Sinapic acid and 3,3'-diindolylmethane potentiate cyclophosphamide antitumor activity through induction of apoptosis and inhibition of metastasis.

AIM: New therapeutic strategies are required to enhance the anticancer efficacy of chemotherapeutic drugs and to reduce their cytotoxicity. The purpose of this study was to assess the anti-tumor, antimetastatic and anti-apoptotic activities of sinapic acid (SA) and 3,3'-diindolylmethane (DIM) in solid Ehrlich carcinoma (SEC) induced in mice and combining SA or DIM compounds with cyclophosphamide (CYP). METHODS: For induction of solid tumor, the right hind limbs of mice were inoculated subcutaneously with Ehrlich carcinoma cells. After 5 days of tumor inoculation, mice were treated with SA (56 mg/kg), DIM (40 mg/kg), CYP (10 mg/kg), and their combinations (SA/CYP) and (SA/DIM) for 21 days. The mRNA levels of Elabela, Serpina3, caspase-3, MMP-2 and MMP-9 were assessed by qPCR. Tumor and liver tissues were stained with hematoxylin and eosin for histological examination. Serum was investigated for ALT and AST activities. MAIN FINDINGS: Treatment of SEC mice with SA and DIM significantly reduced solid tumor weight by 45.6% and 33.2%, respectively. They also reduced tumor size and increased life span of SEC mice. SA and DIM diminished area of metastatic nodules of tumor cells in the liver by 54.1% and 47.4%, respectively. They also reduced serum aminotransferases activities. Both SA and DIM were found to upregulate caspase 3 and downregulate MMP-2 and MMP-9. Furthermore, SA and DIM reduced gene expression of Elabela by (44.8% and 35.1%) and Serpina3 by (30.7% and 23.5%), respectively. SA and DIM were also shown to potentiate the anti-tumor activity CYP. SA and DIM showed promising antitumor effects and enhanced CYP antitumor activity mostly through upregulation of apoptotic caspase 3 and suppressing metastatic enzymes MMP-2 and MMP-9. Additionally, SA and DIM exhibited a hepatoprotective effect. Our results suggest that these natural compounds may be used to improve the efficacy and reduce the adverse effects of chemotherapeutic drugs in the treatment of solid malignancies.

Cord Blood Plasma and Placental Mesenchymal Stem Cells-Derived Exosomes Increase Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells While Maintaining Their Stemness.

BACKGROUND: Mesenchymal stem cells (MSCs) have been used for ex vivo expansion of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) to maintain their primitive characters and long-term reconstitution abilities during transplantation. Therapeutic effects of MSCs mainly rely on paracrine mechanisms, including secretion of exosomes (Exos). The objective of this study was to examine the effect of cord blood plasma (CBP)-derived Exos (CBP Exos) and Placental MSCs-derived Exos (MSCs Exos) on the expansion of UCB HSCs to increase their numbers and keep their primitive characteristics. METHODS: CD34+ cells were isolated from UCB, cultured for 10 days, and the expanded HSCs were sub-cultured in semisolid methylcellulose media for primitive colony forming units (CFUs) assay. MSCs were cultured from placental chorionic plates. RESULTS: CBP Exos and MSCs Exos compared with the control group significantly increased the number of total nucleated cells (TNCs), invitro expansion of CD34+ cells, primitive subpopulations of CD34+38+ and CD34+38-Lin- cells (p < 0.001). The expanded cells showed a significantly higher number of total CFUs in the Exos groups (p < 0.01). CONCLUSION: CBP- and placental-derived exosomes are associated with significant ex vivo expansion of UCB HSCs, while maintaining their primitive characters and may eliminate the need for transplantation of an additional unit of UCB.

Post-cholecystectomy bile reflux gastritis: Prevalence, risk factors, and clinical characteristics.

