that genetic or pharmacologic inhibition of fatty acid synthase (FASN) increased MHC-I levels in HCC cells, promoting antigen presentation and stimulating antigen-specific CD8+ T-cell cytotoxicity. Mechanistically, FASN inhibition reduced palmitoylation of MHC-I that led to its lysosomal degradation. The palmitoyltransferase DHHC3 directly bound MHC-I and negatively regulated MHC-I protein levels. In an orthotopic HCC mouse model, Fasn deficiency enhanced MHC-I levels and promoted cancer cell killing by tumor-infiltrating CD8+ T cells. Moreover, the combination of two different FASN inhibitors, orlistat and TVB-2640, with anti-PD-L1 antibody robustly suppressed tumor growth in vivo. Multiplex IHC of human HCC samples and bioinformatic analysis of The Cancer Genome Atlas data further illustrated that lower expression of FASN was correlated with a higher percentage of cytotoxic CD8+ T cells. The identification of FASN as a negative regulator of MHC-I provides the rationale for combining FASN inhibitors and immunotherapy for treating HCC. SIGNIFICANCE: Inhibition of FASN increases MHC-I protein levels by suppressing its palmitoylation and lysosomal degradation, which stimulates immune activity against hepatocellular carcinoma and enhances the efficacy of immune checkpoint inhibition.

Responses to Crizotinib therapy in five patients with non-small-cell lung cancer who tested FISH negative and Ventana immunohistochemistry positive for ALK fusions.

AIM: Although immunohistochemistry (IHC) and reverse transcription-PCR can detect ALK rearrangements, the ALK break-apart FISH assay is currently considered the standard method. MATERIALS & METHODS: Five patients with advanced non-small-cell lung cancer, who had an ALK-negative FISH result that was later confirmed as positive by the Ventana IHC assay, were studied. Four had previously received chemotherapy or radiotherapy. All five were subsequently treated with Crizoitinib 250 mg twice daily. RESULTS & CONCLUSION: Four patients had a partial response to Crizotinib and one had stable disease. IHC is an efficient technique for diagnosing ALK rearrangements in patients with non-small-cell lung cancer, and may serve as an alternative to FISH in clinical practice.

BTB/POZ domain-containing protein 7: epithelial-mesenchymal transition promoter and prognostic biomarker of hepatocellular carcinoma.

UNLABELLED: Epithelial-mesenchymal transition (EMT) is a critical step in the metastasis of hepatocellular carcinoma (HCC). BTB/POZ domain-containing protein 7 (BTBD7) regulates EMT-associated proteins implicated in HCC progression. However, the role(s) of BTBD7 in HCC have not been identified. Using highly metastatic HCC HCCLM3 cells, immortalized L02 hepatocytes, metastatic HCC animal models, and three independent cohorts of HCC patient specimens, we aimed to determine the involvement of BTBD7 in HCC metastasis. We show that BTBD7 messenger RNA and protein was highly expressed in HCC cells and tumor tissues, with such expression being associated with: enhanced cell motility, venous invasion, and poor prognosis. BTBD7 promoted HCC angiogenesis and metastasis in vitro and in vivo, but did not influence cell proliferation or colony formation. BTBD7 enhancement of HCC invasion and EMT phenotype occurred through activation of a RhoC-Rock2-FAK-signaling pathway, resulting in matrix metalloproteinase-2/9 production and microvessel formation. Applying a predictive risk score model, Cox regression analysis revealed that high BTBD7 expression integrated with high microvessel density was a powerful independent predictive factor of HCC clinical outcome. CONCLUSION: The present study identifies BTBD7 as a novel candidate prognostic factor and a potential therapeutic target of HCC. (HEPATOLOGY 2013; 57:2326-2337).

A reusable capacitive immunosensor for detection of Salmonella spp. based on grafted ethylene diamine and self-assembled gold nanoparticle monolayers.

