Promising xenograft animal model recapitulating the features of human pancreatic cancer.

BACKGROUND: Multiple sites of metastasis and desmoplastic reactions in the stroma are key features of human pancreatic cancer (PC). There are currently no simple and reliable animal models that can mimic these features for accurate disease modeling. AIM: To create a new xenograft animal model that can faithfully recapitulate the features of human PC. METHODS: Interleukin 2 receptor subunit gamma (IL2RG) gene knockout Syrian hamster was created and characterized. A panel of human PC cell lines were transplanted into IL2RG knockout Syrian hamsters and severe immune-deficient mice subcutaneously or orthotopically. Tumor growth, local invasion, remote organ metastasis, histopathology, and molecular alterations of tumor cells and stroma were compared over time. RESULTS: The Syrian hamster with IL2RG gene knockout (named ZZU001) demonstrated an immune-deficient phenotype and function. ZZU001 hamsters faithfully recapitulated most features of human PC, in particular, they developed metastasis at multiple sites. PC tissues derived from ZZU001 hamsters displayed desmoplastic reactions in the stroma and epithelial to mesenchymal transition phenotypes, whereas PC tissues derived from immune-deficient mice did not present such features. CONCLUSION: ZZU001 hamsters engrafted with human PC cells are a superior animal model compared to immune-deficient mice. ZZU001 hamsters can be a valuable animal model for better understanding the molecular mechanism of tumorigenesis and metastasis and the evaluation of new drugs targeting human PC.

Activation of NLRP3 inflammasome promotes the proliferation and migration of esophageal squamous cell carcinoma.

Inflammasomes can identify endogenous danger signals as an inflammatory immune response. As the most common inflammasome, the NLR pyrin family domain containing 3 (NLRP3) inflammasome is associated with the pathogenesis of different tumors. However, the function of the NLRP3 inflammasome in esophageal cancer (EC) has rarely been reported. Herein, the expression levels of the components of NLRP3 inflammasome and Ki­67 were analyzed by immunohistochemistry. Furthermore, correlations between the NLRP3 inflammasome and Ki­67 along with the clinicopathological features of EC patients were evaluated. The components of the NLRP3 inflammasome were also assessed by western blot analysis and quantitative PCR. NLRP3 was silenced or overexpressed in different esophageal squamous cell carcinoma (ESCC) cell lines, and cell viability, migration and invasion were assessed by CCK­8 and Transwell assays. The present results showed that high NLRP3 expression in the tumor specimens was significantly associated with TNM stage and T category. Spearman's correlation analysis revealed a positive correlation between NLRP3 and the Ki­67 proliferation index. The mRNA and protein levels of NLRP3, apoptosis­associated speck­like protein containing a CARD (ASC), cleaved caspase­1, and interleukin (IL)­1ß in tumor tissues were higher than those in non­cancerous tissues. The level of secreted IL­1ß in tumor tissues was also increased, as compared to that in normal tissues. Silencing of NLRP3 in KYSE­70 and TE13 cells strongly attenuated cell viability, decreased cell mobility in wound­healing assays and greatly diminished the ability of cell migration and invasion in the Transwell system. Overexpression of NLRP3 in KYSE­510 and EC9706 cells markedly promoted the proliferation, migration and invasion. Collectively, these results revealed that the the NLRP3 inflammasome is upregulated in human ESCC tissues and promotes ESCC progression. Hence, NLRP3 could be a promising new candidate diagnostic and prognostic target.

AAV/BBB-Mediated Gene Transfer of CHIP Attenuates Brain Injury Following Experimental Intracerebral Hemorrhage.

Cell death is a hallmark of secondary brain injury following intracerebral hemorrhage (ICH). The E3 ligase CHIP has been reported to play a key role in mediating necroptosis-an important mechanism of cell death after ICH. However, there is currently no evidence supporting a function of CHIP in ICH. In the present study, we aimed to determine whether CHIP plays an essential role in brain injury after ICH. Our findings indicated that CHIP expression was increased in the peri-hematomal area in rat models of ICH. The AAV/BBB viral platform enables non-invasive, widespread, and long-lasting global neural expression of target genes. Treatment with AAV/BBB-CHIP ameliorated brain injury and inhibited neuronal necroptosis and inflammation in wild type (WT) rats following ICH. Furthermore, rats with CHIP deficiency experienced severe brain injury and increased levels of neuronal necroptosis and inflammation relative to their WT counterparts. However, treatment with AAV/BBB-CHIP attenuated the effects of CHIP deficiency after ICH. Collectively, our results demonstrate that CHIP inhibits necroptosis and pathological inflammation following ICH, and that overexpression of CHIP may represent a therapeutic intervention for ICH. Moreover, the AAV/BBB viral platform may provide a novel avenue for the treatment of brain injury.

