scholarly journals Resolvin D1 and Resolvin D2 Protect Against Hypoxia/Reoxygenation Induced Lung and Kidney Damage in a Sickle Cell Mouse Model of Acute Vaso-Occlusive Crisis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 966-966
Author(s):  
Alessandro Mattè ◽  
Antonio Recchiuti ◽  
Charles Serhan ◽  
Angela Siciliano ◽  
Enrica Federti ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Inflammatory Cell ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Hexaenoic Acid ◽  
Glomerular Sclerosis ◽  
Inflammatory Cell Infiltrate ◽  
Activation Markers ◽  
Hypoxia Reoxygenation

Abstract Sickle cell disease (SCD) is characterized by hemolytic anemia in association with acute and chronic life-threatening clinical complications. Acute vaso-occlusive crisis (VOCs) are the main cause of hospitalization for SCD patients. In VOCs, amplified inflammatory response plays a key role in acute organ damage. Pro-Resolving lipid mediators such as resolvins (Rv) accelerate resolution of acute inflammation in different models, indicating that stimulation of endogenous resolution of inflammatory processes may be an additional strategy in limiting tissue damage. Recent data suggest beneficial effects of Rvs in hypoxia/reoxygenation (H/R) related tissue injury. Here, we study the effects of Rvs on a model of acute VOCs using humanized SCD mice (Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n =6-7 animals in each group) with RvD1, 7S, 8R, 17S- trihydroxy-docosa-4Z, 9E, 11E, 13Z, 15E, 19Z-hexaenoic acid, 17R- RvD1, 7S, 8R, 17R- trihydroxy-docosa-4Z, 9E, 11E, 13Z, 15E, 19Z-hexaenoic acid; RvD2, 7S, 16R, 17S-trihydroxy-docosa-4Z, 8E, 10Z, 12E, 14E, 19Z-hexaenoic acid. Mice were treated with RvD1 and RvD2 at the dose of 2.5 ug/Kg by gavage 1 hour (hr) before H/R stress (10 hrs 8% oxygen followed by 3 hrs reoxygenation), which we have used in the past to mimic acute VOCs. We found that RvD1 and RvD2 significantly reduced the H/R-induced (i) increase in neutrophil count; (ii) lung inflammatory cell infiltrate, mucus and thrombi formation; (iii) glomerular inflammatory cell infiltration, glomerular sclerosis and thrombi formation. In the lung of H/R SCD mice, RvD1 prevented the H/R induced up-regulation of (i) cytokines such as MCP2, IL-6 and ET-1; (ii) vascular endothelial activation markers (VCAM-1 and ICAM-1; (iii) cytoprotective systems such as Prx6 and HO-1. In the kidney of H/R SCD mice, RvD1 significantly reduced H/R induced expression of IL-6 and ET-1 as well as HO-1. Our data indicate that RvD1 and RvD2 modulating inflammatory responses related to H/R in SCD, protect sickle cell target organs, and foster resolution. Thus, RvD1 and RvD2 might represent a novel therapeutic approach for acute VOCs in SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Imatinib Protects Against Hypoxia/Reoxygenation Induced Lung and Kidney Injury in a Humanized Mouse Model for SCD

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 725-725 ◽  
Author(s):  
Alessandro Mattè ◽  
Enrica Federti ◽  
Antonio Rechiutti ◽  
Amtonella Panataleo ◽  
Roberta Russo ◽  
...  
Keyword(s):  
Inflammatory Cell ◽  
Kinase Inhibitor ◽  
Case Reports ◽  
Inflammatory Cell Infiltrate ◽  
Imatinib Treatment ◽  
Red Cell ◽  
Cell Infiltrate ◽  
Activation Markers ◽  
Phosphorylation State ◽  
Hypoxia Reoxygenation

