In humans, heparin-binding protein (HBP) and the potent chemotactic lipid leukotriene B₄ (LTB₄) are important mediators of innate immune reponses. Here we show that human neutrophils (PMNs) ...challenged with LTB₄ (30 s to 5 min) release HBP as determined by Western blot analysis. This response peaks at 100 nM of agonist and is mediated by the BLT1 receptor. Protein phosphatase-1 (30 μM) and wortmannin (0.5 μM) block the LTB₄-mediated HBP release from PMNs, which suggests involvement of the 1-phosphatidylinositol 3-kinase intracellular pathway during degranulation. Furthermore, postsecretory supernatants from LTB₄-stimulated PMNs induce intracellular calcium mobilization in endothelial cells in vitro and increase in vascular permeability in vivo, as assessed in a mouse model of pleurisy. Selective removal of HBP from the supernatant significantly reduces these activities attributing a key role to HBP in the LTB₄-induced change in vascular permeability. This lipid-protein axis could offer novel opportunities for pharmacological intervention in key steps of the vascular response to inflammation.--Di Gennaro, A., Kenne, E., Wan, M., Soehnlein, O., Lindbom, L., Haeggström, J. Z. Leukotriene B₄-induced changes in vascular permeability are mediated by neutrophil release of heparin-binding protein (HBP/CAP37/azurocidin).
Loss of endothelial barrier function in arterial blood vessels is characteristic of vascular pathologies, including atherosclerosis. Here, we present a near-infrared fluorescence (NIRF) imaging ...methodology for quantifying endothelial permeability and macromolecular uptake in large arteries in the mouse and evaluate its applicability for studying mechanisms of vascular inflammation.
To validate the NIRF methodology, macrovascular inflammation was induced in C57bl/6 mice by local tumor necrosis factor-α stimulation of the carotid artery or in apolipoprotein E-deficient mice by Western diet for 4 weeks. Evans blue dye, serving as plasma protein marker and fluorescent in the near-infrared spectrum, was given intravenously at different doses. Carotids and aorta were excised, and Evans blue dye fluorescence was assessed through whole vessel scan in an infrared imaging system. NIRF correlated to extraction-absorbance methodology for Evans blue dye quantification and was superior at discriminating plasma protein accumulation in tumor necrosis factor-α-stimulated carotids. NIRF allowed for focal quantification of increased arterial wall Evans blue dye uptake in (apolipoprotein E-deficient) mice. Importantly, NIRF left vessels intact for subsequent histological analysis or quantification of leukocyte subpopulations by flow cytometry.
The described NIRF methodology provides a sensitive and rapid tool to locate and quantify macromolecular uptake in the wall of arterial blood vessels in vascular pathologies in mice.
Background:
Acute lung injury (ALI) and respiratory distress can develop as a consequence of sepsis with pathogens such as Group A streptococcus (GAS) including S. pyogenes. In the pathogenesis of ...sepsis‐associated ALI, plasma exudation and leukocyte accumulation in the lungs are central events, and endothelial barrier disruption brought on by degranulation of neutrophil‐derived proteins is considered a causative factor. Heparin and heparin derivatives have been suggested as novel therapeutics in sepsis on the basis of their pleiotropic anti‐inflammatory effects.
Aims:
Here, we study wheter sevuparin, a heparinoid wihich lacks anticoagulant activity, affects neutrophil‐induced lung plasma leakage.
Methods:
A murine model of systemic inflammation evoked by heat‐killed GAS and furthermore, we utilized human neutrophils and endothelial cell monolayers
Results:
We demonstrate that sevuparin inhibits hkGAS‐induced endothelial barrier disruption by neutralizing the activity of neutrophil‐derived proteins. By mass spectrometry of neutrophil secretion we identify candidate proteins, including serprocidins, S100 proteins and histone H4, that interact with sevuparin and that are responsible for the disruptive effect on endothelial integrity.
Summary/Conclusion:
Collectively, our results corroborate the role of neutrophil‐derived proteins in vascular hyperpermeability caused by Group A streptococci, and suggest sevuparin as a potential therapeutic in acute neutrophilic inflammation.
Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on ...muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here we show that oxidative post-translational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spectrometry, we identified the actin residues targeted by oxidative 3-nitrotyrosine (3-NT) or malondialdehyde adduct (MDA) modifications in weakened skeletal muscle from mice with arthritis and patients afflicted by RA. The residues were primarily located to three distinct regions positioned at matching surface areas of the skeletal muscle actin molecule from arthritis mice and RA patients. Moreover, molecular dynamic simulations revealed that these areas, here coined "hotspots", are important for the stability of the actin molecule and its capacity to generate filaments and interact with myosin. Together, these data demonstrate how oxidative modifications on actin promote muscle weakness in RA patients and provide novel leads for targeted therapeutic treatment to improve muscle function.
The aim of this study was to assess the effect of partial body weight support on the oxygen cost of treadmill walking in children and adolescents with spastic cerebral palsy (CP). Five children and ...adolescents (2 girls and 3 boys) with spastic CP (12.4 ± 3.6 years) volunteered for the study. Participants performed three 4-min tread mill walks on three separate days at their comfortable treadmill walking speeds. At each visit a different partial body weight harness setting was used. Significant (
p
< .05) differences in oxygen cost were found when the harness was worn but not connected to the support frame. Partial body weight support reduces the oxygen cost of walking in children and adolescents with spastic CP.
In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of ...monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.
