Attenuated Leukocyte Infiltration Research Articles

Hyperbaric oxygen therapy mitigates left ventricular remodeling, upregulates MMP-2 and VEGF, and inhibits the induction of MMP-9, TGF-β1, and TNF-α in streptozotocin-induced diabetic rat heart

AimsHyperbaric oxygen (HBO) therapy has been widely used for the adjunctive treatment of diabetic wounds, and is currently known to influence left ventricular (LV) function. However, morphological and molecular repercussions of the HBO in the diabetic myocardium remain to be described. We aimed to investigate whether HBO therapy would mitigate adverse LV remodeling caused by streptozotocin (STZ)-induced diabetes. Main methodsSixty-day-old Male Wistar rats were divided into four groups: Control (n = 8), HBO (n = 7), STZ (n = 10), and STZ + HBO (n = 8). Diabetes was induced by a single STZ injection (60 mg/kg, i.p.). HBO treatment (100% oxygen at 2.5 atmospheres absolute, 60 min/day, 5 days/week) lasted for 5 weeks. LV morphology was evaluated using histomorphometry. Gene expression analyzes were performed for LV collagens I (Col1a1) and III (Col3a1), matrix metalloproteinases 2 (Mmp2) and 9 (Mmp9), and transforming growth factor-β1 (Tgfb1). The Immunoexpression of cardiac tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) were also quantified. Key findingsHBO therapy prevented LV concentric remodeling, heterogeneous myocyte hypertrophy, and fibrosis in diabetic rats associated with attenuation of leukocyte infiltration. HBO therapy also increased Mmp2 gene expression, and inhibited the induction of Tgfb1 and Mmp9 mRNAs caused by diabetes, and normalized TNF-α and VEGF protein expression. SignificanceHBO therapy had protective effects for the LV structure in STZ-diabetic rats and ameliorated expression levels of genes involved in cardiac collagen turnover, as well as pro-inflammatory and pro-angiogenic signaling.

Mesenchymal Stem/Stromal Cell Therapy in Blood-Brain Barrier Preservation Following Ischemia: Molecular Mechanisms and Prospects.

Ischemic stroke is the leading cause of mortality and long-term disability worldwide. Disruption of the blood–brain barrier (BBB) is a prominent pathophysiological mechanism, responsible for a series of subsequent inflammatory cascades that exacerbate the damage to brain tissue. However, the benefit of recanalization is limited in most patients because of the narrow therapeutic time window. Recently, mesenchymal stem cells (MSCs) have been assessed as excellent candidates for cell-based therapy in cerebral ischemia, including neuroinflammatory alleviation, angiogenesis and neurogenesis promotion through their paracrine actions. In addition, accumulating evidence on how MSC therapy preserves BBB integrity after stroke may open up novel therapeutic targets for treating cerebrovascular diseases. In this review, we focus on the molecular mechanisms of MSC-based therapy in the ischemia-induced prevention of BBB compromise. Currently, therapeutic effects of MSCs for stroke are primarily based on the fundamental pathogenesis of BBB breakdown, such as attenuating leukocyte infiltration, matrix metalloproteinase (MMP) regulation, antioxidant, anti-inflammation, stabilizing morphology and crosstalk between cellular components of the BBB. We also discuss prospective studies to improve the effectiveness of MSC therapy through enhanced migration into defined brain regions of stem cells. Targeted therapy is a promising new direction and is being prioritized for extensive research.

Lignosus rhinocerotis Cooke Ryvarden ameliorates airway inflammation, mucus hypersecretion and airway hyperresponsiveness in a murine model of asthma.

