Mediated Inhibition Research Articles

TBX21 attenuates colorectal cancer progression via an ARHGAP29/RSK/GSK3β dependent manner.

Previous studies have shown that TBX21 (T-Box Transcription Factor 21) plays a vital role in coordinating multiple aspects of the immune response especially type 1 immune response as well as tumor progression. However, the function of TBX21 in colorectal cancer (CRC) remains unclear. IHC to investigate TBX21 expression in CRC tissues. Cell proliferation and apoptosis assays to validate TBX21 function in vitro and in vivo. RNA-seq assay to explore target genes of TBX21. Human phospho-kinase array assay to explore down-stream signaling of TBX21. We disclosed that the expression of TBX21 was marked decreased in CRC versus normal tissue, and negatively correlated with CRC TNM stages. Surprisingly, we found that the CRC and normal cell lines show no TBX21 expression levels. Ectopic expression of TBX21 inhibited cell proliferation and promoted cell apoptosis in vitro. Moreover, RNA-sequence data first time showed that ARHGAP29 acts as the target gene of TBX21 to mediate down-stream signaling activation. Human phospho-kinase array data first time displayed that ectopic expression of TBX21 reduced kinase RSK and GSK3β activation. In contrast, knocked down the expression of TBX21 or ARHGAP29 alternatively abolished TBX21 mediated cell proliferation suppression, cell apoptosis enhancement and RSK/GSK3β activation. In addition, xenograft model studies demonstrated that TBX21 inhibits colorectal tumor progression via ARHGAP29/ RSK/ GSK3β signaling in vivo. In summary, the aforementioned findings suggest a model of TBX21 in suppressing CRC progression. This may provide a promising target for CRC therapy.

Processing mediated suppression of bitterness causing compounds in ready to serve kinnow beverage

Delayed bitterness in kinnow juice affects its market value as well acceptability rate by consumers. For the purpose to enhance the shelf life of kinnow juice, RTS (ready to serve) beverage was prepared. Further, processing mediated suppression of bitterness causing compounds in ready to serve kinnow beverage was studied. Processing methods used during the study was β-cyclodextrin (2%); lye treatment (LT 1.5%); hot water treatment (HWT); Florisil® (3%), and their suitable combination. Processing significantly modulate the bitterness causing compounds. Combined treatment was observed as best due to presence of less concentration of bitter compounds and higher sensorial score. RTS prepared using different treatments were stored in glass bottles for 90 days and studied in terms of their titratable acidity, pH, TSS and antioxidant properties. Samples were stored in refrigerator at 4 °C and withdrawal after every 15 days with the aim to evaluate the storage time effect on quality and sensorial attributes. Increasing storage time deteriorate the quality attribute such as bioactive compounds, antioxidant properties, sensorial scores and other parameters (titratable acidity, pH and TSS) respectively.

Machine learning algorithms reveal potential miRNAs biomarkers in gastric cancer

Gastric cancer is the high mortality rate cancers globally, and the current survival rate is 30% even with the use of combination therapies. Recently, mounting evidence indicates the potential role of miRNAs in the diagnosis and assessing the prognosis of cancers. In the state-of-art research in cancer, machine-learning (ML) has gained increasing attention to find clinically useful biomarkers. The present study aimed to identify potential diagnostic and prognostic miRNAs in GC with the application of ML. Using the TCGA database and ML algorithms such as Support Vector Machine (SVM), Random Forest, k-NN, etc., a panel of 29 was obtained. Among the ML algorithms, SVM was chosen (AUC:88.5%, Accuracy:93% in GC). To find common molecular mechanisms of the miRNAs, their common gene targets were predicted using online databases such as miRWalk, miRDB, and Targetscan. Functional and enrichment analyzes were performed using Gene Ontology (GO) and Kyoto Database of Genes and Genomes (KEGG), as well as identification of protein–protein interactions (PPI) using the STRING database. Pathway analysis of the target genes revealed the involvement of several cancer-related pathways including miRNA mediated inhibition of translation, regulation of gene expression by genetic imprinting, and the Wnt signaling pathway. Survival and ROC curve analysis showed that the expression levels of hsa-miR-21, hsa-miR-133a, hsa-miR-146b, and hsa-miR-29c were associated with higher mortality and potentially earlier detection of GC patients. A panel of dysregulated miRNAs that may serve as reliable biomarkers for gastric cancer were identified using machine learning, which represents a powerful tool in biomarker identification.

A novel peptide isolated from Aphonopelma chalcodes tarantula venom with benefits on pancreatic islet function and appetite control