Fabrication of a novel capacitive immunosensor based on grafted ethylene diamine and self-assembled gold nanoparticle monolayer on glassy carbon electrode for the detection of Salmonella spp. is described for the first time. In the present study, the Salmonella spp. monoclonal antibodies (denoted as McAbs) was immobilized on gold nanoparticles. Interaction of McAbs and Salmonella spp. was detected directly using the electrochemical impedance spectroscopy (EIS) technique. The experimental results showed that the concentration of antigen was measured through the relative change in capacitance in the corresponding specific binding of Salmonella spp. and McAbs. Under the optimized conditions, the relative changes in capacitance were proportional to the logarithmic values of Salmonella spp. concentrations in the range of 1.0 x 10(2) to 1.0 x 10(5) CFU mL(-1) (r = 0.991) with the detection limit of 1.0 x 10(2) CFU mL(-1). The stability of proposed immunosensor could be estimated by determining the relative change in capacitance, which remained almost the same in two months and decreased gradually to 85.3% of initial value after four months' storage. The used immunosensor could be regenerated repeatedly by immersing in glycine-HCl buffer solution (pH 2.8). Finally, the proposed immunosensor was successfully used for the detection of Salmonella spp. in lab-processed commercial pork samples.

[Safety and efficacy of attenuated Salmonella typhimurium harbouring DNA vaccine against Newcastle disease virus].

A pair of primers were designed and synthesized according to the previously published sequence of fusion protein (F) gene of Newcastle disease virus (NDV) and used to amplify F gene by reverse-transcription polymerase chain reaction (RT-PCR) from the genomic RNA of a NDV strain JS5 isolated from goose. The PCR product was identified by sequencing. Then recombinant eukaryotic expression vector pVAX1-F was constructed through inserting F gene into MCS of pVAX1. The recombinant plasmid pVAX1-F was transfected in COS-7 cells, and identified for the transient expression of F gene by indirect immunofluorescent assay. Finally, the recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was screened and designated as SL7207 (pVAX1-F). It was verified that SL7207 (pVAX1-F) as the oral NDV DNA vaccine was safe for chickens after oral immunization at dosage of 10(10) CFU or below. 1-day-old commercial ISA brown chickens were immunized orally with SL7207 (pVAX1-F) at two different dosages (10(9) CFU and 10(8) CFU) on day 1, 14 and 28. On day 7 after the last immunization, no significant difference was observed in the body weight between these two groups (p > 0.05), and also no significant difference between those two groups and negative control group (p > 0.05). Since there were maternal antibodies, high ELISA titers of serum antibodies against NDV were detected in the chickens of all groups on day 14. However, the levels of serum antibodies were decreased in the chickens of all groups on day 28, but the anti-NDV antibody response detected in the sera of chickens immunized with SL7207 (pVAX1-F) at the dosage of 10(9) CFU were increased and significantly higher than the response induced by immunization with SL7207 (pVAX1) on day 35 (p < 0.05). Intestinal mucosal immune response was observed in chickens immunized with SL7207 (pVAX1-F) at the dosage of 10(9) CFU or 10(8) CFU. The high ELISA titers of antibodies against NDV in small intestinal mucosal samples from immunized chickens were on day 28 and 35. After challenged intranasally with virulent NDV strain F48E8, the chickens immunized with SL7207 (pVAX1-F) at the dosage of 10(9) CFU could be protected with the protective rate of 77.27%, significantly higher than those with SL7207 (pVAX1) (p < 0.05). In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium was safe and has good immunogenicity for chickens. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of ND in the poultry.

[Preparation and characterization of monoclonal antibodies against the hemagglutinin of H9 subtype of avian influenza virus].

AIM: To prepare monoclonal antibodies (mAb) against the hemagglutinin(HA) of H9 subtype of avian influenza virus (AIV). METHODS: 8 week-old female BALB/c mice were immunized with the inactivated vaccine of H9 subtype of AIV. Splenocytes from the immunized mice were fused with Sp2/0 myeloma cells, and positive hybridoma clones were screened by indirect ELISA and hemagglutination inhibition test (HI). The specificity of the mAb was characterized by ELISA, HI test, indirect immunofluorescence (IF) staining and Western blot. RESULTS: Three hybridoma cell lines named 2H1, 2A3 and 1C8 against HA of AIV H9 were obtained. The HI titers of 3 mAbs were 1 x 2(8)-1 x 2(13), and the ELISA titers were 1 x 10(-7), 1 x 10(-5) and 5 x 10(-6), respectively. The immunoglobulin subclass of all 3 mAbs was IgG1. Western blot analysis confirmed that mAb 2H1 could recognize HA and reacted to 31 out of 32 isolates of H9 subtype of AIV. CONCLUSION: Three mAbs recognizing HA of H9 subtype of AIV were obtained, which may provide an useful tool for the antigenic analysis, the serological diagnosis, the epidemiological survey and the evaluation of AIV vaccine.