NFKB1 Research Articles

Bifidobacterium bifidum prevents the IL-1B induced increase in intestinal permeability by a novel mechanism: TLR-2 dependent activation of PPAR-gamma and inhibition of NF-kB signaling pathway

Background: Bifidobacterium bifidum (BB) is one of the most common probiotic bacteria strains that have been shown to have protective effects against intestinal inflammation, primarily by enhancing the intestinal epithelial tight junction (TJ) barrier function. IL-1β contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) in part by causing an increase in intestinal permeability. The IL-1β induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB-dependent activation of myosin light chain kinase (MLCK) gene and disruption of the intestinal TJ barrier. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor in enterocytes which is known to have anti-inflammatory activity by interfering with NF-κB activation. However, the role of BB/PPAR-γ (and the possible mechanism involved) in protecting against IL-1β increase in intestinal permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the IL-1β induced increase in intestinal TJ permeability and the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were used to assess intestinal permeability by using a paracellular marker (dextran-10kDa). Results: IL-1β caused a rapid activation of NF-κB and NF-κB and MLCK-dependent increase in intestinal epithelial TJ permeability in vitro and in vivo. A specific strain of BB (1x108 CFU/ml) referred to as BB1 strain inhibited the IL-1β increase in intestinal TJ permeability while strain BB4 had no effect. Other BB strains examined (5 total strains including BB4) did not inhibit the IL-1β increase in intestinal TJ permeability, suggesting that the BB enhancement of Caco-2 TJ barrier function was strain-specific. BB1, not BB4 enhancement of the TJ barrier was associated with an increase in TLR-2 expression, and PPAR-γ activation; BB1 also inhibited NF-kB activation and MLCK activity in Caco-2 monolayers and in mouse enterocyte. The inhibitory effect of BB on 1L-1β induced increase intestinal permeability, NF-κB activation, MLCK expression and activity was blocked by siRNA-induced knockdown of TLR-2 or PPAR-γ in Caco-2 monolayers. We also generated Villin-cre intestinal epithelial specific PPAR-γ knockout mice to study the involvement of enterocytes TLR-2/PPAR-γ on BB1 effect. BB1 did not inhibit the IL-1β-induced activation of NF-κB, increase in MLCK expression and increase in mouse intestinal permeability in TLR-2 or PPAR-γ intestinal deficient mice. Conclusion: These studies provide a novel insight into the BB the protective mechanism against the IL-1β-induced increase in intestinal TJ permeability in -vitro and in-vivo. Our data show that BB protects against the IL-1β induced increase in intestinal TJ permeability by a novel mechanism involving TLR-2/PPAR-g mediated inhibition of NF-kB and MLCK gene activatiation. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) affects 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to 1) death through pulmonary embolism, 2) the post-thrombotic syndrome, or 3) pulmonary sequelae that dramatically impair quality of life. Thrombosis has long been characterized by the Virchow's triad encompassing hypercoagulability, venous stasis and vascular wall damage. However, inflammatory pathways are now well recognized mechanisms involved in the physiopathology of venous thromboembolism. Endothelial cells play critical roles in regulating immune functions in response to PAMPs and DAMPs. During inflammation, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal by endothelial cells. ATP can interact with the CD39/CD73 system involved in the metabolism of ATP into AMP. Recent data indicated that the CD39/CD73 system might protect against venous thrombosis by downregulating the NLRP3 inflammasome. ATP can also interact with the P2X7 receptor involved in a wide range of responses including NLRP3 inflammasome activation leading to IL1β production. However, the role of P2X7 receptor in VTE is unknown. To determine how the endothelial P2X7 receptor contributes to venous thromboembolism. After pretreatment with TNFα, HUVECs were incubated with BzATP alone or with thrombin. Immunofluorescence, western blot and real-time quantitative PCR analyses were used to study P2X7 expression in endothelial cells, activation of p38 and NFκB signaling pathways and gene expression, respectively. We confirmed that HUVECs expressed P2X7 receptor in vitro and in vivo after induction of venous thrombosis in an experimental model. BzATP and thrombin induced the activation of p38 and NFκB signaling pathways. Interestingly, this was associated with inflammasome assembly and increased IL1β expression. In addition, ICAM-1 and VCAM-1 expression was increased, while thrombomodulin expression was decreased. Our data suggest that ATP released in the extracellular space during venous thrombosis induced inflammasome activation in endothelial cell through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

Hepatoprotective potential of gabapentin in cecal ligation and puncture-induced sepsis; targeting oxidative stress, apoptosis, and NF-kB/MAPK signaling pathways.