Proof-of-concept for therapeutic application of venom-derived compounds in diabetes is exemplified by the incretin mimetic, exenatide, originally extracted from the saliva of the venomous Heloderma suspectum lizard. In this regard, we have isolated and sequenced a novel 28 amino acid peptide named Δ-theraphotoxin-Ac1 (Δ-TRTX-AC1) from venom of the Mexican Blond tarantula spider Aphonopelma chalcodes, with potential therapeutic benefits for diabetes. Following confirmation of the structure and safety profile of the synthetic peptide, assessment of enzymatic stability and effects of Δ-TRTX-AC1 on in vitro beta-cell function were studied, alongside potential mechanisms. Glucose homeostatic and satiety actions of Δ-TRTX-AC1 alone, and in combination with exenatide, were then assessed in C57BL/6 mice. Synthetic Δ-TRTX-AC1 was shown to adopt a characteristic inhibitor cysteine knot (ICK)-like structure and was non-toxic to beta-cells. Δ-TRTX-AC1 evoked glucose-dependent insulin secretion from BRIN BD11 cells with bioactivity confirmed in murine islets. Insulin secretory potency was established to be dependent on KATP and Ca2+ channel beta-cell signalling. In addition, Δ-TRTX-AC1 enhanced beta-cell proliferation and provided significant protection against cytokine-induced apoptosis. When injected co-jointly with glucose in mice at a dose of 250 nmol/kg, Δ-TRTX-AC1 decreased blood-glucose levels and evoked a significant satiating effect. Moreover, whilst Δ-TRTX-AC1 did not enhance exenatide induced benefits on glucose homeostasis, the peptide significantly augmented exenatide mediated suppression of appetite. Together these data highlight the therapeutic potential of tarantula spider venom-derived peptides, such as Δ-TRTX-Ac1, for diabetes and related obesity.

Abstract 6367: BSI-507, a first-in-class bispecific antibody targeting PD1 and PVRIG for cancer immunotherapy

Abstract Introduction: Despite the success of anti-PD1/PDL1 therapies, only a small fraction of patients benefit from these checkpoint inhibitors (CPIs). Novel approaches to improve outcomes for patients who are resistant to current CPIs are needed. PVRIG is expressed on CD4+ and CD8+ T cells, NKT and NK cells. PVRIG binds with high affinity with its ligand PVRL2, which is expressed on tumor cells and some myeloid cells. The PVRIG-PVRL2 axis exerts an inhibitory effect on the cytotoxic activity of lymphocytes. Bispecific antibodies that exhibit dual blockade of PD1 and PVRIG provide a promising strategy to enhance anti-tumor immune response. Methods: An anti-PD1 mAb was identified from PD1 KO mice immunized with PD1-ECD-Fc and screened by our proprietary H3 (High-throughput, High-content and High-efficiency) platform. An anti-PVRIG mAb was identified from rats immunized with recombinant PVRIG-ECD-Fc and screened by the H3 platform. Both anti-PD1 and anti-PVRIG antibodies were humanized, and the anti-PD1 scFv was fused to the N-terminus of the heavy chain of the anti-PD1 antibody via a flexible linker. The binding activities and affinities were evaluated by ELISA, FACS and SPR, and the ligand blocking activities were measured by ELISA and FACS. Cell-based reporter assays were used to evaluate the functions of the anti-PD1 and anti-PVRIG mAbs alone and the bispecific antibody. In addition, the activity of the bispecific antibody to reverse PD1 and PVRIG mediated suppression of CMV pp65495-503 antigen specific CD8+ T-cell cytotoxicity was evaluated. Results: BSI-507, an anti-PD1xanti-PVRIG bispecific antibody demonstrated comparable activity to the parental anti-PD1 antibody regarding PD1 binding and PD1/PDL1 blocking. It also exhibited comparable activity to the parental anti-PVRIG antibody in PVRIG binding and PVRIG/PVRL2 blocking. Based on cell-based reporter assays, BSI-507 was able to reverse either PD1 or PVRIG mediated T-cell suppression and exhibited comparable potency to the parental antibodies. BSI-507 was also able to show enhanced reversal of both PD1 and PVRIG mediated T-cell suppression, much better than anti-PD1 or anti-PVRIG alone. In addition, BSI-507 showed the ability to reverse PD1 and PVRIG mediated suppression of CMV pp65495-503 antigen specific CD8+ T-cell cytotoxicity, stronger than either the anti-PD1 or anti-PVRIG monoclonal antibody. Summary: BSI-507 is a first-in-class anti-PD1xanti-PVRIG bispecific antibody for dual blockade of PD-1 and PVRIG pathways to enhance reversal of T cell inhibition. BSI-507 demonstrates favorable biophysical and functional characteristics, supporting the initiation of development activities including manufacturing and IND-enabling studies. Citation Format: Zeyu Peng, Xiaodong F. Liu, Hongyan Li, Wenwen Dai, Jinyu Liu, Xiaoyao Hao, Shukai Xia, Qun Lv, Hugh M. Davis, Mingjiu Chen, Mark Z. Ma. BSI-507, a first-in-class bispecific antibody targeting PD1 and PVRIG for cancer immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6367.

Abstract 4874: Hyperforin induces apoptosis and inhibits invasion via inhibiting AKT/NF-ΚB signaling pathway in colorectal cancer cells

