Introduction: Triggering receptor expressed on myeloid cells-1 (TREM-1) has important implications in sepsis and inflammation and is a novel receptor for extracellular cold-inducible RNA-binding protein (eCIRP). We hypothesize that the inhibition of TREM-1 via its interaction with eCIRP by novel peptide inhibitor M3 or knockout gene will attenuate the inflammation and injury associated with severe hepatic ischemia/reperfusion (I/R). Methods: Wild-type (WT) C57BL/6 and TREM-1 -/mice underwent 60 min of 70% hepatic ischemia, with 24 h of reperfusion. Additionally, WT mice underwent hepatic I/R and were treated with M3 (10 mg/kg body weight) or vehicle (normal saline) at the start of reperfusion. Blood and ischemic liver tissues were collected, and analysis was performed using enzymatic assays, enzymelinked immunosorbent assay, reverse-transcription quantitative polymerase chain reaction, and pathohistology techniques. For survival surgery, mice additionally underwent resection of non-ischemic lobes of the liver and survival was monitored for 10 days. Results: There was an increase in serum levels of tissue markers including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase as well as cytokine levels (IL-6) and histological scoring of hematoxylin and eosin sections in WT I/R mice. These markers decreased substantially in TREM-1 -/mice. Additionally, neutrophil infiltration markers and markers of local inflammation (myeloperoxidase, macrophage inflammatory protein-2, cyclooxygenase-2) were attenuated in TREM-1 -/mice. Similarly, we show a significant decrease in injury and inflammation markers with M3 treatment. Additionally, we demonstrate decreased apoptosis with TREM-1 inhibition. Finally, M3 treatment improved the survival rate from 42% to 75% after hepatic I/R. Conclusion: TREM-1 is an important eCIRP receptor in the inflammatory response of hepatic I/R, and deficiency of TREM-1 via knockout gene or peptide inhibition attenuated liver injury and inflammation, and improved survival. Inhibition of the TREM-1 and eCIRP interaction in hepatic I/R may have important therapeutic potential.
Background Sepsis is caused by the dysregulated immune response due to an initial infection and results in significant morbidity and mortality in humans. Extracellular cold inducible RNA binding protein (eCIRP) is a novel mediator identified in sepsis. We have previously discovered that microRNA 130b-3p inhibits eCIRP mediated inflammation. As RNA mimics are very unstable in vivo, we hypothesize that an engineered miRNA 130b-3p mimic named PS-OMe miR130, improves stability of the miRNA by protection from nuclease activity. We further hypothesize that PS-OMe miR130 reduces not only eCIRP-mediated inflammation and but also acute lung injury in a murine model of polymicrobial sepsis. Methods Single stranded PS-OMe miR130 was synthesized and the binding affinity to eCIRP was evaluated using surface plasmon resonance (SPR) and computational modeling. Macrophages were treated with PS-OMe miR130 with and without eCIRP and cell supernatant analyzed for cytokines. In vitro stability and the in vivo half-life of PS-OMe miR130 were also assessed. The effect of PS-Ome miR130 on eCIRP’s binding to TLR4 was evaluated by SPR analysis and modeling. Finally, the effect of PS-OMe miR130 on inflammation and injury was assessed in a murine model of sepsis. Results We demonstrate via SPR and computational modeling that PS-OMe miR130 has a strong binding affinity to eCIRP. This engineered miRNA decreases eCIRP induced TNF-α and IL-6 proteins, and it is highly stable in vitro and has a long in vivo half-life. We further demonstrate that PS-OMe miR130 blocks eCIRP binding to its receptor TLR4. Finally, we show that PS-OMe miR130 inhibits inflammation and lung injury, and improves survival in murine sepsis. Conclusion PS-OMe miR130 can be developed as a novel therapeutic by inhibiting eCIRP-mediated inflammation and acute lung injury in sepsis.
INTRODUCTION: Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel mediator of inflammation and tissue injury. It has been shown that miRNA 130b-3p acts as an endogenous inhibitor of eCIRP. Because RNA mimics are unstable after in vivo administration, we have chemically engineered miRNA 130b-3p mimic (named PS-OMe miR130) to improve its stability by protection from nuclease activity. We hypothesize that PS-OMe miR130 reduces eCIRP-mediated injury and inflammation in a murine model of hepatic ischemia/ reperfusion (I/R), a model of sterile inflammation. METHODS:Adult male mice underwent 70% hepatic ischemia for 60 minutes and 24-hour reperfusion. At the start of reperfusion, mice were treated intravenously with vehicle (phosphate-buffered saline) or PS-OMe miR130. Blood and liver tissue were collected after 24 hours for biochemical analysis. Apoptosis in the liver tissue was determined by transferase dUTP nick-end labeling assay. RESULTS:After hepatic I/R, organ injury markers including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase significantly decreased after PS-OMe miR130 treatment. Furthermore, histological analysis of liver sections demonstrated significantly less injury in PS-OMe miR130 treatment mice versus vehicle mice. In addition, tumor necrosis factor α mRNA, interleukin-1β mRNA, and neutrophil infiltration (myeloperoxidase activity and granulocyte receptor 1 immunohistochemistry) were significantly attenuated after PS-OMe miR130 treatment. Finally, apoptosis significantly decreased in liver tissue after treatment. CONCLUSION:PS-OMe miR130 decreases eCIRP-mediated injury and inflammation in a murine model of hepatic I/R.