OBJECTIVE: Bile reflux gastritis is caused by the backward flow of duodenal fluid into the stomach. A retrospective cohort study was performed to estimate the prevalence and risk factors of bile reflux gastritis postcholecystectomy, and to evaluate the endoscopic and histopathologic changes in gastric mucosa. METHODS: Patients with refractory upper abdominal pain right below the ribs with symptoms of bloating, burping, nausea, vomiting, and bile regurgitation during the period from January 2018 to December 2020, submitted to Zagazig University Hospitals were enrolled in this study. The studied 64 patients were divided into two groups; the control group (CG): 30 subjects who had never undergone any biliary interventions, and the post-cholecystectomy group (PCG): 34 patients who had undergone cholecystectomy. RESULTS: The prevalence of bile reflux gastritis was (16.7%) and (61.8%) in CG and PCG, respectively. Diabetes, obesity, elevated gastric bilirubin, and elevated stomach pH were all risk factors for bile reflux gastritis in both groups (r = .28,.48,.78,.57 respectively). Age, sex, epigastric pain, heartburn, vomiting, and the existence of bile reflux gastritis, on the other hand, had no correlation. DISCUSSION: After a cholecystectomy, bile reflux gastritis is prevalent, especially among obese and diabetic patients.

Do obesity and diabetes increase the frequency and risk of bile reflux gastritis post-cholecystectomy?

<br><b>Introduction:</b> Biliary gastropathy is a disease characterized by upper abdominal pain, frequent heartburn, nausea, and vomiting of bile. It is caused by the backward flow of duodenal fluid into the stomach and esophagus.</br> <br><b>Aim:</b> A retrospective cohort study was performed to estimate the prevalence and risk factors of bile reflux gastritis secondary to cholecystectomy and to evaluate the endoscopic and histopathologic changes in gastric mucosa caused by bile reflux gastritis.</br> <br><b>Materials and methods:</b> The study involved 64 patients with epigastric pain and/or dyspeptic symptoms during the period from January 2018 to December 2020 who presented to Zagazig University Hospitals. The subjects were divided into two groups: the control group (CG), with 30 subjects who had never undergone any biliary interventions, and the post-cholecystectomy group (PCG), consisting of 34 patients who had undergone cholecystectomy.</br> <br><b>Results:</b> The prevalence of bile reflux gastritis was 16.7% in the CG and 61.8% in the PCG. In both groups, diabetes, obesity, increased gastric bilirubin, and increased gastric pH were risk factors for bile reflux gastritis (r = 0.28, 0.48, 0.78, and 0.57, respectively). However, there were no correlations between age, sex, epigastric pain, heartburn, vomiting, and the presence of bile reflux gastritis.</br> <br><b>Discussion:</b> Bile reflux gastritis is a common complication following cholecystectomy and is more common among obese and diabetic patients.</br>.

Interferon-ß regulates proresolving lipids to promote the resolution of acute airway inflammation.

Aberrant immune responses, including hyperresponsiveness to Toll-like receptor (TLR) ligands, underlie acute respiratory distress syndrome (ARDS). Type I interferons confer antiviral activities and could also regulate the inflammatory response, whereas little is known about their actions to resolve aberrant inflammation. Here we report that interferon-ß (IFN-ß) exerts partially overlapping, but also cooperative actions with aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 to counter TLR9-generated cues to regulate neutrophil apoptosis and phagocytosis in human neutrophils. In mice, TLR9 activation impairs bacterial clearance, prolongs Escherichia coli-evoked lung injury, and suppresses production of IFN-ß and the proresolving lipid mediators 15-epi-LXA4 and resolvin D1 (RvD1) in the lung. Neutralization of endogenous IFN-ß delays pulmonary clearance of E. coli and aggravates mucosal injury. Conversely, treatment of mice with IFN-ß accelerates clearance of bacteria, restores neutrophil phagocytosis, promotes neutrophil apoptosis and efferocytosis, and accelerates resolution of airway inflammation with concomitant increases in 15-epi-LXA4 and RvD1 production in the lungs. Pharmacological blockade of the lipoxin receptor ALX/FPR2 partially prevents IFN-ß-mediated resolution. These findings point to a pivotal role of IFN-ß in orchestrating timely resolution of neutrophil and TLR9 activation-driven airway inflammation and uncover an IFN-ß-initiated resolution program, activation of an ALX/FPR2-centered, proresolving lipids-mediated circuit, for ARDS.

