Neutrophil Density Research Articles

(189) - Prospective Evaluation of Urine Dipstick for Detection of Significant Hemolysis in Outpatients on CF-LVAD Support

Purpose: The mechanism of left ventricular assist device (LVAD) thrombosis initiation and propagation is unknown. Correctly identifying the thrombi components could aid in directing therapy for patients with suspected LVAD thrombosis. Methods: H&E staining was used for histologic analysis of thrombi removed from HeartMate II (HMII) devices explanted at the University of Louisville. Immunohistochemistry identified platelets and neutrophils using antibodies against CD42b (glycoprotein Ib) and myeloperoxidase, respectively. Age of fibrin was assessed by Martius, Scarlet, Blue staining. Results: 28 thrombi from 17 LVADs were analyzed. Four pumps only had an inlet bearing thrombus, 3 pumps only had a rotor vein thrombus, and 2 pumps only contained an outlet bearing thrombus. Seven pumps had clots in multiple locations. In total, 72% of the thrombi (20/28) were either located around the inlet bearing (9), the rotor veins (5), or the outlet bearing (6). The laminated fibrin rings of the inlet stator thrombi were 150 -2618 microns thick. The inner rings had crosshatched fibrin followed by dense fibrin rings (Figure). Outer rings had mixed fibrin with less organized structure. Rings with higher densities of platelets and neutrophils were more commonly seen in the outer rings of rotor thrombi (Figure). Outlet bearing thrombi varied histologically, but most were similar to the rotor and inlet thrombi with increased areas of nuclear material. Three thrombi from the inlet conduit and three from outlet elbow were similarly structured and composed of loose meshwork with white blood cells and areas of layered fibrin. Platelets were present in dense areas. Conclusion: The fibrin density varied in clots formed in different parts of the HMII. Fibrin of different ages in the inlet bearing thrombi suggests development over time and reinforces the necessity of anticoagulation. The increased frequency of platelets in the outer rings of the inlet bearing thrombi would support further investigation into their role in thrombus propagation.

In Epstein-Barr virus-associated gastric carcinoma a high density of CD66b-positive tumor-associated neutrophils is associated with intestinal-type histology and low frequency of lymph node metastasis.

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is characterized by prominent intratumoral lymphocyte infiltration, with some infiltration by other types of inflammatory cells, such as neutrophils. In this study, the significance of tumor-associated neutrophils (TANs) and cytotoxic T lymphocytes (CTLs) was investigated in EBVaGC. CD66b-positive TANs and CD8-positive CTLs in surgically resected EBVaGC tissues were evaluated by immunohistochemistry and digital imaging analysis. Cut-off values were determined with receiver operating characteristic curve analyses. Forty-two of 77 cases (55%) had some infiltration of TANs (CD66b-positive areas >0.5%, TAN(+)) and 35 (45%) had scant infiltration (TAN(-)). Thirty-five cases (45%) had more CTLs (CD8-positive area >18%, CTL-high) and 42 (55%) had fewer (CTL-low). There was no correlation between CD66b- and CD8-positive areas in the tumor (P=0.453). TAN(+) correlated with intestinal-type histology (P=0.048) and low frequency of lymph node metastasis (P=0.023), while CTL-low with upper location (P=0.030) and advanced invasion depth (pT2 or greater) (P=0.006). Disease-specific survival was not significantly correlated with TANs or CTLs. Multivariate logistic regression analysis revealed TAN(-) to be independently associated with lymph node metastasis (P=0.036). None of the cases of TAN(+) early EBVaGC with submucosal invasion showed lymph node metastasis (95% confidence interval 0-13.3%). When compared with 24 EBV-negative gastric carcinomas, EBVaGC had significantly more CTLs (P<0.001), while there was no difference in the number of TANs. TANs in EBVaGC may suppress lymph node metastasis, and absence of TANs might suggest careful follow-up or additional therapy in cases of post-endoscopic resection.

Shrimp Protein Hydrolysate Modulates the Timing of Proinflammatory Macrophages in Bupivacaine-Injured Skeletal Muscles in Rats.