Sepsis is a severe inflammatory response to infection with an incidence rate exceeding 48 million cases and 11 million sepsis-related deaths yearly. Furthermore, sepsis remains the fifth most common cause of death worldwide. The present study aimed to examine, for the first time, the potential hepatoprotective activity of gabapentin on cecal ligation and puncture (CLP)-induced sepsis in rats at the molecular level. CLP was used as a model of sepsis in male Wistar rats. Histological examination and liver functions were evaluated. Levels of MDA, GSH, SOD, IL-6, IL-1β, and TNF-α were investigated using ELISA. mRNA levels of Bax, Bcl-2, and NF-kB were assessed by qRT-PCR. Western blotting investigated the expression of ERK1/2, JNK1/2, and cleaved caspase 3 proteins. CLP resulted in liver damage, elevated serum levels of ALT, AST, ALP, MDA, TNF-α, IL-6, and IL-1β, increased expression of ERK1/2, JNK1/2, and cleaved caspase 3 proteins, and upregulated Bax and NF-κB genes expression while it down-regulated Bcl-2 gene expression. However, gabapentin treatment significantly reduced the severity of CLP-induced biochemical, molecular, and histopathological changes. Gabapentin attenuated the levels of the proinflammatory mediators, decreased the expression of JNK1/2, ERK1/2, and cleaved caspase 3 proteins, suppressed Bax and NF-κB genes expression and increased the expression of the Bcl-2 gene. Consequently, Gabapentin reduced hepatic injury resulting from CLP-induced sepsis by reducing proinflammatory mediators, attenuating apoptosis, and inhibiting the intracellular MAPK (ERK1/2, JNK1/2)-NF-kB signaling pathway.

Studying the estimation levels of inflammatory markers and NF-κB in MDR and DS-TB patients

Tuberculosis (TB) is one of the most infectious and deadly diseases, especially after the emergence of drug-resistant bacteria. Therefore, in this study, we developed a method to evaluate the ability of using CXCL10, CXCL9, MMP9, suPAR and the expression gene (NF-κB) and their rolesin the diagnosis and treatment of the disease in patients. Also, we used ELISA and qPCR techniques to compare the serum levels of CXCL10, CXCL9, MMP9, suPAR and NF-κB gene in drug resistant and drug sensitive TB. The serum levels of CXCL10, CXCL9, MMP9, suPAR and the expression of NF-κB gene was compared among drug resistant and drug sensitive TB using ELISA and qPCR Techniques, respectively. Our results showed an increasing in the serum levels of CXCL10, CXCL9 and suPAR, in the drug resistant of TB patients but not in the drug sensitive group, however, all of them did not reach the significant level, it was found that expression of NF-κB in drug sensitive group was higher (3.22-fold) than that in the drug resistant-TB patients (2.70-fold).

The Rare and Atypical Diabetes Network (RADIANT) Study: Design and Early Results.

The Rare and Atypical Diabetes Network (RADIANT) will perform a study of individuals and, if deemed informative, a study of their family members with uncharacterized forms of diabetes. The protocol includes genomic (whole-genome [WGS], RNA, and mitochondrial sequencing), phenotypic (vital signs, biometric measurements, questionnaires, and photography), metabolomics, and metabolic assessments. Among 122 with WGS results of 878 enrolled individuals, a likely pathogenic variant in a known diabetes monogenic gene was found in 3 (2.5%), and six new monogenic variants have been identified in the SMAD5, PTPMT1, INS, NFKB1, IGF1R, and PAX6 genes. Frequent phenotypic clusters are lean type 2 diabetes, autoantibody-negative and insulin-deficient diabetes, lipodystrophic diabetes, and new forms of possible monogenic or oligogenic diabetes. The analyses will lead to improved means of atypical diabetes identification. Genetic sequencing can identify new variants, and metabolomics and transcriptomics analysis can identify novel mechanisms and biomarkers for atypical disease.

