Phosphatase PP2A is essential for TH17 differentiation.

Phosphatase PP2A expression levels are positively correlated to the clinical severity of systemic lupus erythematosus (SLE) and IL17A cytokine overproduction, indicating a potential role of PP2A in controlling TH17 differentiation and inflammation. By generating a mouse strain with ablation of the catalytic subunit α of PP2A in peripheral mature T cells (PP2A cKO), we demonstrate that the PP2A complex is essential for TH17 differentiation. These PP2A cKO mice had reduced TH17 cell numbers and less severe disease in an experimental autoimmune encephalomyelitis (EAE) model. PP2A deficiency also ablated C-terminal phosphorylation of SMAD2 but increased C-terminal phosphorylation of SMAD3. By regulating the activity of RORγt via binding, the changes in the phosphorylation status of these R-SMADs reduced Il17a gene transcription. Finally, PP2A inhibitors showed similar effects on TH17 cells as were observed in PP2A cKO mice, i.e., decreased TH17 differentiation and relative protection of mice from EAE. Taken together, these data demonstrate that phosphatase PP2A is essential for TH17 differentiation and that inhibition of PP2A could be a possible therapeutic approach to controlling TH17-driven autoimmune diseases.

Ultrasensitive and Robust Point-of-Care Immunoassay for the Detection of Plasmodium falciparum Malaria.

Plasmodium falciparum malaria is widespread in the tropical and subtropical regions of the world. There is ongoing effort to eliminate malaria from endemic regions, and sensitive point-of-care (POC) diagnostic tests are required to support this effort. However, current POC tests are not sufficiently sensitive to detect P. falciparum in asymptomatic individuals. After extensive optimization, we have developed a highly sensitive and robust POC test for the detection of P. falciparum infection. The test is based on upconverting nanophosphor-based lateral flow (UCNP-LF) immunoassay. The developed UCNP-LF test was validated using whole blood reference panels containing samples at different parasite densities covering eight strains of P. falciparum from different geographical areas. The limit of detection was compared to a WHO-prequalified rapid diagnostic test (RDT). The UCNP-LF achieved a detection limit of 0.2-2 parasites/µL, depending on the strain, which is 50- to 250-fold improvement in analytical sensitivity over the conventional RDTs. The developed UCNP-LF is highly stable even at 40 °C for at least 5 months. The extensively optimized UCNP-LF assay is as simple as the conventional malaria RDTs and requires 5 µL of whole blood as sample. Results can be read after 20 min from sample addition, with a simple photoluminescence reader. In the absence of a reader device at the testing site, the strips after running the test can be transported and read at a central location with access to a reader. We have found that the test and control line signals are stable for at least 10 months after running the test. The UCNP-LF has potential for diagnostic testing of both symptomatic and asymptomatic individuals.

Casamino acids facilitate the secretion of recombinant dengue virus serotype-3 envelope domain III in Pichia pastoris.

BACKGROUND: Dengue is a viral disease spread to humans by mosquitoes. Notably, there are four serotypes of dengue viruses (DENV) that places ~40 % of the global population at risk of infection. However, lack of a suitable drug or a preventive vaccine exacerbates the matter further. Envelope domain-III (EDIII) antigen of dengue virus (DENV) has garnered much attention as a promising vaccine candidate for dengue, in addition to its use as a diagnostic intermediate. Hence developing a method for efficient production of high quality recombinant EDIII is important for research and industrial purpose. RESULTS: In this work, a Pichia pastoris system was optimized for the secretory over-expression of DENV serotype-3 EDIII under the control of methanol inducible AOX1 promoter. Temperature alone had a significant impact upon the amount of secretory EDIII, with 2.5-fold increase upon reducing the induction temperature from 30 to 20 °C. However surprisingly, supplementation of culture media with Casamino acids (CA), further augmented secretory EDIII titer, with a concomitant drop of intracellular EDIII levels at both temperatures. Though, reduction in intracellular retention of EDIII was more prominent at 20 °C than 30 °C. This suggests that CA supplementation facilitates overexpressing P. pastoris cells to secrete more EDIII by reducing the proportion retained intracellularly. Moreover, a bell-shaped correlation was observed between CA concentration and secretory EDIII titer. The maximum EDIII expression level of 187 mg/L was achieved under shake flask conditions with induction at 20 °C in the presence of 1 % CA. The overall increase in EDIII titer was ~9-fold compared to un-optimized conditions. Notably, mouse immune-sera, generated using this purified EDIII antigen, efficiently neutralized the DENV. CONCLUSIONS: The strategy described herein could enable fulfilling the mounting demand for recombinant EDIII as well as lay direction to future studies on secretory expression of recombinant proteins in P. pastoris with CA as a media supplement.

Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma.