This study was designed to determine whether marine-derived proteins other than cod could have beneficial effects on inflammation following muscle injury. Macrophage and neutrophil densities were measured from bupivacaine-injured tibialis anterior muscle of rats fed isoenergetic diets containing either shrimp hydrolysate (Shr), casein hydrolysate (CaH), or whole casein (Ca). In this study, Shr reduced ED1+-macrophages at day 2 (p = 0.013), day 5 (p = 0.006), and day 14 after injury (p = 0.038) compared with Ca, indicating faster resolution of inflammation in Shr. Except for day 2 after injury where Shr led to lower ED1+-macrophages compared with CaH (p = 0.006), both Shr and CaH responded similarly at days 5, 14, and 28 after injury. This findings suggest that beneficial effects of Shr on ED1+-cells might be related to generation of anti-inflammatory peptides through the hydrolysis process, in addition to its high content of anti-inflammatory amino acids. However, while increasing myofiber cross-sectional area in noninjured muscles compared with both Ca and CaH, Shr failed to have a positive effect in corresponding injured muscles. These data indicate that shrimp hydrolysate can facilitate resolution of inflammation after muscle injury mainly through modulating proinflammatory macrophage accumulation but have less effect on optimal recovery in terms of muscle mass and fiber size.

Validación del Índice de Severidad Endoscópico de Colitis Ulcerativa para la población mexicana

Endoscopy is an extremely valuable instrument to evaluate the effectiveness of new treatments for patients with ulcerative colitis; however it is necessary to validate the current endoscopic classifications to facilitate meaningful comparisons between them.The Ulcerative Colitis Endoscopic Index of Severity (UCEIS) recommended by European Crohn's and Colitis Organisation uses 3 parameters (vascular, bleeding, and ulceration), each with 3-4 levels of severity. However, it has not been validated for the Mexican population.In addition to the colonoscopic features, the establishment of disease activity by histology is performed routinely. The Riley scale shows mainly the density and distribution of neutrophils in mucosal defects. ObjectiveTo determine whether there is a correlation between the UCEIS endoscopic classification and the Riley histological classification, and validate their usefulness in the Mexican population. Material and methodsA retrospective, cross-sectional, open study was conducted from January 2012 to December 2014 in the Centro Medico Nacional 20 de Noviembre on patients with a diagnosis of ulcerative colitis sent to the hospital Endoscopy Department. Endoscopic UCEIS and histological Riley classifications were determined and compared using the SPSS program for statistical analysis. ResultsA total of 63 cases were included, of whom 33 were male (52%) and 30 (48%) female, with mean age of 50 years (23-78 years). The Spearman correlation coefficient was 89.8%, with a confidence interval of 83.7%-93.7% (P<.001). Using the Kendall coefficient, a correlation of 80.1% was observed between the variables with a confidence interval ranging from 70.5% to 86.4% (P<.0001. ConclusionIn accordance to the objective of the investigation, it was concluded that the correlation between the variables is significant, therefore the endoscopic and histological classifications used in ulcerative colitis correlate, and both are valid for use in the Mexican population.

Metallothioneins 1 and 2 Modulate Inflammation and Support Remodeling in Ischemic Cardiomyopathy in Mice.

Aims. Repetitive brief ischemia and reperfusion (I/R) is associated with left ventricular dysfunction during development of ischemic cardiomyopathy. We investigated the role of zinc-donor proteins metallothionein MT1 and MT2 in a closed-chest murine model of I/R. Methods. Daily 15-minute LAD-occlusion was performed for 1, 3, and 7 days in SV129 (WT)- and MT1/2 knockout (MT−/−)-mice (n = 8–10/group). Hearts were examined with M-mode echocardiography and processed for histological and mRNA studies. Results. Expression of MT1/2 mRNA was transiently induced during repetitive I/R in WT-mice, accompanied by a transient inflammation, leading to interstitial fibrosis with left ventricular dysfunction without infarction. In contrast, MT−/−-hearts presented with enhanced apoptosis and small infarctions leading to impaired global and regional pump function. Molecular analysis revealed maladaptation of myosin heavy chain isoforms and antioxidative enzymes in MT1/2−/−-hearts. Despite their postponed chemokine induction we found a higher total neutrophil density and macrophage infiltration in small infarctions in MT−/−-hearts. Subsequently, higher expression of osteopontin 1 and tenascin C was associated with increased myofibroblast density resulting in predominately nonreversible fibrosis and adverse remodeling in MT1/2−/−-hearts. Conclusion. Cardioprotective effects of MT1/2 seem to be exerted via modulation of contractile elements, antioxidative enzymes, inflammatory response, and myocardial remodeling.

