Methods for Analysis of Matrix Metalloproteinase Regulation of Neutrophil-Endothelial Cell Adhesion

Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1-32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1-32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1-32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1-32], thus revealing a self-amplifying loop for ET-1[1-32] generation. ET-1[1-32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1-32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1-32] were mediated via activation of ET(A)receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work

Enhancing neuroimaging genetics through meta-analysis for Tourette syndrome (ENIGMA-TS): A worldwide platform for collaboration

Tourette syndrome (TS) is characterized by multiple motor and vocal tics, and high-comorbidity rates with other neuropsychiatric disorders. Obsessive compulsive disorder (OCD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorders (ASDs), major depressive disorder (MDD), and anxiety disorders (AXDs) are among the most prevalent TS comorbidities. To date, studies on TS brain structure and function have been limited in size with efforts mostly fragmented. This leads to low-statistical power, discordant results due to differences in approaches, and hinders the ability to stratify patients according to clinical parameters and investigate comorbidity patterns. Here, we present the scientific premise, perspectives, and key goals that have motivated the establishment of the Enhancing Neuroimaging Genetics through Meta-Analysis for TS (ENIGMA-TS) working group. The ENIGMA-TS working group is an international collaborative effort bringing together a large network of investigators who aim to understand brain structure and function in TS and dissect the underlying neurobiology that leads to observed comorbidity patterns and clinical heterogeneity. Previously collected TS neuroimaging data will be analyzed jointly and integrated with TS genomic data, as well as equivalently large and already existing studies of highly comorbid OCD, ADHD, ASD, MDD, and AXD. Our work highlights the power of collaborative efforts and transdiagnostic approaches, and points to the existence of different TS subtypes. ENIGMA-TS will offer large-scale, high-powered studies that will lead to important insights toward understanding brain structure and function and genetic effects in TS and related disorders, and the identification of biomarkers that could help inform improved clinical practice

Opposing effects of monomeric and pentameric C-reactive protein on endothelial progenitor cells

C-reactive protein (CRP) has been linked to the pathogenesis of atherosclerosis. The dissociation of native, pentameric (p)CRP to monomeric (m)CRP on the cell membrane of activated platelets has recently been demonstrated. The dissociation of pCRP to mCRP may explain local pro-inflammatory reactions at the site of developing atherosclerotic plaques. As a biomarker, pCRP predicts cardiovascular adverse events and so do reduced levels and function of circulating endothelial progenitor cells (EPCs). We hypothesised that mCRP and pCRP exert a differential effect on EPC function and differentiation. EPCs were treated with mCRP or pCRP for 72 h, respectively. Phenotypical characterisation was done by flow cytometry and immunofluorescence microscopy, while the effect of mCRP and pCRP on gene expression was examined by whole-genome gene expression analysis. The functional capacity of EPCs was determined by colony forming unit (CFU) assay and endothelial tube formation assay. Double staining for acetylated LDL and ulex lectin significantly decreased in cells treated with pCRP. The length of tubuli in a matrigel assay with HUVECs decreased significantly in response to pCRP, but not to mCRP. The number of CFUs increased after pCRP treatment. RNA expression profiling demonstrated that mCRP and pCRP cause highly contradictory gene regulation. Interferon-responsive genes (IFI44L, IFI44, IFI27, IFI 6, MX1, OAS2) were among the highly up-regulated genes after mCRP, but not after pCRP treatment. In conclusion, EPC phenotype, genotype and function were differentially affected by mCRP and pCRP, strongly arguing for differential roles of these two CRP conformations. The up-regulation of interferon-inducible genes in response to mCRP may constitute a mechanism for the local regulation of EPC function

Pulmonary complications after hip and knee arthroplasty in the united states, 2004-2014