Modulating insulin secretion and inflammation against sodium arsenite toxicity by levosimendan as a novel pancreatic islets’ protector

Levosimendan (LEVO) is a calcium sensitizer with established inotropic and vasodilator impacts associated with ATP-sensitive potassium channels (KATP). Although LEVO’s role in modulating oxidative stress of cardiac cells has been studied, in the current study, LEVO was used, for the first time, to investigate its preventive and therapeutic roles in pancreatic islets through viability, functionality, and inflammation pathways as well as oxidative stress against the toxicity induced by sodium arsenite (NaAsO2). Following the optimization studies to select the proper concentrations of LEVO and NaAsO2, isolated pancreatic islets were exposed to different combinations of NaAsO2 and LEVO. MTT assay, glucose-dependent insulin secretion test, investigation of oxidative stress and inflammation biomarkers in addition to qualification of the expressions of P16, P38, and NF-κB genes were performed to assess different underlying mechanisms to investigate the protective role of LEVO against NaAsO2-induced toxicity. This study demonstrated that NaAsO2-treated pancreatic islets’ exposure to LEVO improved their viability and functionality while modulating the generation of oxidative stress and inflammatory biomarkers. Therefore LEVO caused significant recoveries in the characteristics of the islets. The main conclusion is that LEVO showed protective impacts in pancreatic islets against exposure to NaAsO2. However, therapeutic uses of LEVO in the field of diabetes need further investigations. HIGHLIGHTS Sodium arsenite is responsible for activating the oxidative stress and inflammation pathways in pancreatic islet cells and consequently decreasing their viability and function. Treatment of pancreatic islets with sodium arsenite results in the loss of glucose-dependent insulin secretion (GDIS) which can be alleviated by levosimendan. Levosimendan modulates the decreased level of secreted insulin by preventing formation of reactive oxygen species (ROS) and inactivating the inflammatory biomarkers. Levosimendan improves the viability and function of sodium arsenite-treated pancreatic islets by modulating insulin secretion and glucose control. Levosimendan acts as a protective agent against the toxicity induced by sodium arsenite in pancreatic islets through modulating the viability, oxidative stress, inflammation, and function pathways.

Preparation and characterization of the farnesiferol C-loaded solid lipid nanoparticles decorated with folic acid-bound chitosan and evaluation of its in vitro anti-cancer and anti-angiogenic activities

Farnesiferol C (FC) has long been used as a cancer prevention agent. In the present study, FC was loaded into solid lipid nanoparticles (SLN) coated with folic acid (FA) bound chitosan (CS), FC-SLN-CS-FA, by homogenization followed by ultrasonication. Then its toxicity effect on cancer cells compared to normal cells was investigated by the MTT method. In the following, the pro-apoptotic effects of synthesized nanoparticles were evaluated by flow cytometry and acridine orange, propidium iodide, and DAPI staining, and the expression of genes involved in apoptosis was evaluated. The anti-oxidant effects of formulations have been evaluated using DPPH and ABTS assay. To investigate the anti-angiogenic effects of nanoparticles, the chicken chorioallantoic membrane (CAM) assay was used and the expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) genes was used by real-time quantitative PCR (qPCR). The formed nanoparticles with a size of 150 nm and a dispersion index of 0.30 had a surface charge of +17 mV. These nanoparticles exhibited significant growth and proliferation inhibitory effects in PANC, AGS, HT-29, MCF7, and A549 human cancer cells from the lowest concentration (15.6 µg/mL, *** p < 0.001). The flow cytometry analysis shows 10.1%, 48.8%, and 74.4% subG1 arrest at the concentrations of 6, 36, and 66 µg/mL of the FC-SLN-CS-FA, respectively. The formulation caused apoptotic cell death by 3-fold overregulating BAX at concentrations of 36 and 66 µg/mL, and 2- and 2.5-fold overexpression of caspase-9 at concentrations of 36 and 66 µg/mL respectively. The treatment with FC-SLN-CS-FA resulted in decreasing BCL2 and NF-κB gene expression. The ABTS and DPPH assays showed the anti-oxidant properties of formulation in a concentration-dependent manner. FC-SLN-CS-FA significantly inhibited angiogenesis in CAM by reducing the length and number of blood vessels. Considering the pro-apoptotic and anti-angiogenic effects of FC-SLN-CS-FA, it can be said that these nanoparticles can effectively suppress the carcinogenesis of multipotent cancer stem cells. Therefore, they have the potential to be studied as an efficient anticancer agent.

In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants

Alpha-protein kinase 1 (ALPK1) is a pathogen recognition receptor that detects ADP-heptose (ADPH), a lipopolysaccharide biosynthesis intermediate, recently described as a pathogen-associated molecular pattern in Gram-negative bacteria. ADPH binding to ALPK1 activates its kinase domain and triggers TIFA phosphorylation on threonine 9. This leads to the assembly of large TIFA oligomers called TIFAsomes, activation of NF-κB and pro-inflammatory gene expression. Furthermore, mutations in ALPK1 are associated with inflammatory syndromes and cancers. While this kinase is of increasing medical interest, its activity in infectious or non-infectious diseases remains poorly characterized. Here, we use a non-radioactive ALPK1 in vitro kinase assay based on the use of ATPγS and protein thiophosphorylation. We confirm that ALPK1 phosphorylates TIFA T9 and show that T2, T12 and T19 are also weakly phosphorylated by ALPK1. Interestingly, we find that ALPK1 itself is phosphorylated in response to ADPH recognition during Shigella flexneri and Helicobacter pylori infection and that disease-associated ALPK1 mutants exhibit altered kinase activity. In particular, T237M and V1092A mutations associated with ROSAH syndrome and spiradenoma/spiradenocarcinoma respectively, exhibit enhanced ADPH-induced kinase activity and constitutive assembly of TIFAsomes. Altogether, this study provides new insights into the ADPH sensing pathway and disease-associated ALPK1 mutants.