Background: Neuroblastoma (NB) is considered an immunologically cold tumor and is usually less responsive to immune checkpoint blockade (ICB). Tumor-associated macrophages (TAMs) are highly infiltrated in NB tumors and promote immune escape and resistance to ICB. Hence therapeutic strategies targeting immunosuppressive TAMs can improve responses to ICB in NB. We recently discovered that spleen tyrosine kinase (Syk) reprograms TAMs toward an immunostimulatory phenotype and enhances T-cell responses in the lung adenocarcinoma model. Here we investigated if Syk is an immune-oncology target in NB and tested whether a novel immunotherapeutic approach utilizing Syk inhibitor together with radiation and ICB could provide a durable anti-tumor immune response in an MYCN amplified murine model of NB. Methods: Myeloid Syk KO mice and syngeneic MYCN-amplified cell lines were used to elucidate the effect of myeloid Syk on the NB tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or in combination with anti-PDL1 mAb and radiation was also determined in murine NB models. The underlying mechanism of action of this novel therapeutic combination was also investigated. Results: Herein, we report that Syk is a marker of NB-associated macrophages and plays a crucial role in promoting immunosuppression in the NB TME. We found that the blockade of Syk in NB-bearing mice markedly impairs tumor growth. This effect is facilitated by macrophages that become immunogenic in the absence of Syk, skewing the suppressive TME towards immunostimulation and activating anti-tumor immune responses. Moreover, combining FDA-approved Syk inhibitor, R788 (fostamatinib) along with anti-PDL1 mAb provides a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in mice bearing small tumors (50 mm3) but not larger tumors (250 mm3). However, combining radiation to R788 and anti-PDL1 mAb prolongs the survival of mice bearing large NB9464 tumors. Conclusion: Collectively, our findings demonstrate the central role of macrophage Syk in NB progression and demonstrate that Syk blockade can "reeducate" TAMs towards immunostimulatory phenotype, leading to enhanced T cell responses. These findings further support the clinical evaluation of fostamatinib alone or with radiation and ICB, as a novel therapeutic intervention in neuroblastoma.

Syk Inhibition Reprograms Tumor-Associated Macrophages and Overcomes Gemcitabine-Induced Immunosuppression in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an insidious disease with a low 5-year survival rate. PDAC is characterized by infiltration of abundant tumor-associated macrophages (TAM), which promote immune tolerance and immunotherapeutic resistance. Here we report that macrophage spleen tyrosine kinase (Syk) promotes PDAC growth and metastasis. In orthotopic PDAC mouse models, genetic deletion of myeloid Syk reprogrammed macrophages into immunostimulatory phenotype, increased the infiltration, proliferation, and cytotoxicity of CD8+ T cells, and repressed PDAC growth and metastasis. Furthermore, gemcitabine (Gem) treatment induced an immunosuppressive microenvironment in PDAC by promoting protumorigenic polarization of macrophages. In contrast, treatment with the FDA-approved Syk inhibitor R788 (fostamatinib) remodeled the tumor immune microenvironment, "re-educated" protumorigenic macrophages towards an immunostimulatory phenotype and boosted CD8+ T-cell responses in Gem-treated PDAC in orthotopic mouse models and an ex vivo human pancreatic slice culture model. These findings illustrate the potential of Syk inhibition for enhancing the antitumor immune responses in PDAC and support the clinical evaluation of R788 either alone or together with Gem as a potential treatment strategy for PDAC. SIGNIFICANCE: Syk blockade induces macrophage polarization to an immunostimulatory phenotype, which enhances CD8+ T-cell responses and improves gemcitabine efficacy in pancreatic ductal adenocarcinoma, a clinically challenging malignancy.

NSC243928 Treatment Induces Anti-Tumor Immune Response in Mouse Mammary Tumor Models.

NSC243928 induces cell death in triple-negative breast cancer cells in a LY6K-dependent manner. NSC243928 has been reported as an anti-cancer agent in the NCI small molecule library. The molecular mechanism of NSC243928 as an anti-cancer agent in the treatment of tumor growth in the syngeneic mouse model has not been established. With the success of immunotherapies, novel anti-cancer drugs that may elicit an anti-tumor immune response are of high interest in the development of novel drugs to treat solid cancer. Thus, we focused on studying whether NSC243928 may elicit an anti-tumor immune response in the in vivo mammary tumor models of 4T1 and E0771. We observed that NSC243928 induced immunogenic cell death in 4T1 and E0771 cells. Furthermore, NSC243928 mounted an anti-tumor immune response by increasing immune cells such as patrolling monocytes, NKT cells, B1 cells, and decreasing PMN MDSCs in vivo. Further studies are required to understand the exact mechanism of NSC243928 action in inducing an anti-tumor immune response in vivo, which can be used to determine a molecular signature associated with NSC243928 efficacy. NSC243928 may be a good target for future immuno-oncology drug development for breast cancer.

(+)-Cyanidan-3-ol inhibits epidermoid squamous cell carcinoma growth via inhibiting AKT/mTOR signaling through modulating CIP2A-PP2A axis.