Consumption of the epidermis: a suggested precursor of ulceration associated with increased proliferation of melanoma cells.

It has recently been demonstrated that the extent of ulceration and the presence of epidermal involvement that theoretically precede ulceration (consumption of epidermis, COE) or seen subsequent to inflammation (reactive epidermal hyperplasia or re-epithelialization) allowed better prognostic stratification of ulcerated melanoma. Understanding why these histopathologic markers have prognostic potential is important, not least because accurate consensual assessment of ulceration lies at the root of proper staging and clinical management. The authors therefore performed immunohistochemical analyses of tumor cell proliferation (Melan-A/Ki67) and infiltration of inflammatory cells (CD66b neutrophils and CD163 macrophages) to better understand the biology of the epidermal changes described. Tumors with a COE configuration showed 37% (95% CI: 4-54, P = 0.0046) increased tumor cell proliferation compared with tumors of normal epidermal configuration. COE is therefore suggested a precursor of ulceration associated with increased proliferation of melanoma cells. There was no observed correlation between COE and an increased inflammatory response (CD163 macrophages or CD66b neutrophils), which supports that the proliferation drive is noninflammatory. In contrast, the presence of re-epithelialization and/or reactive epidermal hyperplasia demonstrated an 18% (95% CI: 6-53, P = 0.0021) increased density of neutrophils compared with tumor with no evidence of these possibly prolonged late-stage or resolved ulcerations. These results further support the relevance of including these epidermal changes into the definition of ulceration and to define ulceration of a primary melanoma as loss of epidermis with evidence of a host response (infiltration of neutrophils or fibrin deposition) and thinning, effacement, or reactive hyperplasia of the surrounding epidermis.

Abstract 213: Deletion of 12/15 Lipoxygenase and Fatty Acids Interaction Alters Leukocyte Kinetics Leading to Improved Healing Following Myocardial Infarction

The 12/15 lipoxygenase (LOX) enzyme catalyzes oxygenation of fatty acids to form lipid mediators leading to non-resolving inflammation. However, how 12/15LOX interacts with PUFA (polyunsaturated fatty acids) in post-myocardial infarction (MI) cardiac healing is unclear. Here we assessed the role of 12/15LOX in post-MI cardiac remodeling in a PUFA (10% w/w, 22 Kcal) enriched environment. C57BL/6J wild-type (WT) and 12/15LOX null (12/15LOX –/– ) male mice of 8-12-weeks age were fed PUFA-enriched diet for 1 month and subjected to permanent coronary artery ligation. Post-MI mice were monitored for day (d)1 or d5 along with standard diet (SD)-fed MI controls. No-MI surgery mice served as d0 controls. 12/15LOX and PUFA+12/15LOX -/- mice reduced infarcted wall thinning with decreased lung edema index at d5 post-MI (all p&lt;0.05) despite comparable infarct area (49-52%). PUFA+WT and 12/15LOX -/- mice showed improved ejection fraction with reduced left ventricle (LV) hypertrophy index than SD+WT at d5 post-MI (p&lt;0.05). The neutrophil density was decreased in both PUFA+WT and 12/15LOX -/- mice at d1 post-MI (P&lt;0.05). The neutrophil clearance was rapid in 12/15LOX -/- alone and PUFA+12/15LOX -/- mice, with increases in expression of formyl peptide receptor 2 at d5 post-MI. The macrophage density was decreased in PUFA+WT and 12/15LOX -/- mice compared with their respective SD+WT control at d5 post-MI. PUFA+12/15LOX -/- mice displayed an increased expression of ccl2 (all p&lt;0.05) in the infarct area. 12/15LOX deletion stimulated 5-LOX and heme oxygenase-1 in PUFA+12/15LOX -/- mice with increased levels of 9-and 13-hydroxyoctadecadienoic acid indicating improved resolution of inflammation at d5 post-MI. The PUFA+12/15LOX -/- mice displayed higher expression of proresolving macrophages ym-1, mrc-1 and arg-1 compared with SD+12/15LOX -/- mice at d5 post-MI (all p&lt;0.05). Further, 12/15LOX -/- mice with or without PUFA showed reduced collagen deposition at d5 post-MI compared to SD+WT. In conclusion, deletion of 12/15LOX and short-term PUFA exposure attenuated cardiac remodeling via shortening the pro-inflammatory window, thus leading to an improved resolution of inflammation and LV function post-MI.