Induction of Innate Inflammatory Pathways in the Corneal Epithelium in the Desiccating Stress Dry Eye Model.

To determine whether 24-hour exposure to the desiccating stress (DS) dry eye model induces NF-kB and NLRP3 inflammasome pathways in the mouse cornea epithelium. Six- to 8-week-old C57BL/6J mice were housed under normal humidity (nonstressed) or subjected to DS from a drafty, low-humidity environment combined with subcutaneous scopolamine four times/day for one day to suppress tear production (DS1). Cornea whole mounts were prepared for immunofluorescent staining, or the corneal epithelium was scraped for NF-kB p-p65 ELISA, Western blot, or real-time PCR to detect NF-kB and inflammasome pathway proteins and gene transcripts, respectively. NF-kB phospho-p65 protein, nuclear NF-kB p-p65, and expression of the NF-kB inducible cytokines (IL-12a, IL-12b, and lymphotoxin b [Ltb]) and chemokine (CCL-2) genes were significantly increased in DS1 compared to nonstressed control. NLRP3 protein and RNA transcripts significantly increased in DS1. NLRP3 and Caspase-1 immunostaining increased in the cornea epithelium at DS1. At DS1 there was no change in IL-18 and a decrease in IL-1β mRNA transcripts; however, levels of bound and total IL-18 protein increased at DS1, and the level of mature IL-1β increased from DS1 to DS5. These findings indicate innate NF-kB and NLRP3 inflammasome inflammatory pathways are induced in the corneal epithelium within one day in the DS dry eye model. NF-kB activation was associated with increased expression of inflammatory mediators involved in dry eye. Induction of these pathways is accompanied by increased bound/total IL-18 and mature IL-1β.

Abstract 6164: NF-κB and NRF2 signaling pathways affect prognosis in HPV-associated head and neck cancer

Abstract Incidence of HPV associated head and neck squamous cell carcinoma (HNSCC) is on the rise, displaying a significantly favorable prognosis and overall better survival as compared to HPV negative HNSCC. Intense radiotherapy is the primary medical care leading to treatment associated morbidity in patients, thereby generating a need for de-intensification strategies. Biomarkers that can identify patients with less aggressive tumors as candidates for de-escalation therapy would revolutionize treatment. We applied an unbiased approach (weighted gene correlation network analysis (WGCNA)) that allows detection of autocorrelated gene sets on 3 independent cohorts of HPV+ HNSCC and created 22 consensus transcriptional modules by selecting genes that grouped together in WGCNA analyses from all 3 cohorts. Interestingly, only 1 module intrinsically divided HPV+ HNSCC into 2 subtypes, displaying different mutational profiles, mutational signatures, HPV gene expression patterns, HPV integration status, and patient survival. Gene set enrichment analysis revealed that this module was enriched in NF-kB genes and strongly associated with NF-κB signaling, confirming our prior findings that defined HPV+ HNSCC subtypes by presence or absence of NF-kB regulators, TRAF3 or CYLD defects and by the NF-κB activity classifier. Our studies show that NF- κB -driven intrinsic tumor characteristics contribute to increased sensitivity of NF- κB active HPV+ head and neck tumors to radiation, providing patients survival benefits. Indeed, TRAF3 or CYLD deletion activated NF-kB and dramatically increased radiation sensitivity of HPV+ head and neck cancer cells. In line with our RNA seq analysis, we found that activation of NF-κB via TRAF3/CYLD deletion significantly correlated with marked downregulation of NRF2 activity and reduced nuclear localization in in HPV+ HNSCC cells. Interestingly, TRAF3 CRISPR KO cells had lower NRF2 protein levels that were restored by MG132 treatment, indicating an involvement of KEAP1/CUL3 mediated proteasomal degradation of NRF2. In summary, our data unveils a unique relation between NF-κB pathway and radiosensitivity in HPV+ HNSCC. The distinct biological characteristics that separate patients into two subclasses associated with favorable prognosis could serve as a biomarker for personalized therapy. Citation Format: Aditi Kothari, Travis Parke Schrank, Wendell Gray Yarbrough, Natalia Isaeva. NF-κB and NRF2 signaling pathways affect prognosis in HPV-associated head and neck cancer [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 6164.