Abstract Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder characterized by chronic hemolytic anemia in association with acute and chronic life-threatening complications mainly related to acute vaso-occlusive events (VOCs) due to amplified inflammatory response and defective pro-resolving events. Imatinib is an oral Tyrosine (Tyr)-kinase inhibitor, developed for the treatment of chronic myeloid leukemia (CML). Few case reports on SCD patients with CML undergoing imatinib treatment highlight the beneficial impact of imatinib on severity and recurrence of acute VOCs in SCD. In red blood cells (RBCs), Imatinib has been shown to interfere with Tyr-phosphorylation state of the integral membrane protein band 3, affecting RBC microparticle formation. Here, we study the actions of Imatinib on a model of acute VOCs using humanized SCD mice (Hbatm1(HBA)TowHbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n=6-7 animals in each group) with Imatinib 50 mg/Kg/d for 2 weeks before hypoxia/Reoxygenation (H/R) stress used to mimic acute VOCs. Under normoxia, we found that in SCD mice Imatinib significantly reduced (i) Tyr-phosphorylation state of sickle red cell membrane proteins; (ii) the amount of phosphatidyl-serine (PS)+ sickle RBCs; and (iii) the release of erythroid microparticles, which was associated with accumulation of hemichromes and a more efficient erythrophagocytosis compared to vehicle treated animals. In SCD mice exposed to H/R stress, imatinib significantly decreased the H/R-induced (i) increase in peripheral neutrophil count; (ii) lung inflammatory cell infiltrate; (iii) kidney inflammatory cell infiltrate. In the lung of H/R SCD mice, Imatinib inhibited the H/R induced NF-kB activation and prevented the up-regulation of (i) pro-inflammatory cytokines such as ET-1; (ii) vascular endothelial activation markers (VCAM-1, ICAM-1); (iii) pro-fibrotic markers such as PDGF-B and (iv) the activation of UPR system. In the kidney of H/R SCD mice, Imatinib significantly reduced H/R induced expression of ET-1, VCAM-1 and E-selectin, which were again associated with inhibition of NF-kB. In addition, Imatinib significantly upregulated the expression of the microRNAs miR200a/b, which has been described to reduce renal fibrosis. Finally, in isolated aorta from Imatinib treated SCD exposed to H/R stress, a significant reduction in vascular activation markers was observed compared to vehicle treated animals. Collectively, our data indicate that Imatinib acts on multiple targets, modulating signal transduction and reducing inflammatory vasculopathy and extracellular matrix remodeling process related to VOC in SCD. Thus, Imatinib might represent a new therapeutic tool in clinical management of SCD patients. Disclosures El Nemer: Imara: Research Funding.

Protective Effect of Fetal Hemoglobin On Inflammation-Induced Expression of Hypoxia-Inducible Factor-1α (HIF-1α) in Sickle Mice: Microvascular Implications

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3250-3250
Author(s):  
Dhananjay K. Kaul ◽  
Mary E. Fabry ◽  
Sandra M Suzuka ◽  
Janki Shah
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Hypoxia Inducible Factor ◽  
Heme Oxygenase 1 ◽  
No Production ◽  
Cell Disease ◽  
Activation Markers ◽  
No Bioavailability

Abstract Abstract 3250 Chronic inflammation is a salient feature of human sickle cell disease (SCD) and transgenic-knockout sickle (BERK) mouse model. Although tissue ischemia is the primary instigator of hypoxia-inducible factor (HIF) activation, a number of inflammatory factors/pathways and oxidative stress can potentially induce expression of HIF-1α. Increased oxidative stress and inflammation are implicated in the activation of HIF-1α under normoxic conditions. HIF can trigger transcription of genes for vasoactive molecules such as vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1) and endothelin, which are implicated in the pathophysiology of SCD. We hypothesize that, in SCD, inflammation coupled with nitric oxide (NO) depletion will induce expression of HIF-1α. To this end, we have examined the expression of HIF-1α in normoxic BERK mice expressing exclusively human α- and βS- globins, and evaluated the effect of HbF in BERK mice (i.e., <1.0%, 20% and 40% HbF). We have previously shown that HbF exerts anti-sickling and anti-inflammatory effects (Kaul et al. J Clin Invest, 2004; Dasgupta et al. Am J Physiol, 2010). Here, we show that HIF-1α is expressed in BERK mice under normoxic conditions (i.e., normal hemoglobin oxygen saturation levels). In BERK mice expressing HbF, HIF-1α expression decreased concomitantly with increasing HbF, commensurately with increased NO bioavailability, and showed a strong inverse correlation with plasma NO metabolites (NOx) levels. Reduced HIF-1α expression in BERK mice expressing HbF was associated with decreased HO-1 and VEGF expression, and reduced serum endothelin-1 (ET-1) levels, which are among the target vasoactive molecules of HIF-1α. Furthermore, the commensurate decrease in HIF-1α expression with increase in HbF levels in BERK mice was accompanied by a distinct decrease in soluble (s) forms of endothelial activation markers such as sP-selectin and vascular cell adhesion molecule-1 (sVCAM-1). Notably, arteriolar dilation, enhanced volumetric blood flow and low blood pressure in normoxic BERK mice all showed a trend toward normalization with the introduction of HbF. Also, arginine treatment reduced HIF-1α expression as well as ET-1 levels in normoxic BERK mice, supporting a role of decreased NO bioavailability in HIF-1α activation. The present in vivo studies show that reduced inflammation and increased NO production in normoxic BERK mice (expressing HbF or treated with arginine) are distinctly associated with suppression of HIF-1α activation and inhibition of vasodilators, resulting in improved microvascular and hemodynamic parameters in the BERK model of sickle cell disease. The unique feature of inflammation in SCD is that it can be ameliorated by increased HbF, thereby coupling HbS polymerization/sickling to NO depletion, HIF-1α expression and inflammation in this disease. Disclosures: No relevant conflicts of interest to declare.

