muramidase and Reperfusion-Injury

The lysozyme 2 (Lyz2 or LysM) cre mouse is extensively used to achieve genetic manipulation in myeloid cells and it has been widely employed in retinal research. However, LysM has been recently described to be expressed in brain neurons and there is a debate on whether it is also expressed by resident microglia in addition to infiltrating macrophages.. We examined LysM-cre recombination in retinal tissue using a LysM-cre/tdTomato reporter mouse together with immunolabeling for several retinal cell markers. We further compared LysM-cre tdTomato recombination with that of Cdh5-cre driver, which is expressed in both endothelial and hematopoietic cells.. LysM-cre was strongly expressed in most microglia/resident macrophages in neonatal retinas (P8) and to a lesser extent in microglia of adult retinas. In addition, there was some neuronal recombination (8 %) of LysM-cre specifically in adult retinal ganglion cells and amacrine cells. After retinal ischemia-reperfusion injury, LysM-cre was strongly expressed in microglia/infiltrating macrophages. Cdh5-cre was expressed in endothelial and myeloid cells of P8 pups retinas. Unexpectedly, Cdh5 showed additional expression in adult mouse retinal ganglion cells and brain neurons.. LysM-cre is expressed in macrophages and a subset of microglia together with a small but significant recombination of LysM-cre in the retinal neurons of adult mice. Cdh5 also showed some neuronal expression in both retina and brain of adult mice. These findings should be taken into consideration when interpreting results from central nervous system research using LysM-cre and Cdh5-cre mice.

Topics: Animals; Animals, Newborn; Antigens, CD; Biomedical Research; Brain; Cadherins; Diagnostic Imaging; Endothelium, Vascular; Female; Genes, Reporter; Integrases; Luminescent Agents; Luminescent Proteins; Macrophages; Male; Mice; Mice, Transgenic; Microglia; Muramidase; Neurons; Recombination, Genetic; Red Fluorescent Protein; Reperfusion Injury; Retinal Ganglion Cells; Retinal Vessels

Ischemia-reperfusion injury is inevitable during intestinal transplantation (ITx) and executes a key role in the evolution towards rejection. Paneth cells (PCs) are crucial for epithelial immune defense and highly vulnerable to ischemia-reperfusion injury. We investigated the effect of ITx on PC after reperfusion (T0), during follow-up, and rejection. Moreover, we investigated whether PC loss was associated with impaired graft homeostasis.. Endoscopic biopsies, collected according to center protocol and at rejection episodes, were retrospectively included (n = 28 ITx, n = 119 biopsies) Biopsies were immunohistochemically co-stained for PC (lysozyme) and apoptosis, and PC/crypt and lysozyme intensity were scored.. We observed a decrease in PC/crypt and lysozyme intensity in the first week after ITx (W1) compared with T0. There was a tendency towards a larger decline in PC/crypt (P = 0.08) and lysozyme intensity (P = 0.08) in W1 in patients who later developed rejection compared with patients without rejection. Follow-up biopsies showed that the PC number recovered, whereas lysozyme intensity remained reduced. This persisting innate immune defect may contribute to the well-known vulnerability of the intestine to infection. There was no clear evidence that PCs were affected throughout rejection.. This study revealed a transient fall in PC numbers in the early post-ITx period but a permanent reduction in lysozyme intensity following ITx. Further research is needed to determine the potential clinical impact of PC impairment after ITx.

Topics: Adolescent; Apoptosis; Cell Count; Child; Child, Preschool; Female; Graft Rejection; Humans; Infant; Intestines; Male; Muramidase; Paneth Cells; Reperfusion Injury; Retrospective Studies

Neutrophils are considered responsible for the pathophysiologic changes during hepatic ischemia-reperfusion (I/R) injury; however, few studies have examined real-time intravital neutrophil recruitment. Here, we show a method for imaging the neutrophil recruitment in hepatic I/R injury using two-photon laser scanning microscopy (TPLSM).. LysM-eGFP mice were subjected to 45 min of partial warm hepatic ischemia followed by reperfusion. Mice received an intravenous injection of tetramethylrhodamine isothiocyanate-labeled albumin to visualize the microvasculature. Using time-lapse TPLSM technique, we directly observed the behavior of neutrophils in I/R injury.. At low magnification, four to six hepatic lobules could be visualized. The number of adherent neutrophils continued to increase for 4 hr after reperfusion, whereas their crawling velocity reached a maximum of 2 hr after reperfusion and then decreased gradually. High-magnification images revealed the presence or absence of blood circulation in sinusoids. Six hours after control operation or reperfusion, circulation was maintained in all sinusoids in the control group, whereas spotty nonperfused areas accompanied by neutrophil infiltration could be observed in the I/R group. Adherent neutrophils in perfused areas in the I/R group had more elongated shapes and moved more quickly than those in nonperfused areas and in the control group. Some hepatocytes affected by I/R injury showed the changes in their size and fluorescent intensity, which could attract neutrophils.. TPLSM was successfully used for intravital imaging of hepatic I/R injury in mice and has potential for a wide range of applications to investigate the mechanism of I/R injury.