Disrupted structure and aberrant function of CHIP mediates the loss of motor and cognitive function in preclinical models of SCAR16.

CHIP (carboxyl terminus of heat shock 70-interacting protein) has long been recognized as an active member of the cellular protein quality control system given the ability of CHIP to function as both a co-chaperone and ubiquitin ligase. We discovered a genetic disease, now known as spinocerebellar autosomal recessive 16 (SCAR16), resulting from a coding mutation that caused a loss of CHIP ubiquitin ligase function. The initial mutation describing SCAR16 was a missense mutation in the ubiquitin ligase domain of CHIP (p.T246M). Using multiple biophysical and cellular approaches, we demonstrated that T246M mutation results in structural disorganization and misfolding of the CHIP U-box domain, promoting oligomerization, and increased proteasome-dependent turnover. CHIP-T246M has no ligase activity, but maintains interactions with chaperones and chaperone-related functions. To establish preclinical models of SCAR16, we engineered T246M at the endogenous locus in both mice and rats. Animals homozygous for T246M had both cognitive and motor cerebellar dysfunction distinct from those observed in the CHIP null animal model, as well as deficits in learning and memory, reflective of the cognitive deficits reported in SCAR16 patients. We conclude that the T246M mutation is not equivalent to the total loss of CHIP, supporting the concept that disease-causing CHIP mutations have different biophysical and functional repercussions on CHIP function that may directly correlate to the spectrum of clinical phenotypes observed in SCAR16 patients. Our findings both further expand our basic understanding of CHIP biology and provide meaningful mechanistic insight underlying the molecular drivers of SCAR16 disease pathology, which may be used to inform the development of novel therapeutics for this devastating disease.

Immune checkpoint inhibitors in cancer therapy.

In recent years immune checkpoint inhibitors have garnered attention as being one of the most promising types of immunotherapy on the horizon. There has been particular focus on the immune checkpoint molecules, cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1) which have been shown to have potent immunomodulatory effects through their function as negative regulators of T cell activation. CTLA-4, through engagement with its ligands B7-1 (CD80) and B7-2 (CD86), plays a pivotal role in attenuating the activation of naïve and memory T cells. In contrast, PD-1 is primarily involved in modulating T cell activity in peripheral tissues via its interaction with PD-L1 and PD-L2. The discovery of these negative regulators of the immune response was crucial in the development of checkpoint inhibitors. This shifted the focus from developing therapies that targeted activation of the host immune system against cancer to checkpoint inhibitors, which aimed to mediate tumor cell destruction through the removal of coinhibitory signals blocking anti-tumor T cell responses.

SMPD1 variants in Chinese Han patients with sporadic Parkinson's disease.

INTRODUCTION: A founder mutation, p.L302P, in sphingomyelin phosphodiesterase 1, acid lysosomal (SMPD1), causing Niemann-Pick disease, a recessive lysosomal storage disorder, was reported to be associated with increased risk of Parkinson's disease (PD) in Ashkenazi Jewish population. Several other studies about the association between SMPD1 variants and PD were performed afterward in other populations. However, the results on the role of SMPD1 mutations for PD have been conflicting. This study aimed to investigate the role of mutations in SMPD1 in Chinese PD patients. METHODS: We sequenced all the exons of this gene in 512 Chinese Han cases with sporadic Parkinson's disease and 495 matched healthy control subjects. RESULTS: We identified Leu-Ala (Val) repeat variants and six known single nucleotide variants (p.A36V, p.D212D, p.P332R, p.G508R, p.P533L, p.T544T) in SMPD1 in both patients and normal controls. Case-control analysis showed the association between Leu-Ala (Val) repeat variants in SMPD1and Chinese Han patients with PD (χ2 = 8.771, p = 0.012), and the allele with less than seven LeuAla (Val) repeats may increase the risk of PD (p = 0.010). CONCLUSION: We identified association between Leu-Ala (Val) repeat variants in SMPD1 and Chinese Han patients with sporadic Parkinson's disease. Our results provide further support for the role of lysosomal pathways in PD development.

