Xiu-Min Zhang

Perilipin 5 improves hepatic lipotoxicity by inhibiting lipolysis.

Abnormal metabolism of nonesterified fatty acids (NEFAs) and their derivatives has been reported to be the main cause of intracellular lipotoxic injury. Normally, NEFAs are stored in lipid droplets (LDs) in the form of triglyceride (TG), which could reduce the lipotoxicity of cytosolic NEFAs. Previous studies have implicated that Perilipin 5 (Plin5), an LD‐binding protein, regulates the storage and hydrolysis of TG in LD. However, its roles and underlying mechanisms in the liver remain unknown. Here we found that Plin5 expression was increased in steatotic livers. Using Plin5 knockout mice, we found that Plin5 deficiency resulted in reduced hepatic lipid content and smaller‐sized LDs, which was due to the elevated lipolysis rate and fatty acid utilization. Plin5‐deficient hepatocytes showed increased mitochondria proliferation, which could be explained by the increased expression and activity of PPARα stimulated by the increased NEFA levels. Meanwhile, Plin5‐deficient livers also exhibited enhanced mitochondrial oxidative capacity. We also found that Plin5 deficiency induces lipotoxic injury in hepatocytes, attributed to lipid peroxidation. Mechanistically, we found that Plin5 blocks adipose triglyceride lipase (ATGL)‐mediated lipolysis by competitively binding to comparative gene identification‐58 (CGI‐58) and disrupting the interaction between CGI‐58 and ATGL. Conclusion: Plin5 is an important protective factor against hepatic lipotoxicity induced by NEFAs generated from lipolysis. This provides an important new insight into the regulation of hepatic lipid storage and relation between lipid storage and lipotoxicity. (Hepatology 2015;61:870–882)

The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following peroral administration route

Previous studies have shown that there are profuse lymphatic tissues under the intestinal mucous membrane. Moreover, vaccine administered orally can elicit both mucous membrane and system immune response simultaneously, accordingly induce tumor-specific cytotoxic T lymphocyte. As a result, the oral route is constituted the preferred immune route for vaccine delivery theoretically. However, numerous vaccines especially protein/peptide vaccines remain poorly available when administered by this route. Nanoemulsion has been shown as a useful vehicle can be developed to enhance the antitumor immune response against antigens encapsulated in it and it is good for the different administration routes. Of particular interest is whether the protein vaccine following peroral route using nanoemulsion as delivery carrier can induce the same, so much as stronger antitumor immune response to following conventional ways such as subcutaneous (sc.) or not. Hence, in the present study, we encapsulated the MAGE1-HSP70 and SEA complex protein in nanoemulsion as nanovaccine NE (MHS) using magnetic ultrasound method. We then immuned C57BL/6 mice with NE (MHS), MHS alone or NE (-) via po. or sc. route and detected the cellular immunocompetence by using ELISpot assay and LDH release assay. The therapeutic and tumor challenge assay were examined then. The results showed that compared with vaccination with MHS or NE (-), the cellular immune responses against MAGE-1 could be elicited fiercely by vaccination with NE (MHS) nanoemulsion. Furthermore, encapsulating MHS in nanoemulsion could delay tumor growth and defer tumor occurrence of mice challenged with B16-MAGE-1 tumor cells. Especially, the peroral administration of NE (MHS) could induce approximately similar antitumor immune responses to the sc. administration, but the MHS unencapsulated with nanoemulsion via po. could induce significantly weaker antitumor immune responses than that via sc., suggesting nanoemulsion as a promising carrier can exert potent antitumor immunity against antigen encapsulated in it and make the tumor protein vaccine immunizing via po. route feasible and effective. It may have a broad application in tumor protein vaccine.