Roles of neutrophil granule proteins in orchestrating inflammation and immunity.

Neutrophil granulocytes form the first line of host defense against invading pathogens and tissue injury. They are rapidly recruited from the blood to the affected sites, where they deploy an impressive arsenal of effectors to eliminate invading microbes and damaged cells. This capacity is endowed in part by readily mobilizable proteins acquired during granulopoiesis and stored in multiple types of cytosolic granules with each granule type containing a unique cargo. Once released, granule proteins contribute to killing bacteria within the phagosome or the extracellular milieu, but are also capable of inflicting collateral tissue damage. Neutrophil-driven inflammation underlies many common diseases. Research over the last decade has documented neutrophil heterogeneity and functional versatility far beyond their antimicrobial function. Emerging evidence indicates that neutrophils utilize granule proteins to interact with innate and adaptive immune cells and orchestrate the inflammatory response. Granule proteins have been identified as important modulators of neutrophil trafficking, reverse transendothelial migration, phagocytosis, neutrophil life span, neutrophil extracellular trap formation, efferocytosis, cytokine activity, and autoimmunity. Hence, defining their roles within the inflammatory locus is critical for minimizing damage to the neighboring tissue and return to homeostasis. Here, we provide an overview of recent advances in the regulation of degranulation, granule protein functions, and signaling in modulating neutrophil-mediated immunity. We also discuss how targeting granule proteins and/or signaling could be harnessed for therapeutic benefits.

Bile reflux gastropathy: Prevalence and risk factors after therapeutic biliary interventions: A retrospective cohort study.

BACKGROUND: Bile reflux gastropathy is caused by the backward flow of duodenal fluid into the stomach. A retrospective cohort study was performed to declare if the therapeutic biliary interventions cause bile reflux gastropathy, and to estimate its prevalence and risk factors, and to evaluate the gastric mucosa endoscopic and histopathologic changes. METHODS: 62 patients, with epigastric pain and/or dyspeptic symptoms, were grouped into, Group 1 : (34) patients that had undergone cholecystectomy and Group 2 : (28) patients who had undergone at least one of the following procedures for the treatment of benign pathology: endoscopic sphincterotomy and endoscopic stenting. Their ages ranged from 27 to 59 years. All participants had undergone gastroscopy for gastric aspirate analysis as well as gastric mucosa biopsy for histopathological examination. RESULTS: the prevalence of bile reflux gastropathy was (21.34%) after therapeutic biliary interventions with a P-value of 0.000. In both groups, diabetes, obesity, increased gastric bilirubin, and increased gastric pH were risk factors for bile reflux gastropathy (r = 0.27, 0.31, 0.68, 0.59 respectively), while age, sex, epigastric pain, heartburn, vomiting were mot. CONCLUSION: bile reflux gastropathy is common after therapeutic biliary interventions being more among obese and diabetic patients.

ß2 Integrin Regulation of Neutrophil Functional Plasticity and Fate in the Resolution of Inflammation.

Neutrophils act as the first line of cellular defense against invading pathogens or tissue injury. Their rapid recruitment into inflamed tissues is critical for the elimination of invading microorganisms and tissue repair, but is also capable of inflicting damage to neighboring tissues. The ß2 integrins and Mac-1 (CD11b/CD18, αMß2 or complement receptor 3) in particular, are best known for mediating neutrophil adhesion and transmigration across the endothelium and phagocytosis of microbes. However, Mac-1 has a broad ligand recognition property that contributes to the functional versatility of the neutrophil population far beyond their antimicrobial function. Accumulating evidence over the past decade has demonstrated roles for Mac-1 ligands in regulating reverse neutrophil transmigration, lifespan, phagocytosis-induced cell death, release of neutrophil extracellular traps and efferocytosis, hence extending the traditional ß2 integrin repertoire in shaping innate and adaptive immune responses. Understanding the functions of ß2 integrins may partly explain neutrophil heterogeneity and may be instrumental to develop novel therapies specifically targeting Mac-1-mediated pro-resolution actions without compromising immunity. Thus, this review details novel insights on outside-in signaling through ß2 integrins and neutrophil functional heterogeneity pertinent to the resolution of inflammation.