BackgroundIntestinal ischemia–reperfusion (I/R) injury is a severe disease associated with high mortality. Stimulator of interferon genes (STING) is an intracellular protein that is activated by cytosolic DNA and is implicated in I/R injury, resulting in transcription of type I interferons (IFN-α and IFN-β) and other proinflammatory molecules. Extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, induces STING activation. H151 is a small molecule inhibitor of STING that has not yet been studied as a potential therapeutic. We hypothesize that H151 reduces inflammation, tissue injury, and mortality after intestinal I/R. Methods: In vitro, RAW264.7 cells were pretreated with H151 then stimulated with recombinant murine (rm) CIRP, and IFN-β levels in the culture supernatant were measured at 24 hours after stimulation. In vivo, male C57BL/6 mice were subjected to 60-minute intestinal ischemia via superior mesenteric artery occlusion. At the time of reperfusion, mice were intraperitoneally instilled with H151 (10 mg/kg BW) or 10% Tween-80 in PBS (vehicle). Four hours after reperfusion, the small intestines, lungs, and serum were collected for analysis. Mice were monitored for 24 hours after intestinal I/R to assess survival. Results: In vitro, H151 reduced rmCIRP-induced IFN-β levels in a dose-dependent manner. In vivo, intestinal levels of pIRF3 were increased after intestinal I/R and decreased after H151 treatment. There was an increase in serum levels of tissue injury markers (lactate dehydrogenase, aspartate aminotransferase) and cytokine levels (interleukin 1β, interleukin 6) after intestinal I/R, and these levels were decreased after H151 treatment. Ischemia-reperfusion–induced intestinal and lung injury and inflammation were significantly reduced after H151 treatment, as evaluated by histopathologic assessment, measurement of cell death, chemokine expression, neutrophil infiltration, and myeloperoxidase activity. Finally, H151 improved the survival rate from 41% to 81% after intestinal I/R. Conclusions: H151, a novel STING inhibitor, attenuates the inflammatory response and reduces tissue injury and mortality in a murine model of intestinal I/R. H151 shows promise as a potential therapeutic in the treatment of this disease.
Coagulum pyelolithotomy is mainly indicated in cases of multiple stones, soft and crumbly stone or stones, small, mobile caliceal stones, and as an aid to fixing caliceal stone or stones in the calix prior to partial resection of the kidney or nephrolithotomy. Its main advantages are:-I. Trauma to the kidney is minimized. 2. All the free stones in the pelvicaliceal system may be removed, thus reducing the incidence of so-called 'recurrent' stones.3. Fragmentation of the stone during removal is avoided. Surgical mobilization of the kidney is notnecessary since it is sufficient to expose the pelvis.A method of coagulum pyelolithotomy is described in detail and discussed. The operation has been performed in 22 cases with good results. Two cases are described in detail. It is suggested that this method could be utilized for common bile-duct stones.THE number of cases with recurrent renal stones after pyelolithotomy and nephrolithotomy reported in the world literature is very high. However, it is very questionable whether these are always recurrent stones. In many cases this 'recurrence' is due to incomplete removal of renal stones during the operation. Oppenheimer (1940) reported incomplete removal of renal stones in 41.4 per cent of nephrolithotomies, in 32'4 per cent of pyelonephrolithotomies, and in 10.6 per cent of pyelolithotomies. Barney (1922) stated that there was incomplete removal of stones in 45 per cent of nephrolithotomies, and the author feels certain that the percentage of incomplete removal of stones is even higher, since postoperative radiographic checks fail to reveal all the stones which are left behind after operation. Several factors may be responsible for the impossibility of removing all the stones present at the time of operation:-I. The shadow of smaller stones could be obscured by that of a bigger one on the radiograph.2. Radiolucent stones and small radio-opaque stones may not be visible on the radiograph.3. The interior of the renal pelvis cannot be completely surveyed through a pyelotomy incision, and the fact that a small stone may move from one calix to another in the course of the operation makes it difficult or impossible to locate the calculus. 4.A blood-clot may cover a small stone, which is then difficult to locate.The technique of overdistending the renal pelvis with an isotonic saline solution with the ureter occluded has served well in washing out elusive stones, but this method leaves much to be desired. The same is true of carrying out routine radiographs on the operating table. Oppenheimer (1940) found that in a series of 63 cases radiographs taken on the operating table showed no evidence of any stones remaining in the kidney, yet a subsequent check revealed stones in 12.7 per cent of these cases.
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