Plasmin Cleaves Complement iC3b, Preventing CR3/4 Mediated Phagocytosis By Macrophages

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1105-1105
Author(s):  
Erica A. Peterson ◽  
Jonathan H Foley ◽  
Michael J Krisinger ◽  
Edward Conway
Keyword(s):  
Flow Cytometry ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Complement Receptors ◽  
Red Cell Membrane ◽  
Western Blots ◽  
Complement Pathways

Abstract Introduction The plasmin(ogen) and complement systems are activated at sites of tissue injury and are involved in hemostasis, wound healing, inflammation and immune surveillance. Although the mechanisms are poorly understood, dysregulation of these systems underlie the pathogenesis and progression of inflammatory and vascular diseases. We aimed to characterize the relevant molecular interactions between the plasmin(ogen) and complement pathways. The three complement pathways converge with formation of C3-convertases that cleave C3 into C3a and C3b. C3a is liberated as an anaphylatoxin while C3b participates in further formation of the C3 and C5 convertases, thereby amplifying complement activation. To dampen the system, negative regulatory mechanisms exist. C3b is degraded to iC3b by the factor I (FI)/FH complex, which in turn is degraded to C3dg by the FI/complement receptor 1 (CR1) complex. iC3b and C3dg induce cellular responses by binding to complement receptors CR3 / CR4 / CR2, and CR2, respectively. Interactions of iC3b with CR3 or CR4 induce phagocytosis by macrophages, and binding of iC3b or C3dg to CR2 promotes B-cell responses. Recent studies show that plasmin proteolyses C3b and iC3b. We further characterized the plasmin cleavage sites in iC3b and evaluated the functional consequences in vitro. Methods and Results Plasmin cleavage of iC3b was examined over a range of concentrations and times. Plasmin (50 nM) generated a 40 kDa iC3b cleavage fragment (946TLD – PSR1303) which was notable for containing both C3dg (1002HLI – PSR1303) and the C3 thioester domain, necessary for opsonic binding to surfaces. We tested the relevance of this cleavage in phagocytosis assays using immunofluorescence and flow cytometry (Figure 1). C3b bound to the surface of fluorescent (Alexa 488) zymosan particles (C3b-zym), was treated with FI/FH to generate iC3b-zym, and subsequently incubated with FI/CR1 or plasmin to yield C3dg-zym or 946TLD – PSR1303-zym, respectively. Western blots confirmed that plasmin generated 946TLD – PSR1303 from iC3b-zym. The C3 fragment-zymosan species (C3b-zym, iC3b-zym, C3dg-zym and 946TLD – PSR1303-zym) were each incubated with macrophages (PMA-differentiated THP-1 cells) for 90 minutes. Cells were washed, stained and fixed for immunofluorescence, or suspended for flow cytometry. Figure 1, panel A shows macrophages stained with CellMask (red, cell membrane) and DAPI (blue, nucleus). Fluorescent zymosan is seen in green. No phagocytosis was detected with zymosan lacking C3 (zym alone), but there was a small amount with C3b-zym. In contrast, iC3b-zym was highly effective in inducing phagocytosis by most macrophages. This effect of iC3b-zym was abolished with FI/CR1 or plasmin, i.e. little phagocytosis was detected with C3dg-zym or 946TLD – PSR1303-zym. Flow cytometry-based quantitative analyses confirmed the preceding findings (Figure 1, panel B), with a similar pattern of phagocytosis induced by the zymosan-bound fragments. No phagocytosis was detected with zymosan lacking C3. Phagocytosis of C3b-zym and iC3b-zym was 7±2% and 17±1% of cells, respectively. C3dg-zym and 946TLD – PSR1303-zym induced phagocytosis was <5%. We also evaluated the role of the complement receptors in mediating the effect of the C3b/iC3b fragments using CR3/4 and CR1 blocking antibodies. These confirmed that phagocytosis of iC3b-zym and C3b-zym is mediated by CR3/4 and CR1, respectively. Conclusions Plasmin cleaves iC3b to form a redundant complement regulatory pathway with the FI/CR1 complex, but which notably does not require a cellular cofactor. Further studies will delineate the role of this and other plasmin-generated complement fragments in modulating innate immune and inflammatory responses. Disclosures: No relevant conflicts of interest to declare.