Abstract Recently, the incidence of colorectal cancer (CRC) has been increasing year by year and has become the first incidence cancer in Taiwan. Epidermal growth factor receptor inhibitor (EGFR inhibitor) has been recognized as an important target of CRC. Therefore, targeting EGFR and its related signaling could be a potential strategy to overcome this disease. Hyperforin is a natural prenylated phloroglucinol and has been identified as the major molecule responsible for the antidepressant effects. Previous studies indicated that hyperforin has other potentially pharmacologically meaningful biological properties, including antibacterial ability and inhibition of inflammatory mediators. Numerous studies have also pointed out the anti-tumor potential of hyperforin is associated with the induction of apoptosis signaling. However, whether hyperforin may regulate CRC progression and its underlying mechanism is unclear. In our study, we used the MTT assay to prove the cytotoxicity of CRC cells (HT29 and HCT116 cells) was induced by hyperforin. We then used the flow cytometry to confirmed that hyperforin may induce both extrinsic/intrinsic apoptotic pathways in CRC cells. We further demonstrated hyperforin-induced apoptosis pathways is associated with mitochondrial-dependent apoptosis and death receptor dependent apoptosis. Then, transwell invasion, migration, and wound healing assay results proved the inhibition effect by hyperforin in HT29 and HCT116 cells. Additionally, we suggested that hyperforin can effectively inhibit EGFR downstream genes MEK-ERK and AKT signaling, as well as inhibit the phosphorylation of NF-ΚB and the nuclear translocation in CRC cells. In sum, our results show that hyperforin can inhibit the apoptosis and metastasis ability of CRC cells by inhibiting both AKT/NF- ΚB and ERK/NF-ΚB signaling pathway. Citation Format: Ying-Tzu Chen, I-Tsang Chiang, Yuan Chang, Fei-Ting Hsu. Hyperforin induces apoptosis and inhibits invasion via inhibiting AKT/NF-ΚB signaling pathway in colorectal cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4874.

Abstract 1618: A highly selective and potent TLR8 agonist ZG0895 in preclinical studies of anti-tumor activities

Abstract Toll-like receptor 8 (TLR8) recognizes ssRNA fragments from pathogens and initiate both innate and adaptive immune responses. TLR8 activation can reverse Treg and myeloid-derived suppressor cells (MDSC) mediated immune suppression by tumors that lead to a restore of immune response to inhibit tumor growth. ZG0895 is a potent and novel TLR agonist that shows a highly selective activation of TLR8 and has a more than 300-fold potency over TLR7 in EC50 values. The agonist screening results further confirmed that ZG0895 without activation of TLR isoforms like TLR2, 4 or 9 in cells. In pharmacokinetics and pharmacodynamics, ZG0895 has a preferable ADME and PK profile and can activate immune system via subcutaneous (s.c.) administration. For in vivo anti-tumor efficacy studies, ZG0895 at 1~30 mg/kg exhibited significant dose-dependent inhibition of tumor growth in immunocompetent myeloid-sufficient murine models with CT26, EMT6 or HH subcutaneous xenografts. Furthermore, ZG0895 administration resulted in a complete tumor regression by ZG0895 at high dose via s.c. injection. Mechanism studies of ZG0895 in ex vivo human PBMC assay showed a strong induction of TLR8 related cytokines. In vivo study in tumor-bearing mouse demonstrated that pro-inflammatory cytokines, such as TNFα, IL12p40, IL6 and IL10, were quickly released after ZG0895 treatment but soon returned to normal that can avoid continuous activation of systemic immunity and reduce the risk of cytokine release storm. Consistently, in vivo cynomolgus monkey study by consecutive treatments with ZG0895 (day 30) resulted in a significant increase of immune system cells including intermediate monocytes, non-classic monocytes, cDCs and NK cells, along with notable decrease of classical monocytes but unchanged pDCs, suggesting a possible reversion of tumor-initiated MDSC via TLR8 activation. All these changes were back to normal after period of recovery (day 57), indicated the immune effect of ZG0895 was restrained. These preclinical results support that ZG0895 as a highly TLR8-selective agonist inhibits tumor growth via the activation of TLR8 eliciting immune responses but under a better safety than other conventional TLR8 agonists, and it is worth to be further explored as an anti-cancer drug candidate. Citation Format: Dawei Cui, Ruifeng Liu, Huanyi Yang, Bing Zhu, Zelin Sheng, Binhua Lv. A highly selective and potent TLR8 agonist ZG0895 in preclinical studies of anti-tumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1618.

Abstract 572: Tumor-activated PD1-directed IL-2 increased antigen specific T cells in tumors and demonstrated anti-tumor activity in mice

