scholarly journals Polymyxin B Attenuates LPS-Induced Death but Aggravates Radiation-Induced Death via TLR4-Myd88-IL-6 Pathway

2017 ◽  
Vol 42 (3) ◽  
pp. 1120-1126 ◽  
Author(s):  
Ying Cheng ◽  
Jicong Du ◽  
Jiaqi Han ◽  
Weimin Sun ◽  
Fu Gao ◽  
...  
Keyword(s):  
Polymyxin B ◽  
Protection Factor ◽  
Knock Out ◽  
Endotoxin Contamination ◽  
Radiation Induced ◽  
Total Body ◽  
Α Level

Background/Aims: Polymyxin B (PMB) is a cyclic cationic polypeptide antibiotic widely used to counteract the effects of endotoxin contamination, both in vitro and in vivo. Lipopolysaccharide (LPS) is an endotoxin that acts as a radiation protection factor. In this study, we focus on the role of PMB in LPS-induced and radiation-induced mortality in mice. Methods: Mice received total-body radiation or were pretreated by LPS or PMB, and the survival of mice was recorded. Elisa were used to detect the cytokines levels. Results: PMB decreased LPS-induced, but increased radiation-induced mortality in mice. Moreover, PMB could block the LPS-induced radioprotective effect. The ELISA and gene knock-out experiments indicated that PMB reduces TNF-α level to block LPS-induced mortality in mice, and inhibits IL-6, G-CSF and IL-10 to increase radiation-induced mortality via the TLR4-Myd88-IL-6 pathway. Conclusions: Our study revealed a role of PMB in LPS-induced endotoxemia and radiation exposure. We infer that the TLR4-Myd88-IL-6 pathway may play a crucial role in the process.

scholarly journals The Extracellular Small Leucine-Rich Proteoglycan Biglycan Is a Key Player in Gastric Cancer Aggressiveness

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1330
Author(s):  
Filipe Pinto ◽  
Liliana Santos-Ferreira ◽  
Marta T. Pinto ◽  
Catarina Gomes ◽  
Celso A. Reis
Keyword(s):  
Clinical Value ◽  
Knock Out ◽  
Angiogenic Potential ◽  
In Vivo Experiments ◽  
Cancer Aggressiveness ◽  
Advanced Stages ◽  
Oncogenic Gene

Biglycan (BGN gene), an extracellular proteoglycan, has been described to be associated with cancer aggressiveness. The purpose of this study was to clarify the clinical value of biglycan as a biomarker in multiple independent GC cohorts and determine the in vitro and in vivo role of biglycan in GC malignant features. We found that BGN is commonly over-expressed in all analyzed cohorts, being associated with disease relapse and poor prognosis in patients with advanced stages of disease. In vitro and in vivo experiments demonstrated that biglycan knock-out GC cells display major phenotypic changes with a lower cell survival, migration, and angiogenic potential when compared with biglycan expressing cells. Biglycan KO GC cells present increased levels of PARP1 and caspase-3 cleavage and a decreased expression of mesenchymal markers. Importantly, biglycan deficient GC cells that were supplemented with exogenous biglycan were able to restore biological features, such as survival, clonogenic and migratory capacities. Our in vitro and in vivo findings were validated in human GC samples, where BGN expression was associated with several oncogenic gene signatures that were associated with apoptosis, cell migration, invasion, and angiogenesis. This study provided new insights on biglycan role in GC that should be taken in consideration as a key cellular regulator with major impact in tumor progression and patients’ clinical outcome.

scholarly journals DR5 Knockout Mice Are Compromised in Radiation-Induced Apoptosis

2005 ◽  
Vol 25 (5) ◽  
pp. 2000-2013 ◽  
Author(s):  
Niklas Finnberg ◽  
Joshua J. Gruber ◽  
Peiwen Fei ◽  
Dorothea Rudolph ◽  
Anka Bric ◽  
...  
Keyword(s):  
Cell Death ◽  
Null Mouse ◽  
Organ Specific ◽  
Radiation Induced ◽  
Induced Apoptosis ◽  
The Brain ◽  
Necrosis Factor