Abstract 2497: Glioma-induced neuronal remodeling promotes regional immunosuppression

Abstract Recent studies have elucidated that gliomas remodel neuronal circuits, and distinct intratumoral regions maintain functional connectivity through a subpopulation of synaptogenic malignant cells expressing Thrombospondin-1 (TSP-1, encoded by the THBS1 gene). Single-cell RNA sequencing analyses of our primary glioblastoma patient samples identified a significant downregulation of immune response signatures in myeloid cells and lymphoid cells in functionally connected intratumoral regions characterized by upregulated THBS1. Understanding the biological significance of immunosuppression within functionally connected intratumoral regions may uncover therapeutic vulnerabilities. Here, we investigate glioma-neuronal-immune crosstalk across clinical tumor specimens and preclinical syngeneic models through bulk and single-cell RNA sequencing (13,670 cells), flow cytometry, and spatial transcriptomics. Using an SB28 murine glioma cell line that endogenously expresses Thbs1 at high levels, we generated a CRISPR Thbs1-knock-out (KO) cell line. In bulk RNA-sequencing data performed on orthotopic tumor models, Thbs1-KO tumors were characterized by the downregulated expression of synapse-associated genes and synaptogenic factors and the recovered expression of genes related to immune response. It highlighted the important role of Thrombospondin-1 in synaptogenesis and immunosuppression consistent with the observation from our primary patient tumor samples. Flow cytometry revealed that tumor-associated macrophages isolated from Thbs1-KO tumors were more polarized toward the pro-inflammatory “M1-like” phenotype (median M1/M2 ratio = 0.6 [WT] vs. 1.34 [KO], p &amp;lt; 0.006). Unbiased gene expression program analysis using spatial transcriptomics for in vivo tumor-harboring mouse brains demonstrated a significant negative correlation between signatures of synaptogenesis (represented by Ntng1 and Nlgn3 genes) and those of immune response (represented by Nfkb1 and Cd83 genes). SB28-Thbs1-KO syngeneic models demonstrated slower tumor growth and significantly longer survival compared to Thbs1-WT counterparts (19 days [WT] vs. 25 days [KO], p &amp;lt; 4.5E-5). The survival difference was abrogated in B6-SCID immunodeficient mice, indicating the critical role of adaptive immunity in the survival advantage associated with Thrombospondin-1 downregulation. Our results identify previously unknown immunosuppression mechanisms in the context of glioma-induced intratumoral connectivity via Thrombospondin-1. Future therapeutic strategies targeting this glioma-neuronal-immune crosstalk may open up new avenues for glioblastoma immunotherapy. Citation Format: Takahide Nejo, Saritha Krishna, Christian Jimenez, Akane Yamamichi, Jacob S. Young, Tiffany Chen, Senthilnath Lakshmanachetty, Payal Watchmaker, Abrar Choudhury, Hirokazu Ogino, David R. Raleigh, Shawn L. Hervey-Jumper, Hideho Okada. Glioma-induced neuronal remodeling promotes regional immunosuppression [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 2497.

Abstract 6156: MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia

Abstract Introduction: Specific B cell malignancies, including CLL and the aggressive non-GCB subtype of DLBCL, are driven by constitutive activation of the B cell receptor (BCR) pathway and the transcription factor NF-κB. Pharmacological inhibition of MALT1 protease, a key mediator of the BCR/NF-κB signal transduction pathway, may therefore provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Experimental Procedures: A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results: Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-κB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton’s tyrosine kinase (BTK) inhibitors. NF-κB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-κB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with B cell malignancies. Citation Format: Joshua P. Plotnik, Raghuveer S. Mali, Velitchka Bontcheva, Colleen Dowell, Jun Chen, Adam E. Richardson, Ryan A. McClure, Paul Jung, Lan Pham, Andrew J. Souers, Jonathan A. Meulbroek, William N. Pappano. MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia [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 6156.

Low vitamin D status is associated with inflammatory response in older patients with cerebral small vessel disease.