Viro-immune therapy: A new strategy for treatment of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease with less than 5% survival at five years. This is largely due to metastatic disease, which is already present in the majority of patients when diagnosed. Even when the primary cancer can be removed by radical surgery, local recurrence occurs within one year in 50%-80% of cases. Therefore, it is imperative to develop new approaches for the treatment of advanced cancer and the prevention of recurrence after surgery. Tumour-targeted oncolytic viruses (TOVs) have become an attractive therapeutic agent as TOVs can kill cancer cells through multiple mechanisms of action, especially via virus-induced engagement of the immune response specifically against tumour cells. To attack tumour cells effectively, tumour-specific T cells need to overcome negative regulatory signals that suppress their activation or that induce tolerance programmes such as anergy or exhaustion in the tumour microenvironment. In this regard, the recent breakthrough in immunotherapy achieved with immune checkpoint blockade agents, such as anti-cytotoxic T-lymphocyte-associate protein 4, programmed death 1 (PD-1) or PD-L1 antibodies, has demonstrated the possibility of relieving immune suppression in PDAC. Therefore, the combination of oncolytic virotherapy and immune checkpoint blockade agents may synergistically function to enhance the antitumour response, lending the opportunity to be the future for treatment of pancreatic cancer.

Exome capture sequencing identifies a novel CCM1 mutation in a Chinese family with multiple cerebral cavernous malformations.

PURPOSE: Cerebral cavernous malformations (CCMs) are vascular anomalies predominantly in the central nervous system but may include lesions in other tissues, such as the retina, skin and liver. The main clinical manifestations include seizures, hemorrhage, recurrent headaches and focal neurological deficits. Previous studies of familial CCMs (FCCMs) have mainly reported in Hispanic and Caucasian cases. Here, we report on FCCMs in a Chinese family further characterized by a novel CCM1 gene mutation. MATERIALS AND METHODS: We investigated clinical and neuroradiological features of a Chinese family of 30 members. Furthermore, we used exome capture sequencing to identify the causing gene. The CCM1 mRNA expression level in three patients of the family and 10 wild-type healthy individuals were detected by real-time quantitative polymerase chain reaction (real-time RT-PCR). RESULTS: Brain magnetic resonance imaging demonstrated multiple intracranial lesions in seven members. The clinical manifestation of CCM was found in five of these cases, including recurrent headaches, weakness, hemorrhage and seizures. Moreover, we identified a novel nonsense mutation c.1159G>T (p. E387*) in the CCM1 gene in the pedigree. Based on real-time RT-PCR results, we have found that the CCM1 mRNA expression level in three patients was reduced by 35% than that in wild-type healthy individuals. CONCLUSIONS: Our finding suggests that the novel nonsense mutation c.1159G>T in CCM1 gene is associated with FCCM, and that CCM1 haploinsufficiency may be the underlying mechanism of CCMs. Furthermore, it also demonstrates that exome capture sequencing is an efficient and direct diagnostic tool to identify causes of genetically heterogeneous diseases.

[Antitumour efficacy of a novel oncolytic adenovirus Ad-TD-RFP for human nasopharyngeal carcinoma].