BACKGROUND: Pulmonary complications after total joint arthroplasty (TJA) are uncommon but have significant cost impact. Total hip arthroplasty (THA) and total knee arthroplasty (TKA) are 2 of the 5 top procedures requiring inpatient stay within the United States. Subsequent pulmonary complications therefore may impose substantial cost burden for US health care. The purpose of this study was to describe the incidence, risk factors, and clinical implications of pulmonary complications (ie, pneumonia, respiratory failure, pulmonary embolism [PE], and aspiration) after TJA in the United States. METHODS: The National Inpatient Sample (NIS) was queried for all patients undergoing primary, elective THA and TKA between years 2004 and 2014. Pulmonary complications were defined as the occurrence of pneumonia, respiratory failure, PE, or aspiration after TJA. Demographic and clinical characteristics, inpatient cost, length of stay (LOS), and mortality were compared between patients with and without documented perioperative pulmonary complications. Given the stratified nature of the NIS database, estimates of incidence throughout the United States were made with application of trend weights to observed database frequencies. Analyses of estimated annual complication rates were made using χ tests. RESULTS: Between 2004 and 2014, an estimated 2,679,351 patients underwent elective primary THA. A total of 5,527,205 patients were estimated to have undergone elective primary TKA. THA 1.42% (95% CI, 1.37%-1.47%) and 1.71% (95% CI, 1.66%-1.76%) of TKA procedures were complicated by pneumonia, respiratory failure, PE, or aspiration. During this time, the incidence of perioperative pulmonary complications decreased from 1.57% (95% CI, 1.41%-1.73%) to 1.01% (95% CI, 0.92%-1.10%) after THA (P \u3c .0001) and from 2.03% (95% CI, 1.88%-2.18%) to 1.33% (95% CI, 1.25%-1.42%) after TKA (P \u3c .0001). The adjusted odds ratio (aOR) of experiencing a pulmonary complication was highest among patients with history of significant weight loss (aOR = 4.77; 99.9% CI, 3.97-5.73), fluid/electrolyte disorders (aOR = 3.33; 99.9% CI, 3.11-3.56), congestive heart failure (CHF; aOR = 3.32; 99.9% CI, 3.07-3.58), preexisting paralytic condition (aOR = 2.03; 99.9% CI, 1.57-2.61), and human immunodeficiency virus infection (aOR = 2.00; 99.9% CI, 1.06-3.78). Perioperative pulmonary complications were associated with increased LOS (THA = 3.03 days; 99.9% CI, 2.76-3.31; TKA = +2.72 days; 99.9% CI, 2.58-2.86), increased hospital costs (THA = +9163 US dollars; 99.9% CI, 8054-10,272; TKA = +7257 US dollars; 99.9% CI, 6650-7865), and increased mortality (THA: aOR = 121; 99.9% CI, 78-187; TKA: aOR = 150; 95% CI, 97-233). CONCLUSIONS: Despite a decline in overall incidence, perioperative pulmonary complications represent a significant potential source of perioperative morbidity and mortality. The current study highlights potential risk factors for pulmonary complications. Recognition of these factors may help to better stratify patients and mitigate risk of potential complications. This is particularly true of respiratory failure as it is associated with the high increases in resource utilization and mortality in this group

Lipoxin A(4) and aspirin-triggered 15-epi-lipoxin A(4) inhibit peroxynitrite formation, NF-κB and AP-1 activation, and IL-8 gene expression in human leukocytes

Lipoxin A(4) (LXA(4)) and aspirin-triggered 15-epi-LXA(4) (ATL) are emerging as endogenous braking signals for neutrophil-mediated tissue injury. Recent studies indicate that peroxynitrite (ONOO(−)) may function as an intracellular signal for the production of IL-8, a potent proinflammatory cytokine in human leukocytes. In this study, we evaluated the impact of the metabolically stable analogues of LXA(4)/ATL on lipopolysaccharide (LPS)-induced ONOO(−) formation and ONOO(−)-mediated IL-8 gene expression in human leukocytes. At nanomolar concentrations, LXA(4) analogues markedly reduced LPS-stimulated superoxide formation, evoked increases in intracellular diamino-fluorescein fluorescence (an indicator of NO formation), and consequently reduced ONOO(−) formation in isolated neutrophils, as well as in neutrophils, monocytes, and lymphocytes, in whole blood. LXA(4)/ATL analogues attenuated nuclear accumulation of activator protein-1 and nuclear factor-κB in both polymorphonuclear and mononuclear leukocytes and inhibited IL-8 mRNA expression and IL-8 release by 50–65% in response to LPS. The LXA(4) inhibitory responses were concentration dependent and were not shared by 15-deoxy-LXA(4). None of the LXA(4) analogues studied affected neutrophil survival, nor reversed the apoptosis delaying action of LPS in neutrophils. In addition, LXA(4) analogues had no significant effect on exogenous ONOO(−)-induced IL-8 gene and protein expression. These findings suggest that by attenuating ONOO(−) formation, LXA(4) and ATL can oppose ONOO(−) signaling in leukocytes and provide a rationale for using stable synthetic analogues as antiinflammatory compounds in vivo

Structural and functional comparison of native pentameric, denatured monomeric and biotinylated C-reactive protein

There are many controversies surrounding the biological activities of native C-reactive protein (nCRP) and its various modified forms such as monomerized and biotinylated CRP (mCRP and bCRP). No simple methods have been described to distinguish among these forms. By adapting established electrophoresis methods, we have developed a useful quality control method with which we have investigated the structural and functional characteristics of these forms of CRP. Under all electrophoresis conditions, biotinylation altered the electrophoretic mobility of CRP. nCRP was sensitive to sodium dodecyl sulphate (SDS)-induced monomerization, and only mCRP was susceptible to digestion by trypsin or neutrophil-derived serine proteases. bCRP and mCRP but not nCRP bound to cells, suggesting that chemical modification by biotin and denaturation had altered the structural integrity of CRP. Neither nCRP nor mCRP had the ability to induce secretion of chemokines, nor did they increase intracellular adhesion molecule 1 (ICAM-1) expression in endothelial cells