Heat shock protein 70/MAGE-3 fusion protein vaccine can enhance cellular and humoral immune responses to MAGE-3 in vivo

MAGE-3, a member of melanoma antigen (MAGE) gene family, is recognized as an ideal candidate for tumor vaccine because it is expressed in a significant proportion of tumors of various histological types and can induce antigen-specific immune response in vivo. There is now substantial evidence that heat shock proteins (HSPs) isolated from cancer cells and virus-infected cells can be used as vaccines to produce cancer-specific or virus-specific immunity. In this research, we investigated whether M. tuberculosis HSP70 can be used as vehicle to elicit immune response to its accompanying MAGE-3 protein. A recombinant protein expression vector was constructed that permitted the production of fusion protein linking amino acids 195–314 of MAGE-3 to the C terminus of HSP70. We found that HSP70-MAGE-3 fusion protein can elicit stronger cellular and humoral immune responses against MAGE-3 expressing murine tumor than those elicited by MAGE-3 protein in vivo, which resulted in potent antitumor immunity against MAGE-3-expressing tumors. Covalent linkage of HSP70 to MAGE-3 was necessary to elicit immune response to MAGE-3. These results indicate that linkage of HSP70 to MAGE-3 enhanced immune responses to MAGE-3 in vivo and HSP70 can be exploited to enhance the cellular and humoral immune responses against any attached tumor-specific antigens.

Dendritic cells fused with allogeneic breast cancer cell line induce tumor antigen-specific CTL responses against autologous breast cancer cells

Dendritic cell (DC)/tumor cell fusion vaccine has been revealed as a promising tool for the antitumor immunotherapy. Previous research has shown that fusion hybrids of human DCs and autologous tumor cells can induce cytotoxic T lymphocyte (CTL) responses against autologous tumor cells in animal models and human clinical trials. However, a major restriction factor for the clinical use is the difficulty for preparation of sufficient amount of autologous tumor cells especially for the patients with metastasis cancer whose primary tumor lesion is not clear or has been resected. In this study, allogeneic breast cancer cell line MCF-7 cells were electrofused to autologous DCs from patient with breast cancer as a strategy to deliver shared breast cancer antigens to DCs. Fusion cells generated by autologous DCs and allogeneic MCF-7 were able to induce autologous T lymphocytes proliferation, high levels of IFN-gamma production and CTL responses. CTLs induced by DCs/allogeneic MCF-7 fusion cells were able to kill autologous breast cancer cells in an antigen specific and HLA restriction manner. Our study may provide the experiment basis for the use of allogeneic breast cancer cell line in the DC/tumor cell fusion cell vaccination strategy.

Heat shock protein 70 / MAGE-1 tumor vaccine can enhance the potency of MAGE-1–specific cellular immune responses in vivo

The cancer-testis antigen encoded by the MAGE-1 gene is an attractive antigen in tumor immunotherapy because it can be processed as a foreign antigen by the immune system and generate tumor-specific cellular immune response in vivo. However, increase of the potency of MAGE-1 DNA vaccines is still needed. The high degree of sequence homology and intrinsic immunogenicity of heat shock protein 70 (HSP70) have prompted the suggestion that HSP70 might have immunotherapeutic potential, as HSP70 purified from malignant and virally infected cells can transfer and deliver antigenic peptides to antigen-presenting cells to elicit peptide-specific immunity. In this research, we evaluated the enhancement of linkage of Mycobacterium tuberculosis HSP70 to MAGE-1 gene of the potency of antigen-specific immunity elicited by naked DNA vaccines. We found that vaccines containing MAGE-1-HSP70 fusion genes enhanced the frequency of MAGE-1–specific cytotoxic T cells in contract to vaccines containing the MAGE-1 gene alone. More importantly, the fusion converted a less effective DNA vaccine into one with significant potency against established MAGE-1–expressing tumors. These results indicate that linkage of HSP70 to MAGE-1 gene may greatly enhance the potency of DNA vaccines, and generate specific antitumor immunity against MAGE-1–expressing tumors.

MAGE-1/Heat shock protein 70/MAGE-3 fusion protein vaccine in nanoemulsion enhances cellular and humoral immune responses to MAGE-1 or MAGE-3 in vivo