Mesenteric Lymphatic-Perilymphatic Adipose Crosstalk: Role in Alcohol-Induced Perilymphatic Adipose Tissue Inflammation

The digestive tract lymphatics transport approximately two-thirds of all lymph produced in the body and have a key role in mucosal immunity through their contribution to antigen transport and immune cell trafficking. Mesenteric lymphatic pumping function integrity is critical for maintaining homeostasis and lipid transport. We previously demonstrated that acute alcohol intoxication (AAI) increases mesenteric lymphatic amplitude of contraction and ejection fraction, enhancing the ability of the lymphatic vessels to pump lymph. AAI has been shown to disrupt intestinal barrier integrity, which would be expected to increase the endotoxin content of mesenteric lymph. In this study, we tested the prediction that AAI increases lymphatic permeability directly affecting perilymphatic adipose tissue (PLAT) milieu. Male Sprague Dawley rats received an intragastric infusion of 2.5g/kg of alcohol. Isovolumic administration of water (vehicle) served as control. PLAT was isolated for the determination of Evans Blue extravasation (permeability), cytokine content, and immunohistochemistry for inflammatory cell infiltration at 30minutes and 24hours after alcohol administration. PLAT isolated from AAI animals had greater Evans Blue concentrations and cytokine expression (24hours post-AAI) and mast cell and neutrophil density than that isolated from controls. AAI resulted in significantly higher plasma lipopolysaccharide (endotoxin) levels, lower plasma adiponectin levels (at 30minutes), and unchanged plasma visfatin levels. The data indicate that AAI induces mesenteric lymphatic hyperpermeability, promotes PLAT inflammatory milieu and disrupts the systemic adipokine profile. These findings suggest an association between alcohol-induced lymphatic hyperpermeability and early manifestations of metabolic dysfunction as a result of alcohol abuse. We propose that crosstalk between lymph and PLAT results in adipose inflammation and adipokine dysregulation during AAI.

How neutrophil extracellular traps orchestrate the local immune response in gout.

Neutrophil granulocytes possess a large arsenal of pro-inflammatory substances and mechanisms that empower them to drive local acute immune reactions to invading microorganisms or endogenous inflammatory triggers. The use of this armory needs to be tightly controlled to avoid chronic inflammation and collateral tissue damage. In gout, inflammation arises from precipitation of uric acid in the form of needle-shaped monosodium urate crystals. Inflammasome activation by these crystals in local immune cells results in a rapid and dramatic recruitment of neutrophils. This neutrophil influx is accompanied by the infamously intense clinical symptoms of inflammation during an acute gout attack. Neutrophilic inflammation however is equipped with a built-in safeguard; activated neutrophils form neutrophil extracellular traps (NETs). At the very high neutrophil densities that occur at the site of inflammation, NETs build aggregates that densely pack the monosodium urate (MSU) crystals and trap and degrade pro-inflammatory mediators by inherent proteases. Local removal of cytokines and chemokines by aggregated NETs explains how acute inflammation can stop in the consistent presence of the inflammatory trigger. Aggregated NETs resemble early stages of the typical large MSU deposits that constitute the pathognomonic structures of gout, tophi. Although tophi contribute to muscosceletal damage and mortality in patients with chronic gout, they can therefore be considered as a payoff that is necessary to silence the intense inflammatory response during acute gout.

Neutrophil biology: an update.