OBJECTIVE: To evaluate the antitumor efficacy of Ad-TD-RFP for human nasopharyngeal carcinoma cells (C666-1) in vitro and in vivo. METHODS: The oncolytic effects of Ad-TD-RFP and control virus dl11520 on C666-1 cells were determined by cytotoxicity assay (MTS assay). Viral replication of Ad-TD-RFP and dl11520 was detected at different time points (24 h, 48 h, 72 h and 96 h) by tissue culture infective dose (TCID(50)) in C666-1 cells implanted subcutaneously into the flank in each of BALB/c nude mice. The xenografts were injected intratumorally with Ad-TD-RFP or dl1520 to investigate their effects on tumor growth. RESULTS: The concentration for 50% of maximal effect (EC(50)) values of Ad-TD-RFP and dl1520 were (107.6 ± 3.2) pt/cell and (174.1 ± 4.0) pt/cell, respectively (t = 22.6, P < 0.001). The Ad-TD-RFP replication was 3-14 folds more than dl1520 replication at four time points (24 h, 48 h, 72 h and 96 h) in C666-1 cells (t values were 33.6, 23.4, 20.8 and 17.3, respectively, P < 0.001). The average tumor volumes of PBS group, dl1520 group and Ad-TD-RFP group were (1765.5 ± 713.9) mm(3), (1036.9 ± 623.8) mm(3), and (420.8 ± 238.7) mm(3), respectively (F = 12.0, P < 0.05) on day 67 after treatment. CONCLUSIONS: The antitumour efficacy of the novel oncolytic adenovirus Ad-TD-RFP for human nasopharyngeal carcinoma C666-1 cells is superior to that of dl1520 in vitro and in vivo. The outcome of this study provides an experimental basis for the treatment of human nasopharyngeal carcinoma by viral gene therapy.

Immunolipoplexes: an efficient, nonviral alternative for transfection of human dendritic cells with potential for clinical vaccination.

Genetic manipulation of dendritic cells (DCs) is important in the context of using either mature DCs to immunize patients or immature DCs to induce tolerance. Here, we describe a novel method of transfecting monocyte-derived human DCs using immunolipoplexes containing anti-CD71 or anti-CD205 monoclonal Abs. This results in up to 20% transfection, which can be increased to 20-30% if the immunolipoplexes are used to transfect CD14+ monocytes prior to differentiation into DCs. Transfected DCs can be substantially enriched using a drug-selection protocol during differentiation. Unlike adenoviral transduction, this nonviral transfection does not alter the expression of costimulatory molecules or the production of proinflammatory cytokines by DCs. In addition, DC function is unaltered, as assessed by mixed lymphocyte reactions. To test the feasibility of the immunolipoplexes and selection protocol for therapeutic intervention, we transfected DCs with the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Allogeneic T cells exposed to IDO-expressing DCs did not proliferate, secreted more IL-10 and less Th1 and Th2 cytokines, and had a higher amount of apoptosis than T cells incubated with control DCs. Furthermore the remaining T cells were rendered anergic to further stimulation by allogeneic DC. These immunolipoplexes, which can be easily and rapidly assembled, have potential for clinical immunization, in particular for tolerance induction protocols.

Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy.

Genetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) alpha/beta production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.

Analysis of gene expression in cancer cell lines identifies candidate markers for pancreatic tumorigenesis and metastasis.

Pancreatic cancer is a highly aggressive type of malignancy and the prognosis for disease presenting typically at a late stage is extremely poor. A comprehensive understanding of its molecular genetics is required in order to develop new approaches to clinical management. To date, serial analysis of gene expression and more recently oligo/cDNA microarray technologies have been employed in order to identify genes involved in pancreatic neoplasia that can be developed as diagnostic markers and drug targets for this dismal disease. This study describes the expression profile obtained from 20 pancreatic cell lines using cDNA microarrays containing 9,932 human gene elements. Numerous genes were identified as being differentially expressed, some of which have been previously implicated in pancreatic adenocarcinoma (S100P, S100A4, prostate stem cell antigen, lipocalin 2, claudins 3 and 4, trefoil factors 1 and 2) as well as several novel genes. The differentially expressed genes identified are involved in a variety of cellular functions, including control of transcription, regulation of the cell cycle, proteolysis, cell adhesion and signaling. Validation of our array results was performed by exploring the SAGEmap database and by immunohistochemistry for a selection of 4 genes that have not previously been studied in pancreatic cancer: anterior gradient 2 homologue (Xenopus laevis), insulin-like growth factor binding protein 3 and 4 and Forkhead box J1. Immunostaining was performed using pancreas-specific tissue microarrays containing core biopsies from 305 clinical specimens. In addition, using statistical group comparison and hierarchical clustering, a selection of genes was identified that may be linked to the site of metastasis from which these cell lines were isolated.