ABSTRACT DR5 (also called TRAIL receptor 2 and KILLER) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called TRAIL and Apo2 ligand). DR5 is a transcriptional target of p53, and its overexpression induces cell death in vitro. However, the in vivo biology of DR5 has remained largely unexplored. To better understand the role of DR5 in development and in adult tissues, we have created a knockout mouse lacking DR5. This mouse is viable and develops normally with the exception of having an enlarged thymus. We show that DR5 is not expressed in developing embryos but is present in the decidua and chorion early in development. DR5-null mouse embryo fibroblasts expressing E1A are resistant to treatment with TRAIL, suggesting that DR5 may be the primary proapoptotic receptor for TRAIL in the mouse. When exposed to ionizing radiation, DR5-null tissues exhibit reduced amounts of apoptosis compared to wild-type thymus, spleen, Peyer's patches, and the white matter of the brain. In the ileum, colon, and stomach, DR5 deficiency was associated with a subtle phenotype of radiation-induced cell death. These results indicate that DR5 has a limited role during embryogenesis and early stages of development but plays an organ-specific role in the response to DNA-damaging stimuli.

scholarly journals BRCC3 Promotes Tumorigenesis of Bladder Cancer by Activating the NF-κB Signaling Pathway Through Targeting TRAF2

2021 ◽  
Vol 9 ◽  
Author(s):  
Huangheng Tao ◽  
Yixiang Liao ◽  
Youji Yan ◽  
Zhiwen He ◽  
Jiajie Zhou ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Signaling Pathway ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Rna Seq ◽  
Knock Out

NF-κB signaling is very important in cancers. However, the role of BRCC3-associated NF-κB signaling activation in bladder cancer remains to be characterized. Western blotting and IHC of tissue microarray were used to confirm the abnormal expression of BRCC3 in bladder cancer. Growth curve, colony formation, soft agar assay and Xenograft model were performed to identify the role of BRCC3 over-expression or knock-out in bladder cancer. Further, RNA-Seq and luciferase reporter assays were used to identify the down-stream signaling pathway. Finally, co-immunoprecipitation and fluorescence confocal assay were performed to verify the precise target of BRCC3. Here, we found that high expression of BRCC3 promoted tumorigenesis through targeting the TRAF2 protein. BRCC3 expression is up-regulated in bladder cancer patients which indicates a negative prognosis. By in vitro and in vivo assays, we found genetic BRCC3 ablation markedly blocks proliferation, viability and migration of bladder cancer cells. Mechanistically, RNA-Seq analysis shows that NF-κB signaling is down-regulated in BRCC3-deficient cells. BRCC3 binds to and synergizes with TRAF2 to activate NF-κB signaling. Our results indicate that high BRCC3 expression activates NF-κB signaling by targeting TRAF2 for activation, which in turn facilitates tumorigenesis in bladder cancer. This finding points to BRCC3 as a potential target in bladder cancer patients.

scholarly journals Microglial phagocytosis dysfunction during stroke is prevented by rapamycin

2021 ◽  
Author(s):  
Sol Beccari ◽  
Virginia Sierra-Torre ◽  
Jorge Valero ◽  
Mikel Garcia-Zaballa ◽  
Alejandro Carretero-Guillen ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Therapeutic Target ◽  
Apoptotic Cell ◽  
Knock Out ◽  
Microglial Phagocytosis ◽  
Cellular Debris ◽  
The Brain