IFN-ß is a macrophage-derived effector cytokine facilitating the resolution of bacterial inflammation.

The uptake of apoptotic polymorphonuclear cells (PMN) by macrophages is critical for timely resolution of inflammation. High-burden uptake of apoptotic cells is associated with loss of phagocytosis in resolution phase macrophages. Here, using a transcriptomic analysis of macrophage subsets, we show that non-phagocytic resolution phase macrophages express a distinct IFN-ß-related gene signature in mice. We also report elevated levels of IFN-ß in peritoneal and broncho-alveolar exudates in mice during the resolution of peritonitis and pneumonia, respectively. Elimination of endogenous IFN-ß impairs, whereas treatment with exogenous IFN-ß enhances, bacterial clearance, PMN apoptosis, efferocytosis and macrophage reprogramming. STAT3 signalling in response to IFN-ß promotes apoptosis of human PMNs. Finally, uptake of apoptotic cells promotes loss of phagocytic capacity in macrophages alongside decreased surface expression of efferocytic receptors in vivo. Collectively, these results identify IFN-ß produced by resolution phase macrophages as an effector cytokine in resolving bacterial inflammation.

Chemoprevention of rat mammary carcinogenesis by spirulina.

Spirulina (SP) (Arthrospira platensis; previously Spirulina platensis) is a filamentous blue-green microalga (cyanobacterium) with potent dietary phytoantioxidant and anticancerous properties. We investigated the chemopreventive effect of SP against 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat breast carcinogenesis, and further studied its underlying mechanisms of action in vitro. Remarkably, SP cleared DMBA-induced rat mammary tumors, which was clearly confirmed by morphological and histological methods. SP supplementation reduced the incidence of breast tumors from 87% to 13%. At the molecular level, immunohistochemical analysis revealed that SP supplementation reduced expression of both Ki-67 and estrogen α. More interestingly, molecular analysis in the in vitro experiments indicated that SP treatment inhibited cell proliferation by 24 hours, which was accompanied by increased p53 expression, followed by increased expression of its downstream target gene, Cdkn1a (alias p21 or p21(Waf1/Cip1)). In addition, SP increased Bax and decreased Bcl-2 expression, indicating induction of apoptosis by 48 hours after SP treatment. To our knowledge, this is the first report of in vivo chemopreventive effect of SP against DMBA-induced breast carcinogenesis in rat, supporting its potential use in chemoprevention of cancer.

Diagnosis of colon cancer differs in younger versus older patients despite similar complaints.

BACKGROUND: Colon cancer is common, affecting mostly older people. Since age is a risk factor, young patients might not be given the same attention as older ones regarding symptoms that could imply the presence of colon cancer. OBJECTIVES: To investigate whether young patients, i.e., under age 50, complain of symptoms for longer than older patients until the diagnosis of colon cancer is established. METHODS: In this retrospective cohort study, patients were divided into two groups: < 50 years old (group 1) and > or = 50 (group 2). All had undergone surgery for left or right-colon cancer during the 10 year period of the study from January 2000 through December 2009 at one medical center. Rectal and sigmoid cancers were excluded. Data collected included age, geander, quantity and quality of complaints, duration of complaints, in-hospital versus community diagnosis, pathological staging, the side of colon involved, and overall mortality. The primary outcome was the quality and duration of complaints. Secondary outcomes were the pathological stage at presentation and the mortality rate. RESULTS: The study group comprised 236 patients: 31 (13.1%) were < 50 years old and 205 (86.9%) were > or = 50. No significant difference was found in the quantity and quality of complaints between the two groups. Patients in group 1 (< 50 years) complained for a longer period, 5.3 vs.2.4 months (P= 0.002). More younger patients were diagnosed with stage IV disease (38.7% vs. 21.5%, P= 0.035) and fewer had stage I disease (3.2% vs. 15.6%, P= 0.06); the mortality rates were similar (41.9% vs. 39%). Applying a stepwise logistic regression model, the duration of complaints was found to be a predictor of mortality (P= 0.03, OR 1.6, 95% CI 1-3.6), independently of age (P= 0.003) and stage (P< 0.001). CONCLUSIONS: Younger patients are more often diagnosed with colon cancer later, at a more advanced stage. Alertness to patients' complaints, together with evaluation regardless of age but according to symptoms and clinical presentation are crucial. Large-scale population-based studies are needed to confirm this trend.