This study aimed to determine the association of the NF-κB inflammatory signaling pathway with vitamin D status in older cerebral small vessel disease (SVD) patients. We measured serum 25(OH)D, pro-and anti-inflammatory cytokines, and mRNA levels of the vitamin D-activating enzyme, CYP27B1, as well as NF-kB, COX-2, the chemokine-CCL2, IL-1β, IL-6, TNF-α, TGF-β, and IL-10, in cerebral SVD patients aged ≥60years presenting with vascular dementia and age and gender-matched healthy controls. Low vitamin D status (insufficiency: serum 25(OH)D 12-20ng/ml; deficiency: ≤12ng/ml) was more prevalent among patients compared to controls. The mRNA levels of NF-kB, COX-2, CCL2, IL-1β, and IL-6, and serum levels of pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, and TNF-α) were significantly higher in cases compared to controls. There was a significant correlation between CYP27B1 and NF-kB, COX-2, CCL2, and IL-1β gene expression. Serum IL-1α, IL-1β, and IL-6 concentrations and the expression of CCL-2, NF-kB2, and NF-kB3 genes were higher in vitamin D-deficient subjects compared to vitamin D-sufficient subjects. There was a significant negative correlation between serum 25(OH)D and IL-1α, IL-6, and TNF-α, and a positive correlation between 25(OH)D and IL-10. Low vitamin D is associated with an inflammatory response via NF-kB signaling, which could play a role in the etio-pathogenesis of SVD. Further large-scale studies are required to validate our findings.

Bone Marrow-Derived Mesenchymal Stem Cells and Pioglitazone or Exendin-4 Synergistically Improve Insulin Resistance via Multiple Modulatory Mechanisms in High-Fat Diet/Streptozotocin-Induced Diabetes in Rats.

Diabetes mellitus (DM) is a metabolic disease, characterized by hyperglycemia resulting from defects in insulin secretion and/or insulin action. The current study was designed to assess the therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) alone and in combination with pioglitazone (Pz) or exendin-4 (Ex) in high-fat diet/streptozotocin (HFD/STZ)-induced diabetes in rats. The rats were subjected to the HFD for three weeks before being injected with a single low dosage of STZ (35 mg/kg bw). The animals were assigned to different treatment groups after type II diabetes mellitus (T2DM) induction was confirmed. Severe insulin resistance was verified in untreated HFD/STZ T2DM rats, along with the exaggeration of oxidative stress, inflammation, apoptosis, and autophagy suppression in the adipose tissues. Monotherapy of HFD/T2DM rats with BM-MSCs and Pz or Ex alleviated diabetic complications by increasing insulin sensitivity, decreasing apoptosis and inflammation as evidenced by a decrease in serum tumor necrosis factor-alpha, caspase-3, and nuclear factor-kappa B (NF-κB) genes expression and Janus kinase (JNK) protein expression, and enhancing autophagy as revealed by upregulation in beclin and LC3, as well as peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) genes expression in the adipose tissues. An augmented ameliorative efficacy was recorded in combined treatments. The biochemical and molecular results were confirmed by histological investigation of pancreatic tissues. Combining Pz or Ex with BM-MSCs is a synergistic therapeutic option that reduces insulin resistance and subsequent complications in T2DM via multiple molecular mechanisms.

Network pharmacology-based analysis on geniposide, a component of gardenia jasminoides, beneficial effects to alleviate LPS-induced immune stress in piglets.

Geniposide is the main medicinal component of Gardenia jasminoides, and its content is approximately 3-8% depending on its origin. Geniposide is a class of cyclic enol ether terpene glucoside compounds with strong antioxidant, free radical quenching and cancer-inhibiting activities. Many studies have reported that geniposide has hepatoprotective, cholestatic, neuroprotective, blood sugar and blood lipid regulation, soft tissue damage treatment, antithrombotic, antitumor and other effects. As a traditional Chinese medicine, gardenia, whether used as gardenia alone, as the monomer geniposide or as the effective part of cyclic either terpenoids, has been reported to have anti-inflammatory effects when used in the right amounts. Recent studies have found that geniposide has important roles in pharmacological activities such as anti-inflammation activity, inhibition of the NF-κB/IκB pathway, and cell adhesion molecule production. In this study, we predicted the anti-inflammatory and antioxidant effects of geniposide in piglets through network pharmacology based on the LPS-induced inflammatory response-regulated signaling pathway. The effects of geniposide on changes in inflammatory pathways and cytokine levels in the lymphocytes of inflammation-stressed piglets were investigated using in vivo and in vitro models of piglet lipopolysaccharide-induced oxidative stress. Network pharmacology identified 23 target genes, of which the main pathways of action were lipid and atherosclerosis, fluid shear stress and atherosclerosis, and Yersinia infection. The main relevant target genes were VEGFA, ROCK2, NOS3, and CCL2. Validation experiments showed that the interventional effects of geniposide reduced the relative expression of NF-κB pathway proteins and genes, restored the expression of COX-2 genes to normal levels, and increased the relative expression of tight junction proteins and genes in IPEC-J2 cells. This indicates that the addition of geniposide can alleviate inflammation and improve the level of cellular tight junctions.