Microglial phagocytosis is rapidly emerging as a therapeutic target in neurodegenerative and neurological disorders. An efficient removal of cellular debris is necessary to prevent buildup damage of neighbor neurons and the development of an inflammatory response. As the brain professional phagocytes, microglia are equipped with an array of mechanisms that enable them to recognize and degrade several types of cargo, including neurons undergoing apoptotic cell death. While microglia are very competent phagocytes of apoptotic cells under physiological conditions, here we report their dysfunction in mouse and monkey (Macaca fascicularis and Callithrix jacchus) models of stroke by transient occlusion of the medial cerebral artery (tMCAo). The impairment of both engulfment and degradation was related to energy depletion triggered by oxygen and nutrients deprivation (OND), which led to reduced process motility, lysosomal depletion, and the induction of a protective autophagy response in microglia. Basal autophagy, which is in charge of removing and recycling intracellular elements, was critical to maintain microglial physiology, including survival and phagocytosis, as we determined both in vivo and in vitro using knock-out models of autophagy genes and the autophagy inhibitor MRT68921. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by tMCAo in vivo but not by OND in vitro. These results suggest a more complex role of microglia in stroke than previously acknowledged, classically related to the inflammatory response. In contrast, here we demonstrate the impairment of apoptotic cell phagocytosis, a microglial function critical for brain recovery. We propose that phagocytosis is a therapeutic target yet to be explored and provide evidence that it can be modulated in vivo using rapamycin, setting the stage for future therapies for stroke patients.

scholarly journals Krüppel-like factor 4 is a radioprotective factor for the intestine following γ-radiation-induced gut injury in mice

2015 ◽  
Vol 308 (2) ◽  
pp. G121-G138 ◽  
Author(s):  
Daniel Talmasov ◽  
Xinjun Zhang ◽  
Bing Yu ◽  
Mandayam O. Nandan ◽  
Agnieszka B. Bialkowska ◽  
...  
Keyword(s):  
Intestinal Epithelium ◽  
Morphological Changes ◽  
Γ Irradiation ◽  
Γ Radiation ◽  
Proliferative Zone ◽  
Acute Response ◽  
Radiation Induced ◽  
Total Body

Gut radiation-induced injury is a concern during treatment of patients with cancer. Krüppel-like factor 4 (KLF4) is expressed in differentiated villous epithelial cells of the small intestine. We previously showed that KLF4 protects cells from apoptosis following γ-irradiation in vitro. We sought to determine whether KLF4 mediates the small intestinal response to γ-irradiation in vivo. Mice with intestinal epithelium-specific deletion of Klf4 ( Klf4 ΔIS) and control ( Klf4 fl/fl) mice were irradiated with total-body γ-radiation. Following irradiation, the Klf4 ΔIS mice had significantly increased mortality compared with irradiated Klf4 fl/fl mice. Immunohistochemistry and immunofluorescence staining were used to assess the morphological changes, levels of proliferation, and apoptosis in the intestinal epithelium. At 96 h following irradiation, there was a regenerative response manifested by an expansion of the proliferative zone in both mouse groups, with the control mice having a higher proliferative activity than the Klf4 ΔIS group. In addition, there was a significant increase in the number of Klf4/Ki67-copositive cells in the irradiated control mice compared with unirradiated mice. Also, the irradiated Klf4 ΔIS mice had a significantly higher number of crypt cells positive for apoptosis, p53, and p21 compared with irradiated Klf4 fl/fl mice. Taken together, our data suggest that Klf4 may function as a radioprotective factor against gastrointestinal syndrome in mice following γ-irradiation by inhibiting apoptosis in the acute response to irradiation and contributing to crypt regeneration.

Carboxypeptidase U (TAFIa): a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease

2005 ◽  
Vol 94 (09) ◽  
pp. 471-487 ◽  
Author(s):  
Judith Leurs ◽  
Dirk Hendriks
Keyword(s):  
Bleeding Tendency ◽  
Knock Out ◽  
Lysine Residues ◽  
Knock Out Mice ◽  
The Moment ◽  
And Function ◽  
Coagulation And Fibrinolysis

SummarySince the discovery of Carboxypeptidase U (CPU) in 1988, considerable information has been gathered about its biochemistry and function in physiological and pathophysiological circumstances. A variety of tools such as assays to measure proCPU and CPU, antibodies raised against (pro)CPU, selective CPU inhibitors and knock-out mice have been developed and are currently being used to explore the role of this metallocarboxypeptidase in different in vivo and in vitro settings. The knowledge that proCPU can be activated by thrombin and plasmin, enzymes with a key function in coagulation and fibrinolysis, and the ability of CPU to remove C-terminal lysine residues has led to the hypothesis that the proCPU/CPU pathway plays a role in the balance between coagulation and fibrinolysis. The maintenance of the equilibrium between coagulation and fibrinolysis is crucial for normal haemostasis and disturbance of this delicate balance can lead either to bleeding tendency or thrombosis. This review provides an update on several aspects of CPU known at the moment, including an extensive overview on the clinical studies performed up till now.J. Leurs is a research assistant of the Fund for Scientific Research Flanders (FWO-Vlaanderen).