The nuclear-encoded mitochondrial protein NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) was recently identified. NDUFAe4L2 is shown to be induced by hypoxia via HIF1α and is ...thought to inhibit production of mitochondrial reactive oxygen species in fibroblasts exposed to hypoxia. Here the aim was to characterize the role of NDUFA4L2 in the mitochondria-rich tissues skeletal and cardiac muscle. We show hypoxia induced NDUFA4L2 expression in isolated muscle fibers and in cardiomyocytes with full activation after ~3-6 h in hypoxia. The half-maximal O 2 level for NDUFA4L2 expression (~4.6 % of ambient O 2 ) suggests sensitivity to changes in O 2 tension that occur under physiological conditions (e.g. exercise, moderate ischemia). We identified that the NDUFA4L2 gene promoter has binding sites for transcription factors other than HIF-1α; repetitive sites for PPARα,γ and one for Nrf2. NDUFA4L2 overexpression resulted in functional effects on skeletal and cardiac muscle; e.g. it alters cellular Ca 2+ signaling and the expression of Ca 2+ handling genes. Further, NDUFA4L2 overexpression reduces muscle mass (~20%), leading to a decreased force production in skeletal muscle. The NDUFA4L2-induced loss of muscle mass was associated with increases in mRNA levels of e.g. MurF1, Mul1, caspase-3 and Bax. Additionally, femoral artery ligation (FAL) induced NDUFA4L2 expression, which correlates with the decreased force production eight days post-FAL in skeletal muscle. Moreover, NDUFA4L2 upregulates antioxidant gene expression and silencing NDUFA4L2 makes cardiac cells less tolerant to hypoxia/re-oxygenation. Our results suggest that NDUFA4L2 expression affects vital functions in muscle cells and at least part of this effect is mediated by a link between NDUFA4L2 and nuclear gene expression. Thus, NDUFA4L2 might act as an integrator of the nutritional, environmental and functional status in muscle cells.
AIMWith an interest in providing knowledge for person-centred care, our overall goal is to contribute a greater understanding of diversity among patients in terms of their preparedness before and up ...to six months after colorectal cancer surgery. Our aim was to describe and provide a tentative explanation for differences in preparedness trajectory profiles.MATERIAL AND METHODSThe study was explorative and used prospective longitudinal data from a previously published intervention study evaluating person-centred information and communication. The project was conducted at three hospitals in Sweden. Patient-reported outcomes measures, including the Longitudinal Preparedness for Colorectal Cancer Surgery Questionnaire, were collected before surgery, at discharge, and four to six weeks, three months, and six months after surgery. Clinical data were retrospectively obtained from patients' medical records. We used latent class growth models (LCGMs) to identify latent classes that distinguish subgroups of patients who represent different preparedness trajectory profiles. To determine the most plausible number of latent classes, we considered statistical information about model fit and clinical practice relevance. We used multivariable regression models to identify variables that explain the latent classes.RESULTSThe sample (N = 488) comprised people with a mean age of 68 years (SD = 11) of which 44% were women. Regarding diagnoses, 60% had colon cancer and 40% rectal cancer. The LCGMs identified six latent classes with different preparedness for surgery and recovery trajectories. The latent classes were predominantly explained by differences in age, sex, physical classification based on comorbidities, treatment hospital, global health status, distress, and sense of coherence (comprehensibility and meaningfulness).CONCLUSIONContrary to the received view that emphasizes standardized care practices, our results point to the need for adding person-centred and tailored approaches that consider individual differences in how patients are prepared before and during the recovery period related to colorectal cancer surgery.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract only
Brain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to ...edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. In this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using anti‐Gr‐1 antibody was compared to that in mice with intact neutrophil count. Brain edema formation was assessed by Evan's blue dye extravasation and analysis of brain water content. Immunohistochemistry was used to assess cell death and microglial activation. Lesion volume was measured 7 and 14 days after CCI. Neutrophil‐depletion did not significantly affect Evan's blue extravasation at 24 or 48 hours after CCI. However, neutrophil‐depleted mice exhibited decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Additionally, neutropenic mice had a decreased number of activated microglia, and brain tissue loss was attenuated in these mice. Furthermore, the number apoptotic cells 24 h after injury was significantly reduced in neutrophil depleted mice. Our results suggest that neutrophils play a role in the edema formation and contribute to the cell death and tissue loss following TBI.
Intravital microscopy is a useful tool for studying leukocyte trafficking in atherosclerosis. However, distinction between various subclasses of leukocytes using this technology is lacking. ...Therefore, we generated ApoE(-/-)/Lysozyme M(EGFP/EGFP) mice and investigated whether targeted cell types could be visualized by in vivo microscopy and whether absence of lysozyme M will influence atherosclerosis.
We crossed male ApoE(-/-) mice with mice homozygous for a knock-in mutation of enhanced green fluorescent protein (EGFP) in the lysozyme M locus (Lys(EGFP/EGFP)) creating ApoE(-/-)/Lys(EGFP/EGFP) mice. Mice were sacrificed at the age of 26 weeks. Blood was collected for serum lipid analysis, differential white blood cell count and flow cytometry. Lesion area was determined on en face mounted aortas and sections from aortic roots were stained for immunohistochemistry. Atherosclerotic lesions were also studied by confocal- and intravital microscopy.
Basic parameters, such as white blood cell count, cholesterol profile, lesion area and plaque composition was unaltered in ApoE(-/-)/Lys(EGFP/EGFP) mice compared to ApoE(-/-) mice. Fluorescent neutrophils and monocytes were clearly visualized by intravital fluorescence and confocal microscopy. Fluorescent cells were distributed primarily in the periphery of atherosclerotic lesions indicating a preference for recruitment in these areas.
ApoE(-/-)/Lys(EGFP/EGFP) mice will serve as a useful model to study leukocyte trafficking in atherosclerosis and how different subsets of leukocytes influence atherogenesis.
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