Hydroxyurea Therapy Reduces The Hypercoagulability State In a Sickle Cell Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2234-2234
Author(s):  
Marina Pereira Colella ◽  
Camila Bononi Almeida ◽  
Nicola Conran ◽  
Flavia Rubia Pallis ◽  
Carla Fernanda Franco-Penteado ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Plasma Levels ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Endothelial Activation ◽  
Activation Markers ◽  
Thrombotic Complications ◽  
Hbf Induction ◽  
Elevated Rate ◽  
Cell Adhesion Molecule 1

Abstract Several lines of evidence show that sickle cell anemia (SCA) is characterized by a hypercoagulable state. SCA patients present an elevated rate of thrombotic complications and increased biological markers of coagulation activation. In a previous study we demonstrated that therapy with hydroxyurea (HU) in SCA patients was associated with reductions in hypercoagulability markers, in addition to reductions in hemolysis, inflammation and endothelial activation markers (Colella et al Journal of Thrombosis and Haemostasis 2012). In the present study, we attempted to further investigate whether the effect of HU on hemostatic activation is dependent on fetal haemoglobin (HbF) expression, hemolysis and/or inflammation. To do so, we used the BERK murine model of SCA, a homozygous model of SCA, incapable of expressing HbF. In this BERK model, treatment with HU results in no improvement in hemolysis, due to the lack of HbF induction (Lebensburger et al Haematologica 2010). The murine SCA model was generated by transplantation of nucleated bone marrow cells from BERK mice into lethally-irradiated C57BL6 mice. Only mice expressing > 97% of human hemoglobin S (HbS) at 10 weeks after transplantation were used in the study. HU therapy at the dose of 50mg/kg (HU group) or saline alone (control mice) was administered through intraperitoneal injections 5 times per week, initiated at 11 weeks after transplantation (approximate time of sickle marrow engraftment). After a treatment period of 16 weeks, blood samples drawn from the inferior cava vein were collected and submitted to evaluation of plasma levels of thrombin-antithrombin III (TAT), a final marker of thrombin generation, in both animal groups. We also evaluated plasma levels of the inflammation marker interleukin 6 (IL6), and the endothelial marker soluble vascular cell adhesion molecule-1 (sVCAM-1). TAT, IL6 and sVCAM-1 were all measured by commercially-available ELISA kits. Statistical analyses were performed using Mann-Whitney’s U test. Treatment with HU resulted in a significant reduction of TAT plasma levels (106.7 μg/L vs 138.5 μg/L; P = 0.05). Plasma levels of IL6 (7.1pg/mL vs 12.4pg/mL; P = 0.18) and sVCAM-1 (765.8 ng/mL vs 848.4 ng/mL; P= 0.43) presented non-significant reductions with HU treatment. Our results show that, in this murine SCA model, long-term HU treatment results in an improvement in the hypercoagulability state, even in the absence of HbF induction, or of a significant improvement of inflammation or endothelial activation. This indicates that HU may have a direct effect on inhibiting the activation of coagulation in SCA. Disclosures: No relevant conflicts of interest to declare.

The relationship between tumour necrosis, circulating IL-6 concentrations, and inflammatory responses in patients undergoing curative resection for colorectal cancer.

2013 ◽  
Vol 31 (4_suppl) ◽  
pp. 404-404
Author(s):  
Graeme JK Guthrie ◽  
Campbell SD Roxburgh ◽  
Colin H Richards ◽  
Paul G. Horgan ◽  
Donald C. Mcmillan
Keyword(s):  
Colorectal Cancer ◽  
Skeletal Muscle ◽  
Inflammatory Response ◽  
Interleukin 6 ◽  
Tumour Necrosis ◽  
Curative Resection ◽  
Inflammatory Cell ◽  
Inflammatory Responses ◽  
Inflammatory Cell Infiltrate ◽  
Cell Infiltrate

404 Background: Cancer-associated inflammation, in the form of systemic and local inflammation, and tumour necrosis are known to have prognostic value in colorectal cancer (CRC). In addition, recent work has reported a direct relationship between the systemic inflammatory response and loss of skeletal muscle in patients with CRC. However, the inter-relationships between these inflammatory responses, tumour necrosis, metabolic upset and circulating biochemical mediators are unclear in CRC. Interleukin-6 and its downstream signalling cascades have been implicated in both cancer-associated inflammation and cancer-associated muscle wasting. The aim of the present study was to examine whether circulating IL-6 concentrations may link tumour necrosis, local and systemic inflammatory responses, and metabolic upset in patients undergoing curative resection for colorectal cancer. Methods: The study included 118 patients undergoing surgery for CRC between 2004 and 2009. Data were collected from pre-operative blood tests. Routine pathology specimens were scored for Klintrup criteria and tumour necrosis. Results: Tumour necrosis was associated with increased T-stage (p<0.01), reduced inflammatory cell infiltrate (p<0.05), increased IL-6 (p<0.001), IL-10 (p<0.01), and VEGF (p<0.001) and with markers of the systemic inflammatory response: mGPS (p<0.001), anaemia (p<0.05); increased white cell (p<0.001), neutrophil (p<0.05) and platelet (p<0.001) counts. Circulating IL-6 was associated with increased IL-10 (p<0.01), VEGF (p<0.001), increased mGPS (p<0.001), increased white cell (p<0.01) and platelet (p<0.01) counts and low skeletal muscle index (p<0.01). On Spearman rank correlation there were significant associations between circulating concentrations of IL-6 and IL-10 (rs= 0.39, p<0.001) and CRP (r= 0.42, p<0.001). Conclusions: Interleukin-6 appears to be associated with systemic inflammation, tumour necrosis, and sarcopenia in colorectal cancer. However, the lack of an association between IL-6 and the local inflammatory response suggests a more complex relationship with the tumour inflammatory cell infiltrate.