Neutrophil extracellular traps (NETs) are involved in bacterial killing as well as autoimmunity, because NETs contain proteases, bactericidal peptides, DNA and ribonucleoprotein. NETs are formed via a novel type of cell death called NETosis. NETosis is distinct from apoptosis, but it resembles necrosis in that both membranes are not intact so that they allow intracellular proteins to leak outside of the cells. Removal of NETs and neutrophils undergoing NETosis by phagocytes and its subsequent response are not completely clarified, as compared with the response after removal of either apoptotic or necrotic neutrophils by phagocytes. How neutrophil density in peripheral blood is kept within a certain range is important for health and disease. Although the studies on severe congenital neutropenia and benign ethnic neutropenia have provided unbiased views on it, the studies are rather limited to human neutropenia, and mice with a mutation of mouse counterpart gene often fail to exhibit neutropenia. Degranulation plays a critical role in bactericidal action. The recent studies revealed that it is also involved in immunomodulation, pain control and estrous cycle control. N1 and N2 are representative of neutrophil subpopulations. The dichotomy holds true in patients or mice with severe trauma or cancer, providing the basis of differential roles of neutrophils in diseases.

Prognostic value of neutrophil distribution in cholangiocarcinoma.

To explore the relationship of clinicopathological features and the distribution of neutrophils in the tumor microenvironment with the prognosis of cholangiocarcinoma. Two hundred and fifty-four formalin-fixed and paraffin embedded tissue blocks were analyzed, including tissues from cholangiocarcinoma (n = 254), and tumor adjacent tissues (n = 238). Tissue sections were stained for CD15 using immunohistochemical staining. CD15 expression was detected to identify the distribution of neutrophils in the local tumor microenvironment. The neutrophil density of the tumor tissues and the adjacent tumor tissues was detected to reflect their inflammatory status. Clinical data and follow-up information of cholangiocarcinoma patients who underwent surgery from January 2004 to December 2010 were analyzed retrospectively. The relationship between clinicopathological features and the distribution of neutrophils with prognosis of the patients were analyzed. The positive expression level of CD15 was only significantly related to the TNM stage. CD15 expression was higher in tumor tissues than in adjacent tissues (73.6% vs 54.6%), with significant differences. Patients with high expression of CD15 had significantly shorter overall survival (OS) than those with low expression of CD15 (median overall survival time 39.77 mo vs 16.87 mo, P = 0.008). Patients with high CD15 expression had significantly shorter disease free survival time (DFS) than those with low expression of CD15 (median DFS 38.27 mo vs 16.83 mo, P = 0.029). COX multivariate analysis indicated that high CD15 expression in tumor tissues was an independent risk factor for predicting OS for patients with cholangiocarcinoma [P = 0.012, relative risk (RR) = 1.601], but it was not an independent risk factor for predicting DFS (P = 0.073, RR = 1.462). Patients with high CD15 expression in cancer tissues had shorter DFS and OS. High expression of CD15 is an independent risk factor for OS.

Expression of ATF3 in mouse protects the liver against sepsis via inhibiting HMGB expression

Lipopolysaccharides are components of Gram-negative enterobacteria that cause septic shock in mammals and triggers innate immunity mainly via TLR4 signaling. HMGB1 play a critical role in regulating innate immunity-induced sepsis. ATF3 is a negative regulator of TLR4 signaling and the mechanism of HMGB1 induced liver injury after sepsis are incompletely understood. In this study, we investigated the protective effects of ATF3 after LPS injection. Adult (4-6 months) C57/BL6 mice and ATF3 knockout mice were treated with a low dose of LPS (0.5 mg/kg, iv) for 6, 12 hrs. Liver enzymes and cytokines (TNF-α, IL-1β and IL-6) are assessed. The neutrophil and mononuclear cells in the liver tissue were examined using immunofluorescent staining. We found that serum HMGB1 levels were 8-fold higher in C57/BL6 mice with sepsis than ATF3 knockout with greater densities of neutrophils and mononuclear cells in the liver tissue, and higher levels of TNF-α, IL-1β and IL-6 in the circulation and liver tissue as well as associated with an increase in the mortality rate. In conclusion, upregulation of ATF3 contributes to the reduced release of HMGB1, and increased the survival rate of mice after LPS treated. Therefore, suppressing LPS-induced inflammation with ATF3 induction may be an important strategy for sepsis therapy.