Abstract Although PD-1/PD-L1 therapies have shown significant activity across a range of tumor types, only a subset of patients (10-30%) achieve durable responses. With the goal of improving response and application to PD-1/PD-L1 therapies, we have leveraged our proprietary Xilio Advanced Cytokine Therapies (X-ACT) platform to develop PD1/IL2-ACT, a PD-1 blocker enhanced with a tumor-activated, engineered IL-2 agonist. The activity of IL-2 is blocked by a protein domain that prevents IL-2Rβγ binding until activated in the tumor microenvironment by matrix metalloproteinases . Tumor selective activation of IL-2 enables dosing of PD1/IL2-ACT at sufficient exposure levels to block immunosuppressive PD1/PD-L1 signaling as well as mitigating systemic IL-2 driven toxicity. PD1/IL2-ACT is designed to enable in cis engagement of IL-2 receptors on antigen experienced PD-1+ CD8+ T cells in the tumor microenvironment (TME), inducing effector T cell functions while preventing regulatory T cell (Treg) mediated immune suppression. In a preclinical model, PD1/IL2-ACT, compared to anti-PD1 alone, induced significant tumor growth inhibition. Further, PD1/IL2-ACT was better tolerated than a PD-1 directed unmasked IL-2Rβγ biased agonist in the same preclinical model. Pharmacodynamic studies showed that PD1/IL2-ACT did not significantly change immune cell profiles in the periphery but increased antigen specific CD8+ T cells in the TME, consistent with preferential engagement of PD1+ effector T cells at the tumor site. Ex vivo protease cleavage assays performed in human tumors demonstrated significant activation of PD1/IL2-ACT by tumors of multiple indications but minimal activation in human plasma. A pharmacokinetic study in nonhuman primates revealed PD1/IL2-ACT can achieve comparable exposure levels to existing PD-1 blocking immunotherapies. Taken together, these data illustrate that PD1/IL2-ACT has the potential to broaden the activity of existing immunotherapies by inhibiting the PD1/PDL1 axis as well as directing an immunostimulatory cytokine to antigen specific T cells in the TME. Citation Format: Ertan Eryilmaz, Wilson Guzman, Dheeraj S. Tomar, Parker Johnson, Stephanie Hsiao, Lisa Quinn, Nancy Chan, Jimit Lakhani, Brendan Whalen, Rosa Quiroz, Kurt Jenkins, Oleg Yerov, Will Scott, Justin Greene, Zhen Liu, Megan McLaughlin, Sallyann Vu, Chelsea Turcotte, Hanumantha R. Madala, Jacob Taylor, Caitlin O'Toole, Magali Pederzoli-Ribeil, Haley Duprey, Natalia Malkova, Damiano Fantini, David Crowe, Sean Yang, Kyle Smith, Joseph Card, Janice Lee, Kerri Smith, Benjamin Nicholson, Jijun Dong, Jennifer O'Neil, Carl U. Bialucha. Tumor-activated PD1-directed IL-2 increased antigen specific T cells in tumors and demonstrated anti-tumor activity in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 572.

Abstract 4088: A neovasculature-inducible CA9 CAR resistant to FASL and TGFb mediated suppression for the treatment of ccRCC

Abstract Clinically effective CAR-T cell therapy for solid tumors, such as clear cell renal cell carcinoma (ccRCC), will require substantial T cell engineering to increase their specificity and potency. We have developed an Integrated Circuit T cell (ICT) that encodes multiple synthetic “modules” in order to overcome diverse barriers to efficacy in ccRCC; ICT cells are generated via CRISPR-mediated, targeted knock-in of a single large transgene into the novel GS94 safe-harbor locus. Both primary and metastatic sites of ccRCC are highly vascularized, with the majority of tumor cells expressing elevated levels of carbonic anhydrase IX (CA9), suggesting CA9 may be an excellent CAR target. However, CA9 is also expressed in healthy bile ducts and stomach tissue which has led to on-target, off-tumor toxicities in patients treated with constitutive CA9 CAR T cells. To improve the therapeutic index of CA9 CAR T cells, we developed an “AND” logic gated ICT cell that requires the presence of two antigens to trigger tumor cell killing, thereby enhancing tumor specificity. Induction of the CA9 CAR is gated on the expression of PSMA found on the tumor neovasculature of ccRCC. Importantly, PSMA and CA9 are not co-expressed in normal tissues. When the anti-PSMA priming receptor (PrimeRTM) binds PSMA, PrimeRTM engagement triggers proteolytic release of a chimeric, fully human transcription factor that induces expression of a CA9 CAR. We confirmed the feasibility of vascular priming using a transwell assay where ICTs were primed by a PSMA expressing endothelial cell line and then migrated across the transwell membrane to kill CA9 expressing RCC cells. In addition, a dual flank xenograft model was used to show logic gated circuits selectively kill tumors that express both CA9 and PSMA, and not tumors that express CA9 alone. Transforming growth factor beta (TGFb) is an immunosuppressive cytokine known to be highly expressed in ccRCC. To further increase the potency and persistence of the ICT cells an shRNA cassette was developed targeting both FAS and TGFBR2, a receptor required for TGFB signaling in T cells. Addition of FAS/TGFBR2 shRNA enhanced antitumor activity of PSMAxCA9 logic gate expressing T cells during in vitro chronic stimulation assays conducted in the presence of exogenous TGFb. Furthermore, FAS/TGFBR shRNA containing ICTs demonstrated enhanced antitumor activity in multiple xenograft RCC models. Collectively, these results demonstrate that PSMAxCA9 ICT cells can (i) selectively target antigens that cannot be safely targeted by conventional CARs and (ii) overcome multiple suppressive mechanisms in the tumor microenvironment. Citation Format: Angela C. Boroughs, Irene Scarfo, Nickolas Attanasio, Thomas Gardner, Jenessa B. Smith, Jennifer McDevitt, Laura Lim, Nishant Mehta, Suchismita Mohanty, James Zhang, Eric Cui, Vibhavari Sail, Amanda Fearon, Samuel Williams, Stephen Santoro, W. Nicholas Haining, Levi Gray-Rupp. A neovasculature-inducible CA9 CAR resistant to FASL and TGFb mediated suppression for the treatment of ccRCC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4088.