scholarly journals Specific macrophage populations promote both cardiac scar deposition and subsequent resolution in adult zebrafish

2019 ◽  
Vol 116 (7) ◽  
pp. 1357-1371 ◽  
Author(s):  
Laura Bevan ◽  
Zhi Wei Lim ◽  
Byrappa Venkatesh ◽  
Paul R Riley ◽  
Paul Martin ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Inflammatory Cell ◽  
Inflammatory Responses ◽  
Tissue Injury ◽  
Cardiac Injury ◽  
Repair Process ◽  
Adult Zebrafish ◽  
Cardiac Damage ◽  
Scar Resolution ◽  
Macrophage Subsets

Abstract Aims A robust inflammatory response to tissue injury is a necessary part of the repair process but the deposition of scar tissue is a direct downstream consequence of this response in many tissues including the heart. Adult zebrafish not only possess the capacity to regenerate lost cardiomyocytes but also to remodel and resolve an extracellular scar within tissues such as the heart, but this scar resolution process remains poorly understood. This study aims to characterize the scarring and inflammatory responses to cardiac damage in adult zebrafish in full and investigate the role of different inflammatory subsets specifically in scarring and scar removal. Methods and results Using stable transgenic lines, whole organ imaging and genetic and pharmacological interventions, we demonstrate that multiple inflammatory cell lineages respond to cardiac injury in adult zebrafish. In particular, macrophage subsets (tnfα+ and tnfα−) play prominent roles with manipulation of different phenotypes suggesting that pro-inflammatory (tnfα+) macrophages promote scar deposition following cardiac injury whereas tnfα− macrophages facilitate scar removal during regeneration. Detailed analysis of these specific macrophage subsets reveals crucial roles for Csf1ra in promoting pro-inflammatory macrophage-mediated scar deposition. Additionally, the multifunctional cytokine Osteopontin (Opn) (spp1) is important for initial scar deposition but also for resolution of the inflammatory response and in late-stage ventricular collagen remodelling. Conclusions This study demonstrates the importance of a correctly balanced inflammatory response to facilitate scar deposition during repair but also to allow subsequent scar resolution, and full cardiac regeneration, to occur. We have identified Opn as having both pro-fibrotic but also potentially pro-regenerative roles in the adult zebrafish heart, driving Collagen deposition but also controlling inflammatory cell resolution.

Abstract T P324: Sex Differences in Ischemic Outcomes and Inflammatory Responses in Neonatal Stroke

Stroke ◽  
2014 ◽  
Vol 45 (suppl_1) ◽  
Author(s):  
Mehwish A Mirza ◽  
Kathryn Bentivegna ◽  
Rodney Ritzel ◽  
Kaitlyn H Hajdarovic ◽  
Louise D McCullough ◽  
...  
Keyword(s):  
Sex Differences ◽  
Inflammatory Response ◽  
Microglial Activation ◽  
Inflammatory Responses ◽  
Tissue Injury ◽  
Activation Markers ◽  
Male And Female ◽  
Hormone Levels ◽  
Time Points ◽  
Cytokine Analysis

Background and Purpose: Neonatal arterial ischemic stroke (NAIS) is an important cause of motor and cognitive impairment in children. Clinically, male infants are more vulnerable to ischemic insult and suffer more long-term deficits than female infants though the mechanisms remain elusive. Inflammatory processes are fundamental in the pathophysiology of ischemia as microglial activation initiates the inflammatory response after ischemia. Recent studies report a sexual dimorphism in microglia numbers and expression of activation markers in neonatal brains under normal conditions. How these basal sex differences in microglia affect NAIS remains largely unexplored. This study investigated sex differences in stroke phenotypes and inflammation triggered by NAIS. We hypothesize that ischemia induces sex-specific tissue injury in male and female neonates, which is related to differences in microglial activation and inflammatory responses. Methods: Male and female C57BL6 mice were subjected to 60-minute Rice-Vanucci Modeling at post-natal day 10 (P10) to induce NAIS. Stroke outcomes were measured at 24 hours, 72 hours and 7 days after stroke. Microglial activation and inflammatory responses were evaluated by flow cytometry, immunohistochemistry, and multiplex cytokine analysis. Results: At 24 hours no difference in infarct volumes (total infarct: male vs. female 46.6±7.2% vs. 43.2±9.3%, n=6/gp) and in Iba-1 staining of the ischemic brain were seen between male and female neonates. However, at 72 hours female neonates exhibited significantly smaller infarct size and improved behavior outcomes compared to males (total infarct: male vs. female 43.1±9.9% vs. 27.1±8.8%, n=6/gp, p <.05). Male animals demonstrated increased microglial activation and up-regulated inflammatory response compared to females at 72 hours. This male-specific phenotype was also seen at 7 days after injury. There was no difference in hormone levels at any of the three time points after stroke. Conclusions: Acute ischemia leads to an equivalent primary brain injury in male and female P10 mice. However, infarct damage worsens in males at sub-acute time points vs. females, as does the immune response. This sex difference independent of hormone levels exists in NAIS.