Endotoxemia-suppress cardiac function through dysregulation of mitochondrial biogenesis in mice model

Sepsis exaggerated proinflammatory response to infection that can progress to carries a significant cardiac dysfunction associated with morbidity and mortality. Mitochondrial biogenesis is involved in the control of cell metabolism, signal transduction, and regulation of mitochondrial reactive oxygen species (ROS) production. We are hypothesis that impairment of biogenesis has been invoked in the pathogenesis of myocardial endotoxemia. C57/BL6 mice were treated with LPS (0.5 mg/kg, iv) for 4 hrs. cardiac function was assessed using a microcatheter. Electronmicroscopy and confocal microscopy revealed that mitochondrial biogenesis is reduced after treatment the mice with LPS compared with control. Further, endotoxemic mice exhibited worse LV function than the control. The exaggerated cardiac contractile depression in LPS treated mice is associated with greater densities of neutrophils and mononuclear cells in the myocardium, and higher levels of TNF-α, IL-1β and IL-6 in the circulation and myocardium. This study will provide insights into mitochondrial biology, the relevance to sepsis, and therapeutic opportunities that possibly emerge.

Abstract 13276: DHA but not Etanercept Expedites Resolution of Inflammation leading to Improved Survival Following Myocardial Infarction in Aging Mice

Dietary intake of n-3 polyunsaturated fatty acids (PUFA) has been touted for cardioprotective benefits. n-3 PUFA contains docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) but their individual effects on left ventricle (LV) remodeling remains unclear compared to anti-inflammatory molecule. We assessed the individual component of n-3 PUFA and their therapeutic potential on LV remodeling post-MI in aging mice (n=150). Male C57BL/6J mice were fed chow diet for first 12 months and then placed on safflower oil (SO; 10% w/w) enriched diet (n-6 PUFA) for 3 months to induce obesity. Then mice were randomized into 5 isocaloric groups: SO or DHA or EPA or DHA+EPA (D+E) or Etanercept (ETAN; 5mg/kg/day) (n=26-30 mice/group) for 2 months. At 17 months of age, mice were subjected to permanent coronary artery ligation resulting in MI and monitored for d1 and d5 survival; maintaining no-MI as d0 naïve controls. Post-MI survival was highest in DHA fed mice compared with ETAN and all other groups (p&lt;0.05). Mice in all groups had lower fractional shortening and extensive ventricular remodeling post-MI compared to d0 control. DHA and D+E mice reduced neutrophil infiltration at d1 compared to SO (p&lt;0.05). The DHA and EPA fed mice showed increased ccr2 and cxcl2 expression at d1 indicating the higher monocyte density, expediting the clearance of dead LV tissues. DHA and EPA groups (both p&lt;0.05) showed early induction of resolving enzyme HO-1 (hemeoxygenase) at d1 which peaked at d5 facilitating resolution of inflammation post-MI. LV immunohistochemistry of DHA fed mice showed enhanced expression of COX-2 (cyclooxygenase-2) at d1 and 5-LOX (5-lipoxygenase) at d5 post-MI compared to d0, and other 4 groups (all p&lt;0.05). Further, DHA fed mice showed the higher expression of timp-1, timp-2 and timp-3 with decrease in MMP-9 levels at d5 stabilizing the matrix maturation within the infarcted LV and thus promoting resolution. In summary, our results suggest that DHA but not ETAN improved post-MI survival in aging and obese mice by reducing neutrophil density, via coordination of COX, LOX and HO-1, leading to enhanced resolution of inflammation post-MI.

Modulation of Inflammatory Responses After Brain Death and Syngeneic Renal Transplantation By Systemic Overexpression of esRAGE.