Cytochrome C suppresses renal accumulation and nephrotoxicity of polymyxin B

2018 ◽  
Vol 38 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Z-D Li ◽  
J Luo ◽  
L-H Jia ◽  
X-Y Wang ◽  
Z-K Xun ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Polymyxin B ◽  
Control Group ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
C 30 ◽  
C 50

The receptor megalin plays an important role in the accumulation of polymyxin B (PMB) in renal cells in vitro. This study aimed to examine the effects of cytochrome c (cyto c), a typical megalin ligand, on renal accumulation and nephrotoxicity of PMB in vivo. Thirty Sprague-Dawley rats were randomly divided into the vehicle control group, PMB group, PMB + cyto c 50, 100, or 200 mg/kg group, respectively, and were treated with intravenous cyto c 30 min before the administration of PMB 4.0 mg/kg once a day for consecutive 5 days. On the 4th day after administration, 24 h urine was collected to determine N-acetyl-β-D-glucosaminidase excretion. Six hours after the last injection on the 5th day, kidneys were harvested to assay PMB concentration and observe pathological alterations, and blood samples were collected to assay serum creatinine (SCr), blood urea nitrogen (BUN), and blood β2-microglobulin (β2-MG) levels. Cyto c 50, 100, and 200 mg/kg decreased the accumulation of PMB in the kidney by 18.5%, 39.1% ( p < 0.01), and 36.8% ( p < 0.01), respectively, and reduced 24 h N-acetyl-β-D- glucosaminidase excretion by 22.5% ( p < 0.05), 40.4% ( p < 0.01), and 40.4% ( p < 0.01), respectively. Kidney pathological damage induced by PMB was markedly reduced by cyto c 100 mg/kg and 200 mg/kg. However, there were no significant differences in SCr, BUN, and blood β2-MG levels among the groups. These results indicated that cyto c may inhibit the renal accumulation and nephrotoxicity of PMB in a rat model, further proving the role of megalin in the accumulation of PMB.

Role of endotoxin in grain dust-induced lung inflammation in mice

1996 ◽  
Vol 270 (6) ◽  
pp. L1052-L1059 ◽  
Author(s):  
P. J. Jagielo ◽  
P. S. Thorne ◽  
J. A. Kern ◽  
T. J. Quinn ◽  
D. A. Schwartz
Keyword(s):  
Inflammatory Response ◽  
Membrane Filtration ◽  
Polymyxin B ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Endotoxin Activity ◽  
Grain Dust

To investigate the role of endotoxin in grain dust-induced airway inflammation, we reduced the endotoxin activity from extracts of corn dust (CDE), using three distinct methods, and determined the effect of endotoxin activity on the in vitro and in vivo inflammatory response to CDE. Escherichia coli lipopolysaccharide solution (LPS) and CDE solution were separated into > 100-kDa and < 100-kDa fractions by ultracentrifugation. Endotoxin activity was predominantly present in the > 100-kDa fractions of the LPS and CDE solutions. Charged-membrane filtration of the > 100-kDa fractions of LPS and CDE resulted in the reduction of endotoxin activity by 99.9 and 80%, respectively. Treatment of the > 100-kDa fractions of LPS and CDE with polymyxin B-coated beads reduced the endotoxin activity by 96 and 89%, respectively. The untreated > 100-kDa fractions of LPS and CDE caused significantly greater (P < 0.01) release of tumor necrosis factor-alpha (TNF-alpha) from THP-1 cells in vitro compared with its respective < 100-kDa fraction or either of the treated (charged filter or polymyxin B) > 100-kDa fractions. Similarly, mice exposed to either of the untreated > 100-kDa fractions of LPS or CDE by inhalation developed significantly greater (P < 0.01) concentrations of lavage neutrophils and TNF-alpha in the lavage fluid compared with mice exposed to the respective < 100-kDa fraction or either of the treated > 100-kDa fractions. These results indicate that endotoxin is primarily responsible for the in vitro and in vivo inflammatory response to CDE.

