Propolis Modulates Inflammatory Mediators and Improves Histopathology in Male Rats with L-arginine-induced Acute Pancreatitis

ABSTRACT: Objectives: This study aimed to determine the effects of propolis on immune mediators and tissue histopathology in rats with L-arginine-induced acute pancreatitis (AP). Methods: This study was conducted at Imam Abdulrahman Bin Faisal University, Dammam, Saudia Arabia between September and November 2017. A total of 24 male albino Wistar rats were divided into three equal groups. Group one was the negative control, group two was the positive control (L-arginine-induced AP) and group three received treatment (L-arginineinduced AP and propolis). The rats in group three were treated with 100 mg/kg propolis for seven days after AP induction. Pancreatic tissue was evaluated histologically and levels of interleukin (IL)-6, IL-22 and IL-1β and tumour necrosis factor-alpha (TNF-α) were measured. Results: Propolis reduced the quanitity of proinflammatory molecules (TNF-α, IL-1β and IL-6) in group three compared to group two, significantly increased the overall anti-inflammatory effect of IL-22 (P <0.005) and reduced interstitial inflammation and neutrophil cell infiltration of the pancreatic tissues. Conclusion: Propolis may exert a therapeutic effect in AP. Further studies are required to demonstrate the mechanisms of propolis in AP.Keywords: Propolis; Arginine; Pancreatitis; Interleukins; Cytokinesis; Rats; Saudi Arabia

Killing of Escherichia coli by Crohn's Disease Monocyte-derived Macrophages and Its Enhancement by Hydroxychloroquine and Vitamin D

BACKGROUND: Crohn's disease (CD) is associated with defective innate immunity, including impaired neutrophil chemotaxis, and mucosal invasion by bacteria, particularly adherent and invasive Escherichia coli that replicate inside macrophage phagolysosomes. We compared CD and healthy control (HC) macrophages for their abilities to kill E. coli and generate neutrophil chemoattractants and also assessed the effects of hydroxychloroquine (HCQ) and vitamin D on killing of phagocytosed E. coli. METHODS: Peripheral blood monocyte-derived macrophages from CD and HC were compared for bacterial killing and generation of neutrophil chemoattractants in response to CD-derived E. coli. Escherichia coli replication was also assessed in the presence and absence of HCQ, alone and with antibiotics, and vitamin D. RESULTS: Monocyte-derived macrophages from patients with CD were similar to HC in allowing replication of phagocytosed CD-derived E. coli: HM605 {CD: N = 10, mean fold replication in 3 hr = 1.08 (95% confidence interval [CI], 0.39–1.78); HC: N = 9, 1.50 (95% CI, 1.02–1.97); P = 0.15} and also in generation of neutrophil chemoattractants in response to E. coli (mean fold chemotaxis relative to control: CD = 2.55 [95% CI, 2.31–2.80]; HC = 2.65 [95% CI, 2.46–2.85], P = 0.42). HCQ and 1,25 OH(2)-vitamin D(3) both caused dose-dependent inhibition of intramacrophage E. coli replication 3-hour postinfection; HCQ: 73.9% inhibition (P < 0.001) at 1 μg/mL, accompanied by raised intraphagosomal pH, and 1,25 OH(2)-vitamin D(3): 80.7% inhibition (P < 0.05) at 80 nM. HCQ had synergistic effects with doxycycline and ciprofloxacin. CONCLUSIONS: CD and HC macrophages perform similarly in allowing replication of phagocytosed E. coli and generating neutrophil chemoattractants. Replication of phagocytosed E. coli was substantially decreased by HCQ and vitamin D. These warrant further therapeutic trials in CD in combination with relevant antibiotics