scholarly journals Combined Administration of Recombinant TGF-β1 and DMSO Decreases the in Vitro Inflammatory Properties of Neutrophils from Sickle Cell Anemia Individuals

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2366-2366
Author(s):  
Lidiane S. Torres ◽  
Lediana I. Miguel ◽  
Merav E. Shaul ◽  
Zvi G. Fridlender ◽  
Wilson A. Ferreira ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Transforming Growth Factor ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Combined Treatment ◽  
Inflammatory Stress ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Tgf Β1

Abstract Sickle cell anemia (SCA) is a chronic inflammatory disease, in which activated neutrophils play a role in initiating vaso-occlusion. Two populations of circulating neutrophils have been described, denominated as low-(LDN) and high-(HDN) density neutrophils. Circulating numbers of LDN (a less inflammatory subset) are normally minimal, but this population augments under inflammatory stress, such as that seen in cancer. Transforming growth factor beta-1 (TGF-β1) is a cytokine with anti-inflammatory properties that is elevated in SCA. In conditions such as Chron's disease, TGF-β1 has protective effects, mediated by its immuno-suppressive functions. In macrophages, it is thought to trigger the polarization from pro- (M1) to anti-inflammatory phenotypes (M2), which hypothetically occur in neutrophils too (N1 and N2). Moreover, dimethyl sulfoxide (DMSO) reportedly increases TGF-β receptors expression on epithelial cells. We aimed to characterize the subsets of circulating neutrophils in SCA patients and investigate the effects of TGF-β1 and DMSO on these cells. Neutrophils from healthy (CON) and SCA individuals, in steady state and without blood transfusion for 90 days, were isolated from peripheral blood by Ficoll-Paque density gradient centrifugation. HDN and LDN were obtained from the granulocyte and mononuclear layers, respectively, and stained with CD66b for neutrophil identification by flow cytometry. As no significant effect of hydroxyurea (HU) therapy on the data obtained was observed, patients' data were grouped together irrespective of HU use. Percentages of LDN were calculated based on the total of gated CD66b+ cells. SCA patients had higher levels of LDN than CON (3.2±0.9%, N=7 vs 1.3±0.3%, N=13; p=0.02). We next investigated the presence of CD66b+/CD206- and CD66b+/CD206+ cells, to infer the presence of N1 and N2 phenotypes, respectively. N2 were more frequent in the LDN than in the HDN subset (CON: 68.1±3.3% vs 52.0±4.4%, N=9, p=0.01; SCA: 77.6±8.9% vs 44.1±5.0%, N=3, p=0.03). To determine whether TGF-β1 and DMSO could shift HDN to a LDN profile, cells were treated (2h) with TGF-β1 (50pM) and/or DMSO (1.5%). Treatments with DMSO alone or combined with TGF-β1 increased the percentage of CD206+ cells in CON (45.7±2.1% vs 61.9±7.6 and 53.6±2.6% respectively, N=6, p=0.04), as well as CD206+ expression on each cell (mean fluorescence intensity, MFI) (137.5±16.9 MFI vs 293.6±71.2 MFI and 210.1±23.9 MFI, respectively, p=0.03). In SCA, only the combined TGF-β1/DMSO treatment increased the MFI of CD206 in HDN (115.7±10.2 vs 255.8±29.7 MFI, N=4, p=0.03). We next investigated whether TGF-β1/DMSO could reduce the adhesion of HDN to fibronectin ligand (FN, 20μg/mL) using static adhesion assays (30 min, 37ºC). HDN from CON and SCA were treated with TGF-β1 and/or DMSO (90min) and stimulated with TNF-α (200ng/mL, 30min). Although TGF-β1 alone did not reduce the adhesion of HDN to FN (p>0.05), the addition of DMSO decreased TNF-α-induced adhesion in CON (16.5±1.8% to 11.3±1.5%, p=0.03, N=10) and SCA HDN (38.9±23.9% to 13.9±1.5%, p=0.04, N=3). Subsequently, HDN were stimulated (4h) with LPS (100ng/mL) and INF-γ (20ng/mL), to induce N2 polarization, in the presence/absence of TGF-β1 and DMSO. The combined treatment again reduced adhesion in both groups (CON: 11.1±1.5% to 4.2±1.2%, p<0.01, N=4; SCA: 47.4±12.3% vs 21.9±4.5%, p=0.04, N=4). To assess whether TGF-β1 and DMSO could affect the production of proinflammatory cytokines by HDN after stimulation with LPS/INF-γ, TNF-α and IL-1β levels in cell supernatants were measured by ELISA. TGF-β1 and DMSO, in combination, decreased both TNF-α and IL-1β release from CON (TNF-α: 39.7±8.7pg/mL to 8.7±0.9pg/mL, p<0.01, N=6; IL-1β: 66.9±10.5pg/mL to 16.5±5.1pg/mL, p=0.02, N=4) and SCA HDN (TNF-α: 174.0±55.5pg/mL to 21.8±6.6pg/mL, p=0.01, N=8; IL-1β: 103.6±25.6pg/mL to 43.1±15.5pg/mL, p=0.01, N=8). Our results demonstrate for the first time the presence of elevated numbers of LDN in SCA patients, indicating an increased basal response to inflammatory stress. However, this shift in the anti-inflammatory subset does not appear enough to control inflammatory responses in the disease, and the use of agents capable of inducing this polarization may be a promising approach. Moreover, the anti-inflammatory effects of TGF-β1 on HDN seem to be enhanced by DMSO and suggest this combination as an effective modulator of the inflammatory profile of neutrophils. Disclosures No relevant conflicts of interest to declare.