Lignosus rhinocerotis Cooke. (L. rhinocerotis) is a medicinal mushroom traditionally used in the treatment of asthma and several other diseases by the indigenous communities in Malaysia. In this study, the effects of L. rhinocerotis on allergic airway inflammation and hyperresponsiveness were investigated. L. rhinocerotis extract (LRE) was prepared by hot water extraction using soxhlet. Airway hyperresponsiveness (AHR) study was performed in house dust mite (HDM)-induced asthma in Balb/c mice while airway inflammation study was performed in ovalbumin (OVA)-induced asthma in Sprague-Dawley rats. Treatment with different doses of LRE (125, 250 and 500 mg/kg) significantly inhibited AHR in HDM-induced mice. Treatment with LRE also significantly decreased the elevated IgE in serum, Th2 cytokines in bronchoalveolar lavage fluid and ameliorated OVA-induced histological changes in rats by attenuating leukocyte infiltration, mucus hypersecretion and goblet cell hyperplasia in the lungs. LRE also significantly reduced the number of eosinophils and neutrophils in BALF. Interestingly, a significant reduction of the FOXP3+ regulatory T lymphocytes was observed following OVA induction, but the cells were significantly elevated with LRE treatment. Subsequent analyses on gene expression revealed regulation of several important genes i.e. IL17A, ADAM33, CCL5, IL4, CCR3, CCR8, PMCH, CCL22, IFNG, CCL17, CCR4, PRG2, FCER1A, CLCA1, CHIA and Cma1 which were up-regulated following OVA induction but down-regulated following treatment with LRE. In conclusion, LRE alleviates allergy airway inflammation and hyperresponsiveness, thus suggesting its therapeutic potential as a new armamentarium against allergic asthma.

Annexin A1 Attenuates Neutrophil Migration and IL-6 Expression through Fpr2 in a Mouse Model of Streptococcus suis-Induced Meningitis.

Streptococcus suis serotype 2 is a crucial pathogenic cause of bacterial meningitis, a life-threatening disease with neurological sequelae and high rates of mortality. Inflammation triggered by S. suis infection must be precisely regulated to prevent further tissue damage. As a glucocorticoid anti-inflammatory mediator, annexin A1 (AnxA1) mainly acts through formyl peptide receptor 2 (Fpr2) to alleviate inflammation in the peripheral system. In this study, we evaluated the roles of AnxA1 and Fpr2 in a mouse model of S. suis meningitis created via intracisternal infection in Fpr2-deficient (Fpr2-/-) and wild-type (WT) mice. We revealed that Fpr2-/- mice were highly susceptible to S. suis meningitis, displaying increased inflammatory cytokine levels, bacterial dissemination, and neutrophil migration compared with WT mice. Additionally, AnxA1 exerted anti-inflammatory effects through Fpr2, such as attenuation of leukocyte infiltration, inflammatory mediator production, and astrocyte or microglial activation in the brain. Importantly, we found that the antimigratory function of AnxA1 decreases neutrophil adherence to the endothelium through Fpr2. Finally, an in vitro study revealed that AnxA1 potentially suppresses interleukin-6 (IL-6) expression through the Fpr2/p38/COX-2 pathway. These data demonstrated that Fpr2 is an anti-inflammatory receptor that regulates neutrophil migration in mice with S. suis meningitis and identified AnxA1 as a potential therapeutic option.

P-selectin glycoprotein ligand 1 deficiency prevents development of acute pancreatitis by attenuating leukocyte infiltration.

BACKGROUNDAcute pancreatitis (AP) is rapid-onset pancreatic inflammation that causes local and systemic inflammatory response syndrome (SIRS) with high morbidity and mortality, but no approved therapies are currently available. P-selectin glycoprotein ligand 1 (PSGL-1) is a transmembrane glycoprotein to initiate inflammatory responses. We hypothesized that PSGL-1 may be involved in the development of AP and would be a new target for the treatment of AP.AIMTo investigate the role and mechanism of PSGL-1 in the development of AP.METHODSThe PSGL-1 expression on leukocytes was detected in peripheral blood of AP patients and volunteers. Pancreatic injury, inflammatory cytokines expression, and inflammatory cell infiltration was measured in AP mouse models induced with PSGL-1 knockout (PSGL-1-/-) and wild-type (PSGL-1+/+) mice. Leukocyte-endothelial cell adhesion was measured in a peripheral blood mononuclear cell (PBMC)-endothelial cell coculture system.RESULTSThe expression of PSGL-1 on monocytes and neutrophils was significantly increased in AP patients. Compared with PSGL-1+/+ mice, PSGL-1-/- AP mice induced by caerulein exhibited lower serum amylase, less Interleukin-1beta (IL-1beta) and Interleukin-6 (IL-6) expression, less neutrophil and macrophage infiltration, and reduced peripheral neutrophil and monocyte accounts. PSGL-1 deficiency alleviated leukocyte-endothelial cell adhesion via IL-6 but not IL-1beta.CONCLUSIONPSGL-1 deficiency effectively inhibits the development of AP by preventing leukocyte-endothelial cell adhesion via IL-6 stimulation and may become a potential therapeutic target for treating AP.