Clinical presentation predicts the outcome of patients with colon cancer.

AIM: To elucidate the relationship between clinical presentation and outcome. METHODS: A single institution retrospective chart review of patients admitted with the diagnosis of colon cancer. We used univariate and a multivariate analysis to identify symptoms association with mortality. An odds ratio based clinical score was created to evaluate the contribution of the quality of symptoms to outcome. Primary measure of outcome was survival. RESULTS: During the study period, 236 patients met the inclusion criteria. Overall survival was 60.6%, mean follow-up 3.0 years. A bivariate analysis showed that increasing number of symptoms is not associated with mortality. However, a symptom-specific analysis performed using a logistic regression model controlling for age, stage and the duration of complaints revealed that the presence of melena was independently associated with mortality [P = 0.04, odds ratio (OR) 7.4], while rectal bleeding was associated with survival (P = 0.004, OR 3.9). Applying the proposed clinical score to an receiver operating characteristic curve showed that score > 1 had a strong association with mortality. The same logistic regression model was applied. The results showed that a score > 1 was an independent predictor of mortality (P < 0.001) and associated with node-positive disease (P = 0.008). CONCLUSION: The quality of symptoms rather than quantity is correlated with outcome among patients with colon cancer. The proposed clinical scoring system may correctly predict the patient's outcome.

12/15-Lipoxygenase-derived lipid metabolites induce retinal endothelial cell barrier dysfunction: contribution of NADPH oxidase.

The purpose of the current study was to evaluate the effect of 12/15-lipoxygenase (12/15-LOX) metabolites on retinal endothelial cell (REC) barrier function. FITC-dextran flux across the REC monolayers and electrical cell-substrate impedance sensing (ECIS) were used to evaluate the effect of 12- and 15-hydroxyeicosatetreanoic acids (HETE) on REC permeability and transcellular electrical resistance (TER). Effect of 12- or 15-HETE on the levels of zonula occludens protein 1 (ZO-1), reactive oxygen species (ROS), NOX2, pVEGF-R2 and pSHP1 was examined in the presence or absence of inhibitors of NADPH oxidase. In vivo studies were performed using Ins2(Akita) mice treated with or without the 12/15-LOX inhibitor baicalein. Levels of HETE and inflammatory mediators were examined by LC/MS and Multiplex Immunoassay respectively. ROS generation and NOX2 expression were also measured in mice retinas. 12- and 15- HETE significantly increased permeability and reduced TER and ZO-1 expression in REC. VEGF-R2 inhibitor reduced the permeability effect of 12-HETE. Treatment of REC with HETE also increased ROS generation and expression of NOX2 and pVEGF-R2 and decreased pSHP1 expression. Treatment of diabetic mice with baicalein significantly decreased retinal HETE, ICAM-1, VCAM-1, IL-6, ROS generation, and NOX2 expression. Baicalein also reduced pVEGF-R2 while restored pSHP1 levels in diabetic retina. Our findings suggest that 12/15-LOX contributes to vascular hyperpermeability during DR via NADPH oxidase dependent mechanism which involves suppression of protein tyrosine phosphatase and activation of VEGF-R2 signal pathway.

High-mobility group box-1 protein activates inflammatory signaling pathway components and disrupts retinal vascular-barrier in the diabetic retina.