Sickle Cell Mice with Hypoxia-Reoxygenation Have Oxidative and Inflammatory Liver Injury - toward a New Experimental Model for Vaso- Occlusive Injury

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 121-121
Author(s):  
Lewis L. Hsu ◽  
Sruti Shiva ◽  
Caroline Mendelsohn ◽  
Michael R Hsu ◽  
Audrey C Noguchi ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Oxidative Damage ◽  
Experimental Model ◽  
Sickle Cell ◽  
Tissue Injury ◽  
Oxygen Species ◽  
Cell Disease ◽  
Hepatocellular Injury ◽  
Aconitase Activity ◽  
Hypoxia Reoxygenation

Abstract INTRODUCTION: Vaso-occlusion causes tissue ischemia and severe pain in patients with sickle cell disease (SCD). The liver is frequently involved in human SCD complications and elevated serum levels of hepatic transaminases often accompany vasoocclusive pain. It is unknown whether in sickle cell disease, the liver sustains oxidative damage after hypoxia-reoxygenation and if so, how long does it take to recover. Both mitochondrial and cytosolic aconitases are potential targets of oxidants in cells due to the oxidant-mediated loss of iron from the [4Fe-4S] cluster. Oxidant damage can occur from the reactive oxygen species generated by dysfunctional mitochondria dysfunction following ischemia-reperfusion and also from peroxidases later released during the inflammatory reaction to tissue injury. A mouse model of SCD can be used to investigate the cellular mechanisms and temporal sequence of vaso-occlusive tissue damage. OBJECTIVES: After experimental vaso-occlusion by exposing sickle cell mice to hypoxia-reoxygenation, we will determine whether hepatocellular injury shows temporal correlation with a marker of oxidative damage (diminished aconitase activity) and a marker of inflammation (increased myeloperoxidase concentration). No changes are predicted to occur in littermate control mice whose erythrocytes do not sickle with hypoxic challenge. METHODS: Mice expressing exclusively human sickle cell hemoglobin (“Berkeley sickle mice”) and non-sickling littermate controls (“hemizygotes”) were exposed to 10% oxygen for 2 hours, and then restored to normoxia. At 6, 18, 48, or 72 hrs after hypoxia, animals were euthanized to harvest blood and liver tissue. Normoxic control mice of both types had blood and liver harvested without hypoxic exposure. Additional sickle cell mice received intraperitoneal injection of nitrite (2.4 millimole/g body weight) or saline control at the end of 2 hours of hypoxia, then had blood and liver harvested 18 hours after they were restored to normoxia. Serum alanine aminotransferase (ALT) level was assayed as a quantitative measure of hepatocellular injury. Aconitase and myeloperoxidase (MPO) results were compared by t-test. RESULTS: Serum ALT level rose 3-fold in sickle mice by 18 hr after hypoxia, and then declined by 48 and 72 hrs after hypoxia. Hemizygotes showed no change in ALT after hypoxia. Liver homogenate aconitase activity was significantly lower in sickle mice than in hemizygotes at the first time point measured, 6 hrs after hypoxia (p=0.003, n=4 per group), suggesting that oxidative damage to the enzyme had occurred early after hypoxia. MPO concentration in livers harvested 48 hr after hypoxia-reoxygenation was 3-fold elevated in sickle mice vs. hemizygotes (p=0.006, n=4 per group), but not at baseline or 72 hr after hypoxia-reoxygenation. Nitrite injection was associated with complete abrogation of rise in ALT in sickle mice challenged with hypoxia-reoxygenation, but was not associated with any differences in aconitase activity or MPO concentration. CONCLUSION: Serum ALT, liver aconitase activity, and liver MPO concentration had different temporal patterns of changes in sickle mice after hypoxia-reoxygenation challenge as an experimental model of vaso-occlusive tissue injury. Oxidative stress appears to be present within hours after hypoxia, followed by tissue injury (serum ALT rise), which is then followed by inflammation may take 48 hr after experimental vasoocclusion. Although the nitrite is not sufficient as antioxidant to protect against reactive oxygen species and inflammatory leukocytes, nitrite may be protective against tissue injury at the mitochondrial level. This experimental model system may be well-suited for pre-clinical testing of therapy for sickle cell vaso-occlusion.