Abstract 6789: The oncolytic virus VET3-TGI both blocks TGF-beta signaling and activates type 2 IFN responses, resulting in potent therapeutic responses in multiple mouse models

Abstract Background: TGFB1 mediated immune resistance is one of the major mechanisms of immune suppression utilized across multiple tumor types. Immune resistance imparted by TGFB1 is mediated through its pleiotropic effects on vasculature, fibrogenesis and regulatory/effector immune cells within the tumor microenvironment. Blockade of TGFB1 (TGFBi) will likely improve response to immunotherapy. IL-12 is a cytokine that through IFNg induction, promotes type 1 inflammatory response, M1 macrophages and effector CD8 T cell response. Combining TGFB1 blockade with IL-12 may maximize therapeutic benefits through simultaneously reducing immunosuppression and enhancing anti-tumor immune response. Current studies have developed a vaccinia-based immunotherapy, combining enhanced systemic virus delivery to CXCR3 ligand rich tumors and locally expressed IL-12 and TGFBi within the tumor microenvironment, for efficient control of multiple tumor models. Methods: An oncolytic vaccinia virus expressing CXCR3, IL-12 and a TGFB1 antagonizing mini-monomer was constructed (VET3-TGI) and the expression and function of the transgenes were confirmed. Using in vivo mouse RENCA, EMT-6 and MC38 tumor models, the functionality and therapeutic efficacy of VET-TGI were tested with comparison to control virus. Post-mortem analysis was used to analyze the impact of VET3-TGI on immune/stromal/endothelial milieu of the tumors and to determine toxicity profile. Results: VET3-TGI infected cells expressed CXCR3 and showed enhanced migration to CXCR3 ligands in vitro and improved systemic delivery to tumors expressing CXCR3 ligands in vivo, even in the face of pre-existing anti-viral immunity. IL-12 expression and TGFBi blockade of TGFB1 mediated suppression of CD8 T cell proliferation were confirmed in vitro. In vivo mouse studies using EMT6, RENCA and MC38 tumor models demonstrated potent therapeutic activity, including 100% CRs, even at doses several logs below equivalent clinical doses and in multiple models. Mechanism of activity studies suggested that the therapeutic efficacy of VET3-TGI is associated with considerable modification of the tumor microenvironment. In addition, preliminary toxicity studies demonstrated the safety of VET3-TGI in mouse models. Conclusions: VET3-TGI demonstrated an ability to reduce immunosuppression and dramatically enhance antitumor immune response leading to safe and potent therapeutic activity in multiple mouse tumor models. This data led to the selection of VET3-TGI as our lead clinical candidate. A human version of the virus is currently undergoing clinical manufacture and toxicology testing. Citation Format: Ravikumar Muthuswamy, Steve Thorne. The oncolytic virus VET3-TGI both blocks TGF-beta signaling and activates type 2 IFN responses, resulting in potent therapeutic responses in multiple mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6789.

Transcriptomic Analysis from Normal Glucose Tolerance to T2D of Obese Individuals Using Bioinformatic Tools

Understanding the role of white adipose tissue (WAT) in the occurrence and progression of metabolic syndrome is of considerable interest; among the metabolic syndromes are obesity and type 2 diabetes (T2D). Insulin resistance is a key factor in the development of T2D. When the target cells become resistant to insulin, the pancreas responds by producing more insulin to try to lower blood glucose. Over time, this can lead to a state of hyperinsulinemia (high levels of insulin in the blood), which can further exacerbate insulin resistance and contribute to the development of T2D. In order to understand the difference between healthy and unhealthy obese individuals, we have used published transcriptomic profiling to compare differences between the WAT obtained from obese diabetics and subjects who are obese with normal glucose tolerance and insulin resistance. The identification of aberrantly expressed messenger RNA (mRNA) and the resulting molecular interactions and signaling networks is essential for a better understanding of the progression from normal glucose-tolerant obese individuals to obese diabetics. Computational analyses using Ingenuity Pathway Analysis (IPA) identified multiple activated signaling networks in obesity progression from insulin-resistant and normal glucose-tolerant (IR-NGT) individuals to those with T2D. The pathways affected are: Tumor Necrosis Factor (TNF), Extracellular signal-Regulated protein Kinase 1/2 ERK1/2, Interleukin 1 A (IL1A), Protein kinase C (Pkcs), Convertase C5, Vascular endothelial growth factor (Vegf), REL-associated protein (RELA), Interleukin1/1 B (IL1/1B), Triggering receptor expressed on myeloid cells (TREM1) and Nuclear factor KB1 (NFKB1) networks, while functional annotation highlighted Liver X Receptor (LXR) activation, phagosome formation, tumor microenvironment pathway, LPS/IL-1 mediated inhibition of RXR function, TREM1 signaling and IL-6 signaling. Together, by conducting a thorough bioinformatics study of protein-coding RNAs, prospective targets could be exploited to clarify the molecular pathways underlying the development of obesity-related type 2 diabetes.

IKBA phosphorylation governs human sperm motility through ACC-mediated fatty acid beta-oxidation

The nuclear factor-κB (NF-κB) signaling pathway regulates specific immunological responses and controls a wide range of physiological processes. NF-κB inhibitor alpha (IKBA) is an NF-κB inhibitory mediator in the cytoplasm that modulates the nuclear translocation and DNA binding activities of NF-κB proteins. However, whether the upstream cascade of the canonical NF-κB signaling pathway has physiological roles independent of IKBA-mediated transcriptional activation remains unclear. Herein we investigated the function of IKBA in mature sperm in which transcriptional and translational events do not occur. IKBA was highly expressed in human sperm. The repression of IKBA phosphorylation by its inhibitor Bay117082 markedly enhanced sperm motility. On the contrary, lipopolysaccharide-stimulated IKBA phosphorylation significantly decreased sperm motility. Nevertheless, Bay117082 treatment did not affect the motility of IKBA-knockout sperm. Further, untargeted metabolomic analysis and pharmacological blocking assays revealed that the Bay117082-induced increase in sperm motility was attributable to fatty acid β-oxidation (FAO) enhancement. In addition, we found that IKBA phosphorylation inhibition resulted in a significant reduction of acetyl-CoA carboxylase levels in the FAO metabolic pathway. Our findings indicate that IKBA-mediated signaling orchestrates sperm motility program and improves our understanding of transcription-independent NF-κB signaling pathway in cells.