Extracellular high-mobility group box-1 (HMGB-1) functions as a pro-inflammatory cytokine and exhibits angiogenic effects. The purpose of this study was to investigate the expression of HMGB-1 signaling pathway components in the retinas of diabetic rats and to examine the effect of intravitreal administration of HMGB-1 on the retinas of rats. The retinas of diabetic and intravitreally injected HMGB-1 rats were studied using immunohistochemistry, Western blotting, co-immunoprecipitation and enzyme-linked immunosorbent assay. The effect of HMGB-1 on retinal endothelial cell barrier function was evaluated using electrical cell-substrate impedance sensing system (ECIS). Diabetes induced significant upregulation of the expression of HMGB-1, receptor for advanced glycation end products (RAGE), ERK(1/2) and nuclear transcription factor Kappa B (NF-κB), whereas the expression of toll-like receptor 2 (TLR2) and occludin was significantly downregulated. Co-immunoprecipitation studies revealed significant increase in interaction between HMGB-1 and RAGE. HMGB-1 reduced transendothelial electrical resistance of bovine retinal endothelial cells. Intravitreal administration of HMGB-1 to normal rats induced significant upregulation of intercellular adhesion molecule-1 (ICAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), HMGB-1, RAGE, ERK(1/2), and NF-κB, and significantly increased retinal vascular permeability, whereas the expression of TLR2 and occludin was downregulated. Oral administration of glycyrrhizin, a specific inhibitor of HMGB-1, attenuated diabetes-induced upregulation of HMGB-1 expression, NF-κB activation and downregulation of occludin expression. Our findings provide evidence that in the diabetic retina, HMGB-1 possibly interacts with RAGE and activates ERK(1/2) and NF-κB to generate an inflammatory response and disrupt retinal vascular barrier.

Targeting Neovascularization in Ischemic Retinopathy: Recent Advances.

Pathological retinal neovascularization (RNV) is a common micro-vascular complication in several retinal diseases including retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration and central vein occlusion. The current therapeutic modalities of RNV are invasive and although they may slow or halt the progression of the disease they are unlikely to restore normal acuity. Therefore, there is an urgent need to develop treatment modalities, which are less invasive and therefore associated with fewer procedural complications and systemic side effects. This review article summarizes our understanding of the pathophysiology and current treatment of RNV in ischemic retinopathies; lists potential therapeutic targets; and provides a framework for the development of future treatment modalities.

Caspase-14: a novel caspase in the retina with a potential role in diabetic retinopathy.

PURPOSE: The purpose of this study was to evaluate caspase-14 expression in the retina under normal and diabetic conditions, and to determine whether caspase-14 contributes to retinal microvascular cell death under high glucose conditions. METHODS: Quantitative real-time polymerase chain reaction and western blot analysis were used to evaluate caspase-14 expression in retinal cells, including pericytes (PCs), endothelial cells (ECs), astrocytes (ACs), choroidal ECs, and retinal pigment epithelium (RPE) cells. We also determined caspase-14 expression in the retinas of human subjects with or without diabetic retinopathy (DR) and in experimental diabetic mice. Retinal ECs and PCs were infected with adenoviruses expressing human caspase-14 or green fluorescent protein. Caspase-14 expression was also assessed in retinal vascular cells cultured under high glucose conditions. The number of apoptotic cells was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling staining and confirmed by determining the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3. RESULTS: Our experiments demonstrated that retinal ECs, PCs, ACs, choroidal ECs, and RPE cells expressed caspase-14, and DR was associated with upregulation and/or activation of caspase-14 particularly in retinal vasculature. High glucose induced marked elevation of the caspase-14 level in retinal vascular cells. There was a significant increase in the apoptosis rate and the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3 in retinal ECs and PCs overexpressing caspase-14. CONCLUSIONS: Our findings indicate that caspase-14 might play a significant role in the pathogenesis of DR by accelerating retinal PC and EC death. Further investigations are required to elaborate the underlying mechanisms.