Introduction: Donor brain death (BD) adversely affects organ quality. Inflammation mediated by engagement of TLR4 and RAGE may contribute to inferior outcomes. Endogenous secretory (es)RAGE is a soluble decoy receptor which sequesters HMGB1 and other RAGE ligands blocking binding to TLRs 2 and 4, as well as to RAGE. Aim: To determine whether in vivo expression of esRAGE can reduce inflammation in syngeneic KTx from BD donors. Methods: Donor and recipient C57BL/6 mice received 5×1011 VG rAAV-esRAGE or ctrl vector 7 days pre-transplant. Donor kidneys were grafted 1 hr after BD induction, and sampled at d1 and 4 post-Tx. Cellular infiltration, proliferation, and gene expression were determined. Serum and kidneys were also collected 3 hrs post-BD. Results: Serum HMGB1 levels in esRAGE-BD mice (˜10ng/ml) were significantly less than those in untreated BD mice (>25ng/ml, p=0.02). There was a trend towards reduced expression of CXCL10, MCP-1, MIP-2, CCL22, IL-10, IL-6, IL-1β and TNF-α in the kidneys of esRAGE-BD mice, but this was not consistently observed after KTx. Neutrophil and macrophage infiltrate density was substantially reduced by esRAGE (nΦ 9.1±2.0/HPF cf 23.1±6.0, p=0.03; F4/80 27.3±3.2/HPF cf 65.1±9.1, p<0.01).Figure: No Caption available.Proliferation of parenchymal cells (PCNA staining) was enhanced in esRAGE-treated mice (7.6±0.5/HPF cf 4.2±1.2, p=0.05).Figure: No Caption available.Conclusions: esRAGE significantly reduced neutrophil and macrophage infiltration following syngeneic renal Tx from BD donors. RAGE is a binding partner for the integrin MAC-1, and reduced leucocyte infiltration may reflect inhibition of this interaction. esRAGE increased proliferation of renal parenchymal cells after Tx from BD donors, similar to documented effects of soluble RAGE in liver injury. These effects may be translated into improved graft function.

Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines.

Gout is characterized by an acute inflammatory reaction and the accumulation of neutrophils in response to monosodium urate (MSU) crystals. Inflammation resolves spontaneously within a few days, although MSU crystals can still be detected in the synovial fluid and affected tissues. Here we report that neutrophils recruited to sites of inflammation undergo oxidative burst and form neutrophil extracellular traps (NETs). Under high neutrophil densities, these NETs aggregate and degrade cytokines and chemokines via serine proteases. Tophi, the pathognomonic structures of chronic gout, share characteristics with aggregated NETs, and MSU crystals can induce NETosis and aggregation of NETs. In individuals with impaired NETosis, MSU crystals induce uncontrolled production of inflammatory mediators from neutrophils and persistent inflammation. Furthermore, in models of neutrophilic inflammation, NETosis-deficient mice develop exacerbated and chronic disease that can be reduced by adoptive transfer of aggregated NETs. These findings suggest that aggregated NETs promote the resolution of neutrophilic inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation.

NF‐κB Activation and Severity of Gastritis in Helicobacter pylori‐Infected Children and Adults

In contrast to adults, Helicobacter pylori gastritis in children is reported as milder and ulcer disease as uncommon, but unequivocal data are lacking. To compare the frequency of gastro-duodenal ulcers in children and adults as well as the proportion of Helicobacter pylori infection in these patients and to study the effect of chronological age on NF-κB activation and on severity of gastritis. Patients referred in one pediatric and one adult facility for upper GI endoscopy were included. Gastric biopsies were obtained in consecutive Helicobacter pylori-infected patients and age-matched negative controls for immunohistochemistry and electrophoresis mobility shift assay. Three age groups were defined: younger than 8years, 8-17years, and adults. Peptic ulcer disease was less frequent in children and less frequently associated with Helicobacter pylori infection. When comparing infected subjects to controls, densities of neutrophils and CD20 cells in the lamina propria increased in all age groups, CD3 cells increasing only in patients older than 8years and CD8 cells only in adults. NF-κB-p65-positive cells were also increased only in infected adults as well as NF-κB-binding activity. A positive correlation was found between age and densities of neutrophils and CD3, but not of CD8 or CD20 cells. Peptic ulcer disease was less frequent in children and less frequently caused by Helicobacter pylori infection. The different clinical outcome of the infection in children can be the consequence of the lower mucosal immune response.