The Related Mechanisms Predicted through Network-Based Pharmacological Analysis and the Anti-Inflammatory Effects of Fraxinus rhynchophylla Hance Bark on Contact Dermatitis in Mice.

Fraxinus rhynchophylla Hance bark has been used to treat patients with inflammatory or purulent skin diseases in China, Japan, and Korea. This study was undertaken to determine the mechanism responsible for the effects of F. rhynchophylla and whether it has a therapeutic effect in mice with contact dermatitis (CD). In this study, the active compounds in F. rhynchophylla, their targets, and target gene information for inflammatory dermatosis were investigated using network-based pharmacological analysis. Docking analysis was conducted using AutoDock Vina. In addition, the therapeutic effect of an ethanolic extract of F. rhynchophylla (EEFR) on skin lesions and its inhibitory effects on histopathological abnormalities, inflammatory cytokines, and chemokines were evaluated. Finally, its inhibitory effects on the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathways were observed in RAW 264.7 cells. In our results, seven active compounds were identified in F. rhynchophylla, and six were associated with seven genes associated with inflammatory dermatosis and exhibited a strong binding affinity (<-6 kcal/mol) to prostaglandin G/H synthase 2 (PTGS2). In a murine 1-fluoro-2,4-dinitrobenzene (DNFB) model, topical EEFR ameliorated the surface symptoms of CD and histopathological abnormalities. EEFR also reduced the levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-6, and monocyte chemotactic protein (MCP)-1 in inflamed tissues and inhibited PTGS2, the nuclear translocation of NF-κB (p65), and the activation of c-Jun N-terminal kinases (JNK) in RAW 264.7 cells. In conclusion, the bark of F. rhynchophylla has potential use as a therapeutic or cosmetic agent, and the mechanism responsible for its effects involves the suppression of inflammatory mediators, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB)-α degradation, the nuclear translocation of NF-κB, and JNK phosphorylation.

Berberine Suppresses Gestational Diabetes in Streptozotocin-induced Diabetes Mellitus Rats by Suppression of Inflammatory Mediators

Abstract: Background: Gestational diabetes mellitus (GDM) is a serious health condition witnessed among females who experience insulin resistance and glucose intolerance with the onset of pregnancy. Multiple risk factors prevail both to the gestating mother and the growing fetus at the time of pregnancy which may prolong even postpartum. Aim: Berberine, a natural plant extract known for its anti-inflammatory and potent antidiabetic activity was used to clinically suppress the risk factors involved in Gestational diabetes mellitus. Materials and Methods: Female Wistar rats were used as the models for this study. Streptozotocin was administered to induce diabetes in the female rat model. Berberine was administered to the test animals and monitored with regular analysis of body weight, fetal-placental weight and index, Foetal Blood Glucose (FBG), Serum advanced glycation end products (AGEs), and antioxidant enzyme concentrations. Biochemical parameters, lipids, and pro-inflammatory cytokine levels were assayed to study the influence of Berberine. Results: Upon investigating, it was observed that Berberine produced remarkable activity in suppressing glucose intolerance and insulin resistance by targeting multiple criteria, including inflammatory mediators enlisted above. Conclusion: From this study, it is evident that Berberine can be used as a therapeutic agent to treat gestational diabetes. Keywords: Gestational diabetes mellitus, Inflammation, Berberine, Antioxidant, Antidiabetic.

Inhibition of Astrocytic Carbohydrate Sulfotransferase 15 Promotes Nerve Repair After Spinal Cord Injury via Mitigation of CSPG Mediated Axonal Inhibition.

Nerve tissue regeneration is a significant problem. After neural diseases and damage such as spinal cord injury (SCI), the accumulation of chondroitin sulfate proteoglycans (CSPG) comprising axonal inhibitory glycosaminoglycan chains in the microenvironment is a major barrier that obstructs nerve repair. Interfering with the production of glycosaminoglycans, especially the critical inhibitory chains, could be a potential therapeutic strategy for SCI, which is, however, poorly defined. This study identifies Chst15, the chondroitin sulfotransferase controlling the generation of axonal inhibitory chondroitin sulfate-E, as a therapeutic target of SCI. Using a recently reported small molecular Chst15 inhibitor, this study investigates the effects of Chst15 inhibition on astrocyte behaviors and the associated consequences of in vivo disruption of the inhibitory microenvironment. Deposition of CSPGs in the extracellular matrix and migration of astrocytes are both significantly impaired by Chst15 inhibition. Administration of the inhibitor in transected spinal cord tissues of rats effectively promotes motor functional restoration and nerve tissue regeneration by a mechanism related to the attenuation of inhibitory CSPGs, glial scar formation and inflammatory responses. This study highlights the role of Chst15 in the CSPG-mediated inhibition of neural recovery after SCI and proposes an effective neuroregenerative therapeutic strategy that uses Chst15 as a potential target.