BACKGROUND: Despite recent advances in the treatment of squamous cell skin cancer (SCSC), the disease persists, and treatment resistance develops. Thus, identifying new targets and developing new therapeutic approaches showing low vulnerability to drug resistance is highly needed. PURPOSE: This study aimed to reveal a novel targeted phytotherapeutic strategy for SCSC treatment alone or in combination with standard targeted anticancer molecules. STUDY DESIGN: A library of natural products was utilized to identify molecules that inhibit the growth of skin cancer cells. The anticancer potential of the selected compound was evaluated in human skin squamous carcinoma models, in vitro and in vivo. A comprehensive ingenuity pathway analysis (IPA) strategy and molecular biology technology was adopted to investigate the therapeutic mechanisms in human SCSC. METHODS: The Matrigel invasion chamber, foci formation and soft agar colony formation assays were employed to study the cells invasion and migration potential in vitro. In vivo antitumor effects were evaluated in DMBA/TPA-induced skin papilloma and A431 human skin squamous carcinoma xenograft tumor models. An integrative IPA was employed to identify mechanisms and protein targets in human SCSC.Compounds synergies were determined by the bliss model and evaluated using human SCSC cell lines and xenograft tumors. Histological staining, immunofluorescence imaging, real-time PCR, Western blots, and flow cytometric analyses were employed to analyze apoptosis and cell signaling mechanisms. RESULTS: We identified (+)-cyanidan-3-ol (CD-3) as a selective compound for inhibiting the growth of SCSC cell lines. CD-3 inhibited tumor growth and burden without apparent toxicity and prolonged the survival of tumor-bearing mice. CD-3 inhibitory effects on SCSC growth are mediated via cell cycle arrest and caspase-dependent apoptosis induction. Mechanistic studies showed that CD-3 activates PP2A via inhibiting CIP2A and produces tumor growth inhibitory effects via promoting dephosphorylation of oncogenic AKT/mTOR signaling proteins in SCSC cells and xenograft tumors in a PP2A dependent manner. Furthermore, the combination of CD-3 and mTOR inhibitors (mTORi) synergistically reduced oncogenic phenotypes. CONCLUSIONS: Our study suggests that PP2A activation is an effective strategy for SCSC treatment and the CD-3 and mTORi combination may serve as a promising treatment for SCSC.

Effect of Long-Term Treatment with C60 Fullerenes on the Lifespan and Health Status of CBA/Ca Mice.

Several studies claimed C60 fullerenes as a prospective geroprotector drug due to their ability to capture free radicals effectively and caused a profound interest in C60 in life extension communities. Multiple additives are already sold for human consumption despite a small body of evidence supporting the beneficial effects of fullerenes on the lifespan. To test the effect of C60 fullerenes on lifespan and healthspan, we administered C60 fullerenes dissolved in virgin olive oil orally to 10-12 months old CBA/Ca mice of both genders for 7 months and assessed their survival. To uncover C60 and virgin olive effects, we established two control groups: mice treated with virgin olive oil (vehicle) and mice treated with drinking water. To measure healthspan, we conducted daily monitoring of health condition and lethality and monthly bodyweight measurements. We also assessed physical activity, glucose metabolism, and hematological parameters every 3 months. We did not observe health deterioration in the animals treated with C60 compared with the control groups. Treatment of mice with C60 fullerenes resulted in an increased lifespan of males and females compared with the olive oil-treated animals. The lifespan of C60-treated mice was similar to the mice treated with water. These results suggest that the lifespan-extending effect in C60-treated mice appears due to the protective effect of fullerenes in opposition to the negative effect of olive oil in CBA/Ca mice.

Functional Characterization of Ly49+CD8 T-Cells in Both Normal Condition and During Anti-Viral Response.

The role of Ly49+CD8 T-cells in the immune system is not clear. Previously, several papers suggested Ly49+CD8 T-cells as immunosuppressors, while multiple studies also suggested their role as potent participants of the immune response. The mechanism of Ly49 expression on CD8 T-cells is also not clear. We investigated phenotype, functions, and regulation of Ly49 expression on murine CD8 T-cells in both normal state and during LCMV infection. CD8 T-cells express different Ly49 receptors compared with NK-cells. In intact mice, Ly49+CD8 T-cells have a phenotype similar to resting central memory CD8 T-cells and do not show impaired proliferation and cytokine production. Conventional CD8 T-cells upregulate Ly49 receptors during TCR-induced stimulation, and IL-2, as well as IL-15, affect it. At the same time, Ly49+CD8 T-cells change the Ly49 expression profile dramatically upon re-stimulation downregulating inhibitory and upregulating activating Ly49 receptors. We observed the expression of Ly49 receptors on the virus-specific CD8 T-cells during LCMV infection, especially marked in the early stages, and participation of Ly49+CD8 T-cells in the anti-viral response. Thus, CD8 T-cells acquire Ly49 receptors during the T-cell activation and show dynamic regulation of Ly49 receptors during stimulation.