Leukotriene B4 receptor 1 exacerbates inflammation following myocardial infarction.

Leukotriene B4 receptor 1 (BLT1), a high-affinity G-protein-coupled receptor for leukotriene B4 (LTB4 ), is expressed on various inflammatory cells and plays critical roles in several inflammatory diseases. In myocardial infarction (MI), various inflammatory cells are known to be recruited to the infarcted area, but the function of BLT1 in MI is poorly understood. Here, we investigated the role of BLT1 in MI and the therapeutic effect of a BLT1 antagonist, ONO-4057, on MI. Mice with infarcted hearts showed increased BLT1 expression and LTB4 levels. BLT1-knockout mice with infarcted hearts exhibited attenuated leukocyte infiltration, proinflammatory cytokine production, and cell death, which led to reduced mortality and improved cardiac function after MI. Bone-marrow transplantation studies showed that BLT1 expressed on bone marrow-derived cells was responsible for the exacerbation of inflammation in infarcted hearts. Furthermore, ONO-4057 administration attenuated the inflammatory responses in hearts surgically treated for MI, which resulted in reduced mortality and improved cardiac function after MI. Our study demonstrated that BLT1 contributes to excessive inflammation after MI and could represent a new therapeutic target for MI.

Inhibition of Matrix Metalloproteinase with BB-94 Protects against Caerulein-Induced Pancreatitis via Modulating Neutrophil and Macrophage Activation.

Methods AP was induced in Balb/C mice by ten hourly intraperitoneal injections of caerulein (100 μg/kg) and LPS (5 mg/kg). The MMP inhibitor, BB-94 (20 mg/kg) was intraperitoneally administered 30 min before AP induction. Pancreatitis was confirmed by histology and serum amylase and lipase. Expression of pancreatic proinflammatory mediators and NF-κB activation were assessed. Bone marrow-derived neutrophils (BMDNs) and macrophages (BMDMs) were isolated. BMDNs were activated by phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) and neutrophil reactive oxygen species (ROS) production was recorded. BMDMs were stimulated with 10 ng/ml IFN-γ and 100 ng/ml LPS to induce M1 macrophage polarization. Results Pancreatic MMP-9 was markedly upregulated and serum MMP-9 was increased in caerulein-induced pancreatitis. Inhibition of MMP with BB-94 ameliorated pancreatic tissue damage and decreased the expression of proinflammatory cytokines (TNFα and IL-6) or chemokines (CCL2 and CXCL2) and NF-κB activation. Furthermore, using isolated BMDNs and BMDMs, we found that inhibition of MMP with BB-94 markedly decreased neutrophil ROS production, inhibited inflammatory macrophage polarization and NF-κB activation. Conclusions Our results showed that inhibition of MMP with BB-94 protected against pancreatic inflammatory responses in caerulein-induced pancreatitis via modulating neutrophil and macrophage activation.