Sodium Nitroprusside Inhibits Detrusor Muscle Contraction through G-protein Mediated Inhibition of Intracellular Ca2+ Release

Background: Nitric oxide (NO) physiology is very complex in detrusor smooth muscle (DSM). While NO relaxes detrusor in some species, in contrary, it produces contraction in other. NO donor sodium nitroprusside (SNP) was found to inhibit CCh-induced contraction in goat DSM in earlier studies, but it is not known how SNP produces inhibitory action on goat DSM. Therefore, the aim of this in vitro study was to investigate the mechanism of inhibitory action of SNP pertaining to involvement of G- protein and other second messenger on goat DSM. Methods: Goat detrusor muscle strips collected from local abattoir were mounted in a thermostatically controlled (37o±0.5oC) organ bath (20 ml capacity) containing physiological solution. Following 1 hr of equilibration, carbachol (CCh) (10-5 M) induced sub-maximal contraction was elicited both in the absence and presence of SNP (10-5 M). Involvement of NO and guanylyl cyclase was assessed using nitric oxide synthase inhibitor L-NAME (10-5 M) and guanylyl cyclase inhibitor ODQ (10-5 M). Experiments were also carried out in the presence of G-protein activator aluminium fluoride (AlF) (10-5 M), protein kinase C (PKC) activator phorbol-12-myristate (PMA) (10-5 M), nifedipine, low Ca2+ PSS, theophylline and Zero Ca2+ PSS. Result: Inhibitory effect of SNP (10-5 M) on CCh-induced contraction on goat DSM was reversed by nitric oxide synthase inhibitor L-name (10-5 M) and guanylyl cyclase inhibitor ODQ (10-5 M). Prior incubation of the tissues with AlF and PMA also reversed the SNP mediated inhibition. On the other hand, nifedipine and low Ca2+ PSS; and theophylline and zero Ca2+ PSS potentiated the SNP-mediated inhibition. Thus, the present study shows that SNP elicited inhibitory effect on goat DSM is both NO-dependent and cGMP-dependent. In addition, the result shows that SNP inhibits G-protein coupled PKC leading to inhibition of both Ca2+ entry and intracellular Ca2+ release with a resultant decrease in intracellular Ca2+.

Octyl Gallate, a Potential Therapeutic Candidate for Psoriasis: An In vitro and In silico Anti-Inflammatory approach

Background: Psoriasis is an inflammatory skin disease characterized by hyper-proliferating epidermal membrane and accumulation of dermal inflammatory cells. A profound understanding of mechanistic studies has revealed the potential role of TNF-α and IL-17a in disease pathogenesis. background: Psoriasis is an inflammatory skin disease labelled with hyper-proliferating epidermal membrane and accumulation of dermal inflammatory cells. Profound understanding of mechanistic studies revealed the potential role of TNF-α and IL-17a in the disease pathogenesis. Aim: The study aims to evaluate the inhibitory potential of octyl gallate on IL-17a through in silico analysis and validate its anti-inflammatory effects against oxidative stress and proinflammatory cytokines in vitro. Aim: The study aims to evaluate the inhibitory potential of octyl gallate on IL-17a through in silico analysis and validate its anti-inflammatory effects against oxidative stress and proinflammatory cytokines in vitro. Objective: The objective of the study is to evaluate the potential of octyl gallate for the treatment of psoriasis by targeting inflammatory mediators using in vitro and in silico approaches. Materials and Methods: The anti-oxidant potential of octyl gallate was evaluated through chemiluminescence and the Griess method. Cytotoxicity was evaluated via MTT assay. TNF-α levels were quantified through ELISA. Mechanistic studies were performed to recognize the inhibition of strong inflammatory mediators, such as TNF-α, IL-1β, IL-6, NCF-1, and NF-κB through gene expression analysis. Molecular docking was performed to study the underlying binding pattern of gallate inhibitor with IL-17a. result: Octyl gallate potently inhibited the TNF-α, reactive oxygen and nitrogen species while significantly reduces the expression of inflammatory genes. The docking analysis revealed that octyl gallate resides well in the binding pocket of IL17a. The physiochemical properties of gallate resulted good ADME profile. Results: Octyl gallate potently inhibited TNF-α, reactive oxygen, and nitrogen species while significantly reducing the expression of inflammatory genes. The docking analysis revealed that octyl gallate resides well in the binding pocket of IL17a. The physicochemical properties of gallate resulted in a good ADME profile. conclusion: Octyl gallate revealed significant antioxidant potential and downregulation of inflammatory genes principally involved in psoriasis. A new inhibitory target IL-17a of octyl gallate has been identified that together with TNF-α develops a feed forward state in disease pathogenesis. This study signifies the potential of octyl gallate to be a prospective lead molecule for treatment of psoriasis. Conclusion: Octyl gallate revealed a significant antioxidant potential and downregulation of inflammatory genes principally involved in psoriasis. A new inhibitory target IL-17a of octyl gallate has been identified that, together with TNF-α, develops a feed-forward state in disease pathogenesis. This study signifies the potential of octyl gallate to be a prospective lead molecule for the treatment of psoriasis. other: NA

Azilsartan suppresses the antiapoptotic biomarker and pro-inflammatory cytokines in rat model of cisplatin-induced retinal and optic nerve toxicity.