Propolis consumption by asymptomatic HIV-individuals: Better redox state? A prospective, randomized, double-blind, placebo-controlled trial

Propolis is a natural product has many biological properties of clinical interest, such as anti-inflammatory and antioxidant. Considering that people living with HIV/aids (PLWHA) on effective combined antiretroviral therapy (cART) present early aging due to an intense immune activation, inflammation, and redox imbalance, propolis consumption could offer a benefit to such patients. This double-blind longitudinal study evaluated whether Brazilian green propolis pills intake (500 mg/day for three months) would decrease the oxidative stress of virological suppressed HIV-individuals. To compare each group (propolis, n = 20 versus placebo, n = 20) in both moments (M0, before and M1, after the intervention), the following markers were assessed: plasma malondialdehyde (MDA), carbonylation, total oxide nitric, total antioxidant capacity (TAP), superoxide dismutase, catalase, and NFkB and NRF2 gene expression. Data were analyzed using Poisson, Gamma distribution and ANOVA followed by Tukey-Kramer. The groups were homogeneous regarding age, gender, time of diagnosis/ treatment, cART scheme, CD4+ T cell count, and no changes were observed in the diet food, or patients’ lifestyles. A decreased MDA concentration was seen in the propolis group (M0 = 0.24 ± 0.13, M1 = 0.20 ± 0.10 protein nmol/mg; p = 0.005) as well as a slight but non-significant increase of TAP (M0 = 49.07 ± 13.26, M1 = 52.27 ± 14.86%; p = 0.06). One may conclude that propolis promoted a lower lipid peroxidation and improved the antioxidant system, suggesting that its use may be beneficial to PLWHA in an attempt to contain the intense inflammatory and oxidant activity.

Quercetin’in HepG2 hücrelerinde, TNF-Alfa ve LPS ile indüklenen enflamasyonda, eritropoietin, HIF-1α, HIF-2α ve piroptoz ile ilgili genlerin ekspresyon seviyeleri üzerine etkileri

Purpose: TNF-α, which increases in the circulation as a result of systemic inflammation, suppresses the production of the erythropoiesis regulating hormone, erythropoietin (EPO) and causes inflammation anemia. The anti-inflammatory activity of quercetin has been reported in different studies. However, no study has been found showing the effect of this situation on EPO gene expression. For this purpose, we evaluated the effects of quercetin on inflammation and, more importantly, its effects on EPO, suppressed in the inflammatory environment and Hypoxia-inducible factors (HIF), which are stimulators of EPO synthesis. In addition, we aimed to evaluate the effect of quercetin on pyroptosis, which is defined as pro-inflammatory programmed cell death.&#x0D; Materials and Methods: We created inflammation models with TNF-α or LPS using HepG2 cells in vitro. In these inflammation models, we evaluated the effects of quercetin on proinflammatory mediators TNF-α, IL-1α, NF-κB gene expressions, EPO, HIF-1α, HIF-2α gene expressions, as well as pyroptosis-related caspase 1 and IL-18 gene expressions. &#x0D; Results: Quercetin showed inflammatory effects by increasing TNF-α, IL-1α, and NF-κB mRNA levels. Consistent with this inflammatory effect, EPO mRNA expression was suppressed. HIF-1α and HIF-2α mRNA levels were increased. &#x0D; Conclusion: These results suggested that the increase in HIFs could not prevent the suppressive effect of inflammatory cytokines and NF-κB on EPO production. However, it has been observed that it tends to suppress proinflammatory cell death by decreasing caspase 1 and IL-18 mRNA levels. These results show that quercetin may show conflicting effects, and further studies are needed to test its safety.

Role of NF-κB/IL-1β Pathway and Caspase 3 in Mediating the Hepatoprotective Effect of Rutin against Paraquat-Induced Liver Toxicity in Male Rats.