Calcium signalling in mast cells

Mast cells play a central role in many allergic and in ammatory conditions. These cells are activated following an intracellular rise in calcium, such as that which occurs after the activation of cell-surface receptors. One such important receptor is cysteinyl leukotriene (CysLT) receptor type 1 (CysLT1), which is activated by lipid mediators such as CysLTs LTC4, LTD4, and LTE4. CysLT1 stimulation leads to the hydrolysis of membrane phospholipids such as phosphatidylinositol 4 5-bisphosphate (PIP2) via phospholipase C-β , which results in the generation of diacylglycerol and inositol trisphosphate. Inositol trisphosphate transiently increases cytosolic calcium levels by releasing calcium from its internal stores. This transient phase is followed by an influx of external calcium caused by the opening of store-operated calcium release-activated calcium (CRAC) channels in the plasma membrane. To understand how CRAC channels are involved in receptor- driven calcium responses, I investigated whether the opening of CRAC channels regulates the production of cellular phosphoinositide. Using cytoplasmic calcium ion (Ca2+) imaging in the mast cell line RBL-2H3, I found that LTC4 induced repetitive calcium oscillations that ran down in the absence of external calcium and were sustained by calcium entry through CRAC channels. The molecular characterisation of CRAC channel components in RBL-2H3 cells revealed that LTC4 -mediated calcium oscillations were maintained through calcium entry via Orai1 and that the calcium signal could not be maintained by Orai3 or other calcium- permeable channels. Furthermore, STIM1 (but not STIM2) was the only homologue that supported calcium oscillations in RBL-2H3 cells. The inhibition of the cellular phosphoinositide pool by lithium chloride (LiCl) reduces calcium oscillations. Adding the substrate inositol rescued these oscillations, but only when external calcium was present. Pharmacologically blocking CRAC channels with a low concentration of CRAC channel blockers prevented the recovery of oscillations in LiCl-treated cells, even when inositol was present. To further understand how calcium entry contributes to the production of PIP2, I investigated whether PI4P- or PI5P-specific pools support the oscillatory calcium signal induced by LTC4. Accordingly, by using pharmacological blockers, concluded that PIP2 used in LTC4 -mediated calcium signalling is produced via the conversion of PI4P into PIP2 by PI5K1 kinases and that the cellular PI5P pool does not contribute to the calcium signal. Moreover, the conversion of PI4P into PIP2 was possible only when there was calcium entry via CRAC channels. Characterisation of the expressed PI5K1 kinases in RBL-2H3 cells revealed expression of only PIP5K1α and PIP5K1γ and that both kinases are needed to maintain the oscillatory calcium signal induced by LTC4 and to provide an overlapping function. To further expand current understanding of how calcium regulates PI5K1 kinases, I specifically investigated how calcium entry regulates PIP5K1γ. This was accomplished by looking into PIP5K1γ-regulating proteins, of which talin is a focal adhesion protein shown to activate PIP5K1γ. In this thesis, I show that the cleavage and activation of talin depend on calcium entry via CRAC channels, thereby elucidating a possible mechanism in how CRAC channels mediating calcium entry are involved in phosphoinositide production. This thesis identifies a new role for CRAC channels in mast cell activation. The opening of CRAC channels and calcium entry are required for PIP2 production and thus the maintenance of agonist-mediated calcium signalling.</p

Selective antitumor activity of datelliptium toward medullary thyroid carcinoma by downregulating ret transcriptional activity

Medullary thyroid carcinoma (MTC) is a rare aggressive form of thyroid cancer with high rates of metastasis. Sporadic and hereditary MTC are strongly driven by somatic and germline mutations, respectively, in the transmembrane REarranged during Transfection (RET) proto-oncogene, which encodes a receptor tyrosine kinase. Our previous study identified datelliptium as a novel RET transcription inhibitor, which stabilizes the RET G-quadruplex structures and suppresses RET oncogene transcription. The present study aimed to elucidate the effect of datelliptium on the suppression of epithelial-to-mesenchymal transition (EMT) and metastasis-related behaviors of MTC cells, including cell migration and formation of cancer stem cells (CSCs). Our results demonstrated that datelliptium downregulated the expression of the mesenchymal markers, including N-cadherin, vimentin, slug, snail, and claudin-1. Compared to untreated cells, datelliptium significantly decreased the migration of TT cells in a dose-dependent manner in a wound healing assay. Additionally, datelliptium significantly reduced the size of preformed spheroids from TT cells over the time course. Finally, datelliptium inhibited approximately 75% of MTC xenograft growth with minimal systemic toxicity. In conclusion, datelliptium exerts its antitumor activity against MTC cells by reducing the EMT program, migratory ability, and self-renewal capacity of TT cells, thus preventing invasive and metastatic behavior of MTC. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer

Objective: Colonic mucosa-associated Escherichia coli are increased in Crohn's disease (CD) and colorectal cancer (CRC). They variously haemagglutinate, invade epithelial cell lines, replicate within macrophages, translocate across M (microfold) cells and damage DNA. We investigated genes responsible for these effects and their co-association in colonic mucosal isolates. Design: A fosmid library yielding 968 clones was prepared in E coli EPI300-T1 using DNA from a haemagglutinating CRC isolate, and resulting haemagglutinating clones were 454-pyrosequenced. PCR screening was performed on 281 colonic E coli isolates from inflammatory bowel disease (IBD) (35 patients), CRC (21) and controls (24; sporadic polyps or irritable bowel syndrome). Results: 454-Pyrosequencing of fosmids from the haemagglutinating clones (n=8) identified the afimbrial adhesin afa-1 operon. Transfection of afa-1 into E coli K-12 predictably conferred diffuse adherence plus invasion of HEp-2 and I-407 epithelial cells, and upregulation of vascular endothelial growth factor. E coli expressing afaC were common in CRC (14/21, p=0.0009) and CD (9/14, p=0.005) but not ulcerative colitis (UC; 8/21) compared with controls (4/24). E coli expressing both afaC and lpfA (relevant to M-cell translocation) were common in CD (8/14, p=0.0019) and CRC (14/21, p=0.0001), but not UC (6/21) compared with controls (2/24). E coli expressing both afaC and pks (genotoxic) were common in CRC (11/21, p=0.0015) and UC (8/21, p=0.022), but not CD (4/14) compared with controls (2/24). All isolates expressed dsbA and htrA relevant to intra-macrophage replication, and 242/281 expressed fimH encoding type-1 fimbrial adhesin. Conclusions: IBD and CRC commonly have colonic mucosal E coli that express genes that confer properties relevant to pathogenesis including M-cell translocation, angiogenesis and genotoxicity