Tumor vaccines, which can elicit antigen-specific antitumor immunity and play an important role in prevention and therapy of tumor, are regarded as the most attractive method. Its underlying mechanism is that tumor antigen, especially the tumor specific antigen (TSA), can induce tumor-specific cytotoxic T lymphocyte (CTL) accordingly to damage tumor cells. The melanoma antigen (MAGE) was the first reported example of TSAs. MAGE-1 and MAGE-3 are two important members of MAGE. Whereas the expressions of MAGE family proteins deviate in different tumors, MAGE-1 and MAGE-3 are two most common tumor antigens. They are expressed in most tumors, respectively or jointly but not in normal tissues except the testes and placenta. Moreover, MAGE-1 and MAGE-3 antigens are termed as tumorrejection antigens because tumors expressing these antigens on appropriate human leukocyte antigen (HLA) class I molecules are rejected by host CTLs [16]. Heat shock proteins (HSPs), named as ‘‘molecular chaperone’’, participate in the folding, transporting and assembling of proteins. Besides, HSPs are important in the processing and presenting of antigens, which could conjugate proteins or peptides and improve their antigenicity. Moreover, the HSP receptors expressed on the surface of antigen-presenting cells (APCs) can facilitate APCs uptaking tumor antigens and improve the presentation. Related research had shown that HSP70 could be exploited to enhance the cellular and humoral immune responses against any attached tumor-specific antigens [7]. To find more effective and more comprehensive vaccine, our colleagues have constructed and purified a recombinant MAGE-1/HSP70/MAGE-3 (MHM) fusion protein successfully. In recent years, significant effort has been devoted to develop nanotechnology for drug delivery, since it offers a suitable means of delivering drugs with small molecular weights, as well as macromolecules such as proteins, peptides or genes by either localized or targeted delivery to the tissue of interest [10]. A nanoemulsion is a system of water, oil and amphiphile, which is a single optically isotropic and thermodynamically stable liquid solution. Therefore, a nanoemulsion is capable of encapsulating significant amounts of drugs. When compared to available traditional emulsions, a nanoemulsion is characterized by its smaller size (limited to 1–100 nm), higher efficiency, and elegant compatibility with tissues as well as with proteins. In addition, it can be prepared with oil and surfactant components that are generally regarded as safe, having less side effect, and toxicity. Moreover, W/O (water-in-oil) nanoemulsions can improve absorption of water-soluble drugs or peptides following intraduodenal administration. In this study, we used this novel artificial lipoprotein delivery system – nanoemulsion as delivery vehicle of the MHM fusion protein vaccine and determined its humoral and cellular immune responses and the antitumor effects. The goal was to investigate the potential benefits of using W/O nanoemulsion as an alternative carrier of the vaccine. W. Ge Æ Y.-F. Sui (&) Æ Y.-J. Sun Æ G.-S. Chen Æ Z.-S. Li S.-Y. Si Æ P.-Z. Hu Æ Y. Huang Æ X.-M. Zhang State Key Laboratory of Cancer Biology, Department of Pathology, Xi Jing Hospital, Fourth Military Medical University, Xi’an, 710032 Shaanxi Province, China E-mail: suiyanf@fmmu.edu.cn Tel.: +86-29-83374597 Fax: +86-29-83255697

Experimentally created unilateral anterior crossbite induces a degenerative ossification phenotype in mandibular condyle of growing Sprague-Dawley rats

The effect of unilateral anterior crossbite on the remodelling of mandibular condyle needs to be investigated. This study aimed to investigate the effects of experimentally created unilateral anterior crossbite on the remodelling of mandibular condyle and explore the changes in the expression of relevant transcription factors and growth factors. The experimental unilateral anterior crossbite was created in 6-week-old female growing rats by bonding metal tubes to the left pairs of incisors. Remodelings of mandibular condylar cartilage was assessed histologically at 2, 4 and 8 weeks. Protein and mRNA levels of Sox9, runt-related transcription factor 2 (Runx2), Osterix (Osx), transforming growth factor beta 1 (TGFβ1), transforming growth factor beta receptor 2 (TGFβr2) and type X collagen (ColX) were investigated by immunohistochemistry and real-time PCR, while alkaline phosphatise (ALP) by histochemistry and real-time PCR. Decreased ratio of hypertrophic cartilage layer was noticed in the 4w experimental group versus controls. At all the time points, the expression of Sox9 and ALP increased but that of TGFβ1 and TGFβr2 decreased in experimental groups (P < 0·05). The expression of Runx2, Osx and Col X increased at 2w, but decrease at 4w (P < 0·05). The results that obvious cartilage degradation and altered expression of related transcription factors and growth factors were detected in the mandibular condyles of the experimental group suggested that the present unilateral anterior crossbite plays an adverse role in the TMJ, and thus leading to the degenerative endochondral ossification.