Topics: Animals; Cell Death; Chemokine CXCL1; Chemokine CXCL2; Disease Models, Animal; Fluorescent Dyes; Green Fluorescent Proteins; Immunity, Innate; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Microscopy, Fluorescence, Multiphoton; Muramidase; Neutrophil Infiltration; Neutrophils; Promoter Regions, Genetic; Reperfusion Injury; Rhodamines; Time Factors; Warm Ischemia

We have synthesized a renal-specific drug carrier, 14-succinyl triptolide-lysozyme (TPS-LZM) conjugate for targeted delivery of TP to the PTECs. TPS-LZM could be taken up by HK-2 cells, free TP would be degraded and released, mainly from basolateral side of the cells. Compared with TP, the overall targeting efficiency (TE) of TPS-LZM was significantly enhanced from 11.74% to 95.54% and its MRT was moderately prolonged from 3.08h to 4.10h. At very low concentration, TPS-LZM could significantly reverse the disease progression in renal ischemia-reperfusion (I/R) injury animal models, while the mixture of free TP and LZM was ineffective. Further, TPS-LZM conjugate presented much lower hepatotoxicity (0.78 folds lower than TP) and no adverse effect on the immune (1.13 folds higher than TP) and genital system. Thus, TPS-LZM represents a very effective drug candidate for specific treatment of immunological renal diseases with low adverse side effect.

Topics: Animals; Diterpenes; Drug Carriers; Epithelial Cells; Epoxy Compounds; Humans; Kidney; Kidney Diseases; Kidney Tubules, Proximal; Male; Molecular Structure; Muramidase; Phenanthrenes; Rats; Rats, Wistar; Reperfusion Injury; Succinates; Tissue Distribution

The Rho kinase pathway plays an important role in dedifferentiation of epithelial cells and infiltration of inflammatory cells. For testing of the hypothesis that blockade of this cascade within the kidneys might be beneficial in the treatment of renal injury the Rho kinase inhibitor, Y27632 was coupled to lysozyme, a low molecular weight protein that is filtered through the glomerulus and is reabsorbed in proximal tubular cells. Pharmacokinetic studies with Y27632-lysozyme confirmed that the conjugate rapidly and extensively accumulated in the kidney. Treatment with Y27632-lysozyme substantially inhibited ischemia/reperfusion-induced tubular damage, indicated by reduced staining of the dedifferentiation markers kidney injury molecule 1 and vimentin, and increased E-cadherin relative to controls. Rho kinase activation was inhibited by Y27632-lysozyme within tubular cells and the interstitium. Y27632-lysozyme also inhibited inflammation and fibrogenesis, indicated by a reduction in gene expression of monocyte chemoattractant protein 1, procollagen Ialpha1, TGF-beta1, tissue inhibitor of metalloproteinase 1, and alpha-smooth muscle actin. Immunohistochemistry revealed reduced macrophage infiltration and decreased expression of alpha-smooth muscle actin, collagen I, collagen III, and fibronectin. In contrast, unconjugated Y27632 did not have these beneficial effects but instead caused systemic adverse effects, such as leukopenia. Neither treatment improved renal function in the bilateral ischemia/reperfusion model. In conclusion, the renally targeted Y27632-lysozyme conjugate strongly inhibits tubular damage, inflammation, and fibrogenesis induced by ischemia/reperfusion injury.

Topics: Actins; Amides; Animals; Cell Adhesion Molecules; Chemokine CCL2; Collagen Type I; Collagen Type I, alpha 1 Chain; Drug Carriers; Gene Expression; Kidney; Male; Muramidase; Protein Kinase Inhibitors; Pyridines; Rats; Rats, Wistar; Reperfusion Injury; rho-Associated Kinases; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta1

During renal injury, activation of p38 mitogen-activated protein kinase (MAPK) in proximal tubular cells plays an important role in the inflammatory events that eventually lead to renal fibrosis. We hypothesized that local inhibition of p38 within these cells may be an interesting approach for the treatment of renal fibrosis. To effectuate this, we developed a renal-specific conjugate of the p38 inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] and the carrier lysozyme. First, we demonstrated that SB202190 inhibited the expression of albumin-induced proinflammatory (monocyte chemoattractant protein-1) and transforming growth factor (TGF)-beta1-induced profibrotic (procollagen-Ialpha1) genes over 50% in renal tubular cells (normal rat kidney-52E). Next, we conjugated SB202190 via a carbamate linkage to lysozyme. However, this conjugate rapidly released the drug upon incubation in serum. Therefore, we applied a new platinum(II)-based linker approach, the so-called universal linkage system (ULS), which forms a coordinative bond with SB202190. The SB202190-ULS-lysozyme remained stable in serum but released the drug in kidney homogenates. SB202190-ULS-lysozyme accumulated efficiently in renal tubular cells and provided a local drug reservoir during a period of 3 days after a single intravenous injection. Treatment with SB202190-ULS-lysozyme inhibited TGF-beta1-induced gene expression for procollagen-Ialpha1 by 64% in HK-2 cells. Lastly, we evaluated the efficacy of a single dose of the conjugate in the unilateral renal ischemia-reperfusion rat model. A reduction of intrarenal p38 phosphorylation and alpha-smooth muscle actin protein expression was observed 4 days after the ischemia-reperfusion injury. In conclusion, we have developed a novel strategy for local delivery of the p38 MAPK inhibitor SB202190, which may be of use in the treatment of renal fibrosis.

Topics: Animals; Biological Transport; Cisplatin; Fibrosis; Imidazoles; Kidney; Kidney Tubules, Proximal; Male; Muramidase; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Pyridines; Rats; Rats, Wistar; Reperfusion Injury