Neural anti-inflammatory action mediated by two types of acetylcholine receptors in the small intestine

Gastrointestinal prokinetic agents function as serotonin-4 receptor (5-HT4R) agonists to activate myenteric plexus neurons to release acetylcholine (ACh), which then induce anti-inflammatory action. Details of this pathway, however, remain unknown. The aim of this study is to clarify the anti-inflammatory mechanism underlying the 5-HT4R agonist, mosapride citrate (MOS)-induced anti-inflammatory action on postoperative ileus (POI). POI models were generated from wild-type C57BL6/J (WT), 5-HT4R knock-out (S4R KO), α7 nicotinic AChR KO (α7 R KO), and M2 muscarinic ACh receptor KO (M2R KO) mice. MOS attenuated leukocyte infiltration in WT. MOS-induced anti-inflammatory action was completely abolished in both S4R KO and S4R KO mice upon wild-type bone marrow transplantation. MOS-induced anti-inflammatory action against macrophage infiltration, but not neutrophil infiltration, was attenuated in α7 R KO mice. Selective α7nAChR agonists (PNU-282987 and AR-R17779) also inhibited only macrophage infiltration in POI. MOS-mediated inhibition of neutrophil infiltration was diminished by atropine, M2AChR antagonist, methoctramine, and in M2R KO mice. Stimulation with 5-HT4R inhibits leukocyte infiltration in POI, possibly through myenteric plexus activation. Released ACh inhibited macrophage and neutrophil infiltration likely by activation of α7nAChR on macrophages and M2AChR. Thus, macrophage and neutrophil recruitment into inflamed sites is regulated by different types of AChR in the small intestine.

A Mouse Model of Post-Stroke Pneumonia Induced by Intra-Tracheal Inoculation with Streptococcus pneumoniae

Background: Stroke-induced immunodeficiency increases the risk of infectious complications, which adversely affects neurological outcome. Among those, pneumonia affects as many as one third of stroke patients and is the main contributor to mortality in the post-acute phase of stroke. Experimental findings on post-stroke susceptibility to spontaneous pneumonia in mice are contradictory. Here, we established a mouse model inducing standardized bacterial pneumonia and characterized the impaired pulmonary cellular and humoral immune responses after experimental stroke. Methods: Bacterial pneumonia was induced by intra-tracheal inoculation with Streptococcus pneumoniae at different time points after transient middle cerebral artery occlusion (MCAO). Bacterial counts in lungs and blood, histological changes, and cytokine production in the lungs were assessed. Furthermore, we investigated the effect of pneumonia on stroke outcome. Results: Intra-tracheal inoculation resulted in reproducible pneumonia and bacteraemia, and demonstrated post-stroke susceptibility to streptococcal pneumonia developing with a delay of at least 24 h after MCAO. Higher bacterial counts in mice infected 3 days after stroke induction correlated with reduced neutrophil and macrophage infiltration in the lungs and lower levels of pro-inflammatory cytokines in the broncho-alveolar lavage compared to sham-operated animals. Pneumonia increased mortality without affecting brain-infiltrating leukocytes. Conclusions: In this standardized mouse model of post-stroke pneumonia, we describe attenuated leukocyte infiltration and cytokine production in response to bacterial infection in the lungs that has a profound effect on outcome.

Increasing Regulatory T Cells With Interleukin-2 and Interleukin-2 Antibody Complexes Attenuates Lung Inflammation and Heart Failure Progression.

Congestive heart failure (CHF) is associated with an increase of leukocyte infiltration, proinflammatory cytokines, and fibrosis in the heart and lung. Regulatory T cells (Tregs, CD4(+)CD25(+)FoxP3(+)) suppress inflammatory responses in various clinical conditions. We postulated that expansion of Tregs attenuates CHF progression by reducing cardiac and lung inflammation. We investigated the effects of interleukin-2 (IL-2) plus IL-2 monoclonal antibody clone JES6-1 complexes (IL2/JES6-1) on induction of Tregs, transverse aortic constriction-induced cardiac and lung inflammation, and CHF progression in mice. We demonstrated that end-stage CHF caused a massive increase of lung macrophages and T cells, as well as relatively mild left ventricular (LV) leukocyte infiltration. Administration of IL2/JES6-1 caused an ≈6-fold increase of Tregs within CD4(+) T cells in the spleen, lung, and heart of mice. IL2/JES6-1 treatment of mice with existing transverse aortic constriction-induced LV failure markedly reduced lung and right ventricular weight and improved LV ejection fraction and LV end-diastolic pressure. Mechanistically, IL2/JES6-1 treatment significantly increased Tregs; suppressed CD4(+) T-cell accumulation; dramatically attenuated leukocyte infiltration, including decreasing CD45(+) cells, macrophages, CD8(+) T cells, and effector memory CD8(+); and reduced proinflammatory cytokine expressions and fibrosis in the lung of mice. Furthermore, IL2/JES6-1 administered before transverse aortic constriction attenuated the development of LV hypertrophy and dysfunction in mice. Our data indicate that increasing Tregs through administration of IL2/JES6-1 effectively attenuates pulmonary inflammation, right ventricular hypertrophy, and further LV dysfunction in mice with existing LV failure, suggesting that strategies to properly expand Tregs may be useful in reducing CHF progression.