scholarly journals OS6.3 The complex role of lactate dehydrogenases in glioblastoma development

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii12-iii13
Author(s):  
T Daubon ◽  
J Guyon ◽  
T Chouleur ◽  
H Espedal ◽  
C Leon ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Tumor Development ◽  
Vascular Density ◽  
Knock Out ◽  
Hypoxic Conditions ◽  
Extracellular Compartment ◽  
Vessel Development

Abstract BACKGROUND Glioblastomas are among the most malignant primary brain tumors. GBMs are highly angiogenic, exhibit invasive growth, and elevated glycolysis. Under glycolytic conditions, glucose from the blood is metabolized in astrocytes into lactate by LDHA, and exported by MCT4 into the extracellular compartment, inducing a concomitant acidification of the microenvironment. LDHB, generally expressed in oligodendrocytes or neurons, metabolizes lactate into pyruvate for generating ATP in mitochondria. LDH expression was reported to be linked to phenotypic modifications in vitro in GBMs but the mechanisms and the precise role in vivo have not yet been investigated. MATERIAL AND METHODS We designed LDHA and LDHB Crispr-Cas9 constructs for infecting glioblastoma stem-like cells. Results: In vitro tumor cell invasion was not significantly impaired in sgLDHA glioblastoma cells, even under extreme hypoxic conditions. Tumor development was moderately impacted in terms of invasion or vascular density. We then explored the role of LDHB in these processes. LDHB knock-out cells had decreased invasive properties in vitro but surprisingly tumors were highly hemorrhagic and angiogenic, supporting a role of tumor-derived LDHB in blood vessel development. We furthermore evaluated the consequences of a double LDHA and LDHB knock-out in the glioma cells. Under hypoxic conditions, sgLDHA/B cell invasion was dramatically decreased in comparison to control cells, and apoptosis was also increased. Tumor development was dramatically impaired for LDHA/LDHB knockout tumors. CONCLUSION These results indicate the complex role of LDH enzymes in glioblastoma development. It constitutes the basis for further mechanistical studies linking lactate metabolism to brain tumor development and perturbation of the neuro-vascular microenvironment.

Are all granzymes cytotoxic in vivo?

2014 ◽  
Vol 395 (2) ◽  
pp. 181-202 ◽  
Author(s):  
Lars T. Joeckel ◽  
Phillip I. Bird
Keyword(s):  
Serine Proteases ◽  
Ex Vivo ◽  
Cytotoxic Lymphocytes ◽  
Cytotoxic Effects ◽  
Knock Out ◽  
Granzyme A ◽  
Knock Out Mice

Abstract Granzymes are serine proteases mainly found in cytotoxic lymphocytes. The most-studied member of this group is granzyme B, which is a potent cytotoxin that has set the paradigm that all granzymes are cyototoxic. In the last 5 years, this paradigm has become controversial. On one hand, there is a plethora of sometimes contradictory publications showing mainly caspase-independent cytotoxic effects of granzyme A and the so-called orphan granzymes in vitro. On the other hand, there are increasing numbers of reports of granzymes failing to induce cell death in vitro unless very high (potentially supra-physiological) concentrations are used. Furthermore, experiments with granzyme A or granzyme M knock-out mice reveal little or no deficit in their cytotoxic lymphocytes’ killing ability ex vivo, but indicate impairment in the inflammatory response. These findings of non-cytotoxic effects of granzymes challenge dogma, and thus require alternative or additional explanations to be developed of the role of granzymes in defeating pathogens. Here we review evidence for granzyme cytotoxicity, give an overview of their non-cytotoxic functions, and suggest technical improvements for future investigations.

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