The local renin-angiotensin system has been discovered in the eyes; thus, this study evaluates the Azilsartan effect in the retina and optic nerve toxicity induced by Cisplatin in vivo. Forty-eight male rats were randomly assigned into six groups of 8 animals. Group 1 was healthy control that received 0.5mL/day of 0.5% carboxymethyl cellulose (CMC) orally (PO). Group 2 received a single dose of the 7.0mg/kg CIS intraperitoneally with 0.5mL/day of 0.5% CMC-PO. Groups 3 and 4 received 3.5 and 7.0mg/kg/day of AZIL-PO, respectively. Groups 5 and 6 received 3.5 and 7.0mg/kg/day of AZIL-PO, respectively together with a single dose of 7.0mg/kg of CIS-IP. The ocular tissue and serum estimated the TNF-α, NF-kβ, and Casp-3. A complete blood count was also measured, and the eye was sent for histological examination. The administration of the 3.5mg/kg AZIL significantly (p < 0.05) reduced the ocular tissue and serum TNF-α, NF-kB, and Casp-3 levels, when given to CIS treated group, while the 7.0mg/kg AZIL does not. Additionally, azilsartan shows no negative impact on the CBC in rats. Finally, the eye histological examination showed a significant (p < 0.05) drop in the signs of inflammation and cellular degeneration, particularly after administration of the 3.5mg/kg AZIL to the CIS-treated group. A low dose of AZIL exerts an anti-inflammation and an anti-apoptotic effect through significant suppression of the pro-inflammatory mediators and an apoptotic biomarker by blocking the local angiotensin II type.

Engineering pancreatic islets with a novel form of thrombomodulin protein to overcome early graft loss triggered by instant blood-mediated inflammatory reaction

The instant blood-mediated inflammatory reaction (IBMIR) is initiated by innate immune responses that cause substantial islet loss after intraportal transplantation. Thrombomodulin (TM) is a multifaceted innate immune modulator. In this study, we report the generation of a chimeric form of thrombomodulin with streptavidin (SA-TM) for transient display on the surface of islets modified with biotin to mitigate IBMIR. SA-TM protein expressed in insect cells showed the expected structural and functional features. SA-TM converted protein C into activated protein C, blocked phagocytosis of xenogeneic cells by mouse macrophages and inhibited neutrophil activation. SA-TM was effectively displayed on the surface of biotinylated islets without a negative effect on their viability or function. Islets engineered with SA-TM showed improved engraftment and established euglycemia in 83% of diabetic recipients when compared with 29% of recipients transplanted with SA-engineered islets as control in a syngeneic minimal mass intraportal transplantation model. Enhanced engraftment and function of SA-TM–engineered islets were associated with the inhibition of intragraft proinflammatory innate cellular and soluble mediators of IBMIR, such as macrophages, neutrophils, high-mobility group box 1, tissue factor, macrophage chemoattractant protein-1, interleukin-1β, interleukin-6, tumor necrosis factor-α, interferon-γ. Transient display of SA-TM protein on the islet surface to modulate innate immune responses causing islet graft destruction has clinical potential for autologous and allogeneic islet transplantation.

Brexanolone therapeutics in post-partum depression involves inhibition of systemic inflammatory pathways.

Brexanolone has rapid, long-lasting, and remarkable efficacy in the treatment of post-partum depression (PPD). We test the hypothesis that brexanolone inhibits proinflammatory modulators and macrophage activation in PPD patients, which may promote clinical recovery. PPD patients (N=18) provided blood samples before and after brexanolone infusion according to the FDA-approved protocol. Patients were unresponsive to prior treatment before brexanolone therapy. Serum was collected to determine neurosteroid levels and whole blood cell lysates were examined for inflammatory markers and invitro responses to the inflammatory activators lipopolysaccharide (LPS) and imiquimod (IMQ). Brexanolone infusion altered multiple neuroactive steroid levels (N=15-18), reduced levels of inflammatory mediators (N=11) and inhibited their response to inflammatory immune activators (N=9-11). Specifically, brexanolone infusion reduced whole blood cell tumor necrosis factor-α (TNF-α, p=0.003), and interleukin-6 (IL-6, p=0.04) and these effects were correlated with HAM-D score improvement (TNF-α, p=0.049; IL-6, p=0.02). Furthermore, brexanolone infusion prevented LPS and IMQ-induced elevation of TNF-α (LPS: p=0.02; IMQ: p=0.01), IL-1β (LPS: p=0.006; IMQ: p=0.02) and IL-6 (LPS: p=0.009; IMQ: p=0.01), indicating inhibition of toll-like receptor (TLR)4 and TLR7 responses. Finally, inhibition of TNF-α, IL-1β and IL-6 responses to both LPS and IMQ were correlated with HAM-D score improvements (p<0.05). Brexanolone actions involve inhibition of inflammatory mediator production and inhibition of inflammatory responses to TLR4 and TLR7 activators. The data suggest that inflammation plays a role in post-partum depression and that inhibition of inflammatory pathways contributes to the therapeutic efficacy of brexanolone. The Foundation of Hope, Raleigh, NC and UNC School of Medicine, Chapel Hill.