Autophagy involved in lipopolysaccharide-induced foam cell formation is mediated by adipose differentiation-related protein

BackgroundAutophagy is an essential process for breaking down macromolecules and aged/damaged cellular organelles to maintain cellular energy balance and cellular nutritional status. The idea that autophagy regulates lipid metabolism is an emerging concept with important implications for atherosclerosis. However, the potential role of autophagy and its relationship with lipid metabolism in foam cell formation remains unclear. In this study, we found that autophagy was involved in the lipopolysaccharide (LPS)-induced the formation of foam cells and was at least partially dependent on adipose differentiation-related protein (ADRP).MethodFoam cell formation was evaluated by Oil red O staining. Autophagic activity was determined by immunofluorescence and Western blotting. ADRP gene expression of ADRP was examined by real-time PCR (RT-PCR). The protein expression of ADRP and LC3 was measured using Western blotting analysis. Intracellular cholesterol and triglyceride levels in foam cells were quantitatively measured by enzymatic colorimetric assays.ResultsLPS promoted foam cell formation by inducing lipid accumulation in macrophages. The activation of autophagy with rapamycin (Rap) decreased intracellular cholesterol and triglyceride levels, whereas the inhibition of autophagy with 3-methyladenine (3MA) enhanced the accumulation of lipid droplets. Overexpression of ADRP alone increased the formation of foam cells and consequently autophagic activity. In contrast, the inhibitory effects of ADRP activity with siRNA suppressed the activation of autophagy. Taken together, we propose a novel role for ADRP in the regulation of macrophage autophagy during LPS stimulation.ConclusionWe defined a new molecular pathway in which LPS-induced foam cell formation is regulated through autophagy. These findings facilitate the understanding of the role of autophagy in the development of atherosclerosis.

5-hydroxymethylcytosine loss is associated with poor prognosis for patients with WHO grade II diffuse astrocytomas

Currently, the reliable prognostic biomarkers for WHO grade II diffuse astrocytomas (DA) are still limited. We investigated the relations between the level of 5-Hydroxymethylcytosine (5hmC), an oxidated production of 5-methylcytosine (5mC) by the ten eleven translocated (TET) enzymes, and clinicopathological features of glioma patients. With an identified anti-5hmC antibody, we performed immunohistochemistry in 287 glioma cases. We detected that 5hmC variably reduced in most gliomas and 5hmC reduction was closely associated with higher pathological grades and shortened survival of glioma patients. In multivariate analysis, 5hmC had no independent prognostic value in the entire patient cohort. However, multivariate analysis within subtypes of gliomas revealed that 5hmC was still a prognostic marker confined to DA. In addition, we detected that IDH1 mutation by DNA sequencing was associated with favorable survival within DA. Lastly, we detected that the combination of 5hmC/KI67 was a useful prognostic marker for restratification of DA.

In vivo tumor co-transfection with superantigen and CD80 induces systemic immunity without tolerance and prolongs survival in mice with hepatocellular carcinoma

Background: Since transfection of established tumors with immunostimulatory genes can elicit antitumor immunity, we treat mouse HCC with in vivo transfection of superantigen SEA and/or costimulatory molecule CD80 and evaluated the safety and efficacy. Methods Mice with HCC were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin A and/or CD80. Then the mice were evaluated for tumor regression, systemic immunologic responses, survival times and treatment-associated toxicity. Results Of all treated mice, the overall response rates (complete or partial remission) for SEA, CD80 and SEA/CD80 treated mice in this study were 65%, 60% and 75% separately, and were significantly higher than that of untreated mice. Most of the treat mice completed the therapy without any significant reaction. CTL activity increased with time of treatment and correlated temporally with an objective tumor response. Also our results indicated that local intratumoral expression of SEA did not lead to detectable deletion or anergy of SEA-reactive spleen T cells. Survival times for hepatoma mice in this study treated by intratumoral injection of SEA, CD80 and SEA/CD80 were prolonged significantly (P < 0.01) compared with the control mice. Conclusions Thus, local tumor transfection with superantigen and CD80 genes was capable of inducing strong systemic antitumor immunity.