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Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity.

Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay’s ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay’s combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.

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Gelsolin-mediated activation of PI3K/Akt pathway is crucial for hepatocyte growth factor-induced cell scattering in gastric carcinoma.

In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC.

Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides.

Cancer drugs often fail due to the emergence of clinical resistance. This can manifest through mutations in target proteins that selectively exclude drug binding whilst retaining aberrant function. A priori knowledge of resistance-inducing mutations is therefore important for both drug design and clinical surveillance. Stapled peptides represent a novel class of antagonists capable of inhibiting therapeutically relevant protein-protein interactions. Here, we address the important question of potential resistance to stapled peptide inhibitors. HDM2 is the critical negative regulator of p53, and is often overexpressed in cancers that retain wild-type p53 function. Interrogation of a large collection of randomly mutated HDM2 proteins failed to identify point mutations that could selectively abrogate binding by a stapled peptide inhibitor (PM2). In contrast, the same interrogation methodology has previously uncovered point mutations that selectively inhibit binding by Nutlin, the prototypical small molecule inhibitor of HDM2. Our results demonstrate both the high level of structural p53 mimicry employed by PM2 to engage HDM2, and the potential resilience of stapled peptide antagonists to mutations in target proteins. This inherent feature could reduce clinical resistance should this class of drugs enter the clinic.

A unique role for p53 in the regulation of M2 macrophage polarization.

P53 is critically important in preventing oncogenesis but its role in inflammation in general and in the function of inflammatory macrophages in particular is not clear. Here, we show that bone marrow-derived macrophages exhibit endogenous p53 activity, which is increased when macrophages are polarized to the M2 (alternatively activated macrophage) subtype. This leads to reduced expression of M2 genes. Nutlin-3a, which destabilizes the p53/MDM2 (mouse double minute 2 homolog) complex, promotes p53 activation and further downregulates M2 gene expression. In contrast, increased expression of M2 genes was apparent in M2-polarized macrophages from p53-deficient and p53 mutant mice. Furthermore, we show, in mice, that p53 also regulates M2 polarization in peritoneal macrophages from interleukin-4-challenged animals and that nutlin-3a retards the development of tolerance to Escherichia coli lipopolysaccharide. P53 acts via transcriptional repression of expression of c-Myc (v-myc avian myelocytomatosis viral oncogene homolog) gene by directly associating with its promoter. These data establish a role for the p53/MDM2/c-MYC axis as a physiological ‘brake’ to the M2 polarization process. This work reveals a hitherto unknown role for p53 in macrophages, provides further insight into the complexities of macrophage plasticity and raises the possibility that p53-activating drugs, many of which are currently being trialled clinically, may have unforeseen effects on macrophage function.

PharmaCyte Biotech’s Encapsulation Facility for Pancreatic Cancer Therapy Deemed Suitable by Thai FDA

On April 08, 2016 PharmaCyte Biotech, Inc. (OTCQB:PMCB), a clinical stage biotechnology company focused on developing targeted treatments for cancer and diabetes using its signature live-cell encapsulation technology, Cell-in-a-Box, reported that the encapsulation facility located in Bangkok, Thailand, that will be used to encapsulate the live cells used for PharmaCyte’s pancreatic cancer therapy has recently been inspected by the Food and Drug Administration of Thailand (Thai FDA) (Press release, PharmaCyte Biotech, APR 8, 2016, View Source [SID:1234510768]). In its report on that inspection, the Thai FDA stated that, "The facility is built according to the pre-approved floor plan and is now deemed suitable for the manufacture of pharmaceutical products."

PharmaCyte Biotech’s Chief Executive Officer, Kenneth L. Waggoner, commented, "We are very pleased that our partner, Austrianova, has received a positive opinion from the Thai FDA for Austrianova’s live-cell encapsulation facility. This is an important milestone in the development of PharmaCyte’s treatment for pancreatic cancer and a pre-requisite for our upcoming pancreatic cancer clinical trial."

The main role of the Thai FDA is to protect consumer’s health, especially, to ensure safety, quality and efficacy of health products within its purview. These products include: foods, drugs, psychotropic substances, narcotics, medical devices, volatile substances, cosmetics and hazardous substances. It is an absolute requirement that the construction and operation of Austrianova’s live-cell encapsulation facility be in accordance with both Thai national legislation as well as international agreements.

PharmaCyte’s product for pancreatic cancer will be manufactured in Austrianova’s facility in Bangkok. It consists of live human cells that have been genetically engineered to convert the anticancer prodrug ifosfamide into its "cancer-killing" form and then encapsulated using the Cell-in-a-Box encapsulation technology. In PharmaCyte’s upcoming clinical trial, these encapsulated live cells will be used with low doses of ifosfamide (one third the normal dose) as a "consolidation therapy" with the current standard of care for advanced pancreatic cancer when a patient no longer benefits from first line therapy. PharmaCyte’s therapy will be compared with the combination of the anticancer drug capecitabine plus radiation in patients with locally advanced, non-metastatic, inoperable pancreatic cancer whose tumors are stable or progressing after 4-6 cycles of treatment with either the combination of Abraxane plus gemcitabine or the four-drug combination known as FOLFIRINOX.