Liver Hypoxia and Tissue Injury Are Specific to Sickle Cell Mice in An Experimental Model of Sickle Cell Vaso-Occlusion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2569-2569
Author(s):  
Lewis L. Hsu ◽  
Audrey C Noguchi ◽  
Constance Noguchi ◽  
Katherine C Wood ◽  
James G Taylor ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Tissue Damage ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Elevated Serum ◽  
Quantitative Measure ◽  
Tissue Hypoxia ◽  
Hypoxic Exposure ◽  
Wild Type ◽  
Tissue Ischemia

Abstract Abstract 2569 Poster Board II-546 Sickle cell vaso-occlusion causes tissue ischemia, injury, and severe pain in patients with sickle cell disease (SCD). Investigations of the pathophysiology of vaso-occlusion have largely focused on the mechanisms for microcirculatory obstruction, rather than the tissue damage. The liver is frequently involved in human SCD complications and elevated serum levels of hepatic transaminases often accompany vaso-occlusive pain. Animal models can help to study mechanisms of how vaso-occlusive microcirculatory abnormalities cause tissue damage. Sickle cell mice originally developed by Pastzy (“Berkeley sickle mice”) expressing exclusively human sickle hemoglobin have severe hemolytic anemia and physiologic fragility, and are well-suited for examining an acute vaso-occlusive challenge. HYPOTHESIS: Moderate hypoxic exposure will cause liver hypoxia and tissue injury only in homozygote sickle mice, as signs of vaso-occlusive injury. METHODS: We examined vaso-occlusion by challenging male sickle mice with moderate normobaric hypoxia (10% oxygen) for up to 2 hours then restoring them to normoxia and assaying plasma alanine aminotransferase (ALT) as a quantitative measure of hepatic injury. Pimonidazole (HypoxyProbe 60mg/kg, NPI, Inc) was injected intraperitoneally before hypoxia, in order to provide irreversible labeling of cells with pO2 less than 10 torr. Controls included sickle mice without hypoxia, and non-sickle mice with the same hypoxic exposure. Mice were euthanized for blood and tissue harvest. Liver, kidney, and lungs were fixed in formalin, and processed for immunohistochemical staining (HP-100 kit, NPI). A veterinary pathologist unaware of the treatment group scored histologic sections under light microscopy for the extent of staining on a scale of 1 to 4, and intensity of staining on a scale of 1 to 3. RESULTS: Sickle cell mice have significantly greater extent of Hypoxyprobe labeling of liver than wild-type mice with the same hypoxia-reoxygenation challenge (2.6 + 0.5 vs 1.3 + 0.5, p=0.027 by t-test). At normoxic baseline, liver HypoxyProbe staining from sickle mice and wild-type controls had the same extent (1 vs 1) and intensity of liver staining. Kidney had a higher baseline of Hypoxyprobe staining in all normoxic mice, and this did not increase with hypoxia in sickle mice. Lungs had very little Hypoxyprobe staining at baseline, and a few bronchial cell stained under hypoxia in sickle mice, but the change was not statistically significant. ALT was mildly elevated in sickle mice at normoxic baseline, and rose 3-fold to a peak by 18 hours after hypoxia, then declined back to baseline by 48 – 72 hours. CONCLUSION: The extent of HypoxyProbe staining shows that sickle mice suffer significantly greater liver tissue hypoxia with hypoxic exposure than controls with the same mild hypoxia. Sickle mice in normoxia have no greater extent of HypoxyProbe staining than control mice. These results are consistent with hypoxia-triggered vaso-occlusion as the cause of ALT elevation in the sickle mice. The two outcome measures after hypoxic challenge of sickle mice, HypoxyProbe staining for tissue hypoxia and plasma ALT for tissue injury, provide a bioassay system for pre-clinical screening of antisickling therapy. Disclosures: No relevant conflicts of interest to declare.

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