Neutrophils Infiltration in the Tongue Squamous Cell Carcinoma and Its Correlation with CEACAM1 Expression on Tumor Cells

ObjectiveThe present study aimed to explore the clinical significance of neutrophils infiltration and carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1) expression in the tongue squamous cell carcinoma (TSCC), and to probe the possible relationship between them.Materials and MethodsTissue microarray and immunohistochemistry were used to detect neutrophils density and CEACAM1 expression in 74 cases of primary TSCC specimens and 17 cases of corresponding peritumoral tissues. The relationship of CEACAM1 expression and neutrophils density with clinicopathologic parameters and cancer-related survival of TSCC patients were evaluated. The correlation between CEACAM1 expression and neutrophils density was also evaluated. Real-time quantitative transcription polymerase chain reaction (qRT-PCR) was used to explore the possible molecular mechanisms between CEACAM1 expression and neutrophils infiltration.ResultsImmunohistochemistry evaluation revealed that there was more neutrophils infiltration in TSCC tissues than in peritumoral tissues. High neutrophil density was associated with LN metastasis (P = 0.01), higher clinical stage (P = 0.037) and tumor recurrence (P = 0.024). CEACAM1 overexpression was also associated with lymph node metastasis (P = 0.000) and higher clinical stage (P = 0.001). Survival analysis revealed that both neutrophils infiltration and CEACAM1 overexpression were associated with poorer cancer-related survival of TSCC patients (P<0.05), and neutrophils infiltration was an independent prognostic factor for TSCC (P<0.05). Furthermore, overexpression of CEACAM1 was correlated with more neutrophils infiltration in TSCC tissues (P<0.01). qRT-PCR results showed that CEACAM1-4L can upregulate the mRNA expression of IL-8 and CXCL-6, which were strong chemotactic factors of neutrophils.ConclusionOur results demonstrated that more neutrophils infiltration and overexpression of CEACAM1 were associated with poor clinical outcomes in TSCC tissues. Overexpression of CEACAM1 on tumor cells correlated with more neutrophils infiltration to some extent through upregulating mRNA expression of IL-8 and CXCL-6.

Acute alcohol intoxication increases lymphatic permeability and promotes a perilymphatic adipose tissue inflammatory milieu

Acute alcohol intoxication increases lymphatic permeability and promotes a perilymphatic adipose tissue inflammatory milieu

Cardioprotection via preserved mitochondrial structure and function in the mPer2-mutant mouse myocardium

We have previously shown that myocardial infarct size in nonreperfused hearts of mice with a functional deletion of the circadian rhythm gene mPer2 (mPer2-M) was reduced by 43%. We hypothesized that acute ischemia-reperfusion injury (I/R = 30 min I/2 h R) would also be reduced in these mice and that ischemic preconditioning (IPC) (3 × 5 min cycles) before I/R, which enhances protection in wild-type (WT) hearts, would provide further protection in mPer2-M hearts. We observed a 69 and 75% decrease in infarct size in mPer2-M mouse hearts compared with WT following I/R and IPC, respectively. This was coincident with 67% less neutrophil infiltration and 57% less apoptotic cardiomyocytes. IPC in mPer2-M mice before I/R had 48% less neutrophil density and 46% less apoptosis than their WT counterparts. Macrophage density was not different between WT and mPer2-M I/R, but it was 45% higher in mPer2-M IPC mouse hearts compared with WT IPC. There were no baseline differences in cardiac mitochondrial function between WT and mPer2-M mice, but, following I/R, WT exhibited a marked decrease in maximal O₂ consumption supported by complex I-mediated substrates, whereas mPer2-M did not, despite no difference in complex I content. Moreover, cardiac mitochondria from WT mice exhibited a very robust increase in ADP-stimulated O₂ consumption in response to exogenously added cytochrome c, along with a high rate of reactive oxygen species production, none of which was exhibited by cardiac mitochondria from mPer2-M following I/R. Taken together, these findings suggest that mPer2 deletion preserves mitochondrial membrane structure and functional integrity in heart following I/R injury, the consequence of which is preservation of myocardial viability. Understanding the mechanisms connecting cardiac events, mitochondrial function, and mPer2 could lead to preventative and therapeutic strategies for at risk populations.