One of the most common bipyridinium herbicides that can lead to liver toxicity is paraquat. Rutin is a bioflavonoid with antioxidant, anti-inflammatory, anti-hepatotoxic, and antimicrobial properties. The effect of rutin on paraquat-induced liver toxicity was examined in this study. 48 male rats were divided into six groups: the control group was given a normal diet; the non-treated group was given paraquat; the positive control group was given paraquat, and silymarin and the treatment groups were given paraquat and rutin at doses of 25, 50, and 100 mg/kg. After fourteen days, the rats were anesthetized by xylazine-ketamine, and fasting blood samples were obtained from their hearts to measure alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), malondialdehyde (MDA), creatinine, lipid profile, antioxidant capacity, and carbonyl protein. The liver tissue was removed to measure the levels of catalase (CAT), superoxide dismutase (SOD), total protein, vitamin C, plus NF-κB, IL1β, and caspase-3 gene expressions. Paraquat gavage in the untreated group (group 2) for 14 days in comparison with the control group induced a significant augmentation (p<0.05) in levels of lipid profile, AST, ALP, ALT, MDA, carbonyl protein, and also NF-κB, IL1β, Caspase3 expressions. Treatment with rutin reduced the factors as mentioned above. Paraquat poisoning induced a substantial decline (p<0.05) in HDL content, FRAP level, CAT, and SOD activity of the liver compared to the control group. However, rutin oral treatment led to a substantial increase (p<0.05) in the level of these factors compared to the paraquat-only treated group. Based on the findings of the present study, it was found that rutin can be significantly effective in improving hepatotoxicity caused by paraquat.

Effect of Aerobic Exercise on IL-35 Anti-Inflammatory Cytokine and Behavioral Pain Responses in a Model of Diabetic Neuropathy Rats

Introduction: Nerve inflammation plays an important role in the occurrence and development of diabetic neuropathy pain. The aim of this study was to evaluate the effect of aerobic exercise on anti-inflammatory cytokine IL-35 and behavioral pain responses in a model of diabetic neuropathy rats.&#x0D; Methods: In this experimental study, twenty four 8-week-old male Wistar rats (weight range: 204 ± 11.3 grams) were randomly divided into three groups (n = 8): diabetic neuropathy, diabetic neuropathy exercise and control. After confirmation of diabetic neuropathy pain by hot plate behavioral tests and mechanical allodynia, the aerobic exercise protocol was implemented. IL-35 and NF-κB gene expression in spinal cord tissue was measured by real-time PCR. One-way analysis of variance and Tukey's post hoc test with SPSS version 16 software were used for statistical analysis (P &lt;0.05).&#x0D; Results: Exercise reduced the response of behavioral tests of diabetic neuropathic pain in the exercise group compared to the diabetic neuropathy group (P &lt;0.05). Induction of diabetes caused a significant decrease in IL-35 gene expression and a significant increase in NF-kB gene (P=0.001) (P=0.021).There was also a significant increase in IL-35 gene expression and a significant decrease in NF-κB gene expression in the exercise group compared to the diabetic neuropathy group (P=0.007) (P=0.034).&#x0D; Conclusion: It seems that aerobic training has reduced the sensitivity of nociceptors to pain agents by activating the anti-inflammatory cytokine IL-35 in spinal cord microglia. It is recommended to use aerobic exercise to reduce neuropathic pain.&#x0D;

Chrysin Encapsulated Copper Nanoparticles with Low Dose of Gamma Radiation Elicit Tumor Cell Death Through p38 MAPK/NF-κB Pathways.

Improving radiation effect on tumor cells using radiosensitizers is gaining traction for improving chemoradiotherapy. This study aimed to evaluate copper nanoparticles (CuNPs) synthesized using chrysin as radiosensitizer with γ-radiation on biochemical and histopathological approaches in mice bearing Ehrlich solid tumor. CuNPs were characterized with irregular round sharp shape with size range of 21.19-70.79nm and plasmon absorption at 273nm. In vitro study on MCF-7 cells detected cytotoxic effect of CuNPs with IC50 of 57.2 ± 3.1μg. In vivo study was performed on mice transplanted with Ehrlich solid tumor (EC). Mice were injected with CuNPs (0.67mg/kg body weight) and/or exposed to low dose of gamma radiation (0.5Gy). EC mice exposed to combined treatment of CuNPs and radiation showed a marked reduction in tumor volume, ALT and CAT, creatinine, calcium, and GSH, along with elevation in MDA, caspase-3 in parallel with inhibition of NF-κB, p38 MAPK, and cyclin D1 gene expression. Comparing histopathological findings of treatment groups ends that combined treatment was of higher efficacy, showing tumor tissue regression and increase in apoptotic cells. In conclusion, CuNPs with a low dose of gamma radiation showed more powerful ability for tumor suppression via promoting oxidative state, stimulating apoptosis, and inhibiting proliferation pathway through p38MAPK/NF-κB and cyclinD1.