Human adult stem cells derived from adipose tissue and bone marrow attenuate enteric neuropathy in the guinea-pig model of acute colitis.

IntroductionMesenchymal stem cells (MSCs) have been identified as a viable treatment for inflammatory bowel disease (IBD). MSCs derived from bone marrow (BM-MSCs) have predominated in experimental models whereas the majority of clinical trials have used MSCs derived from adipose tissue (AT-MSCs), thus there is little consensus on the optimal tissue source. The therapeutic efficacies of these MSCs are yet to be compared in context of the underlying dysfunction of the enteric nervous system innervating the gastrointestinal tract concomitant with IBD. This study aims to characterise the in vitro properties of MSCs and compare their in vivo therapeutic potential for the treatment of enteric neuropathy associated with intestinal inflammation.MethodsBM-MSCs and AT-MSCs were validated and characterised in vitro. In in vivo experiments, guinea-pigs received either 2,4,6-trinitrobenzene-sulfonate acid (TNBS) for the induction of colitis or sham treatment by enema. MSCs were administered at a dose of 1x106 cells via enema 3 hours after the induction of colitis. Colon tissues were collected 24 and 72 hours after TNBS administration to assess the level of inflammation and damage to the ENS. MSC migration to the myenteric plexus in vivo was elucidated by immunohistochemistry and in vitro using a modified Boyden chamber assay.ResultsCells exhibited multipotency and a typical surface immunophenotype for validation as bona fide MSCs. In vitro characterisation revealed distinct differences in growth kinetics, clonogenicity and cell morphology between MSC types. In vivo, BM-MSCs were comparatively more effective than AT-MSCs in attenuating leukocyte infiltration and neuronal loss in the myenteric plexus. MSCs from both sources equally ameliorated body weight loss, gross morphological damage to the colon, changes in the neurochemical coding of neuronal subpopulations and the reduction in density of extrinsic and intrinsic nerve fibres innervating the colon. MSCs from both sources migrated to the myenteric plexus in in vivo colitis and in an in vitro assay.ConclusionsThese data from in vitro experiments suggest that AT-MSCs are ideal for cellular expansion. However, BM-MSCs were more therapeutic in the treatment of enteric neuropathy and plexitis. These characteristics should be considered when deciding on the MSC tissue source.

Characterization of Haemanthus coccineus extract and its bioactive component: A promising and novel anti-inflammatory approach

Haemanthus coccineus (Amaryllidaceae) extracts (HCEs) have been used in traditional African medicine against febrile colds and asthma. The main ingredient of HCE, the non-basic alkaloid narciclasine, was recently reported to induce apoptosis in tumor cell lines [1]. Beyond this anti-cancer action, we hypothesized that HCE and narciclasine could exhibit an anti-inflammatory potential. Dried bulbs of H. coccineus were extracted using 60% (w/w) ethanol. The ethanol was largely removed and the remaining solution was partitioned with ethyl acetate. The organic phase was separated and dried (DER 50:1). The resulting HCE contained 2.2% narciclasine. In vitro, HCE (3 ng/ml to 10 µg/ml) concentration-dependently inhibited the proliferation of lymphocytes and the synthesis of pro-inflammatory cytokines (TNF-α, IL-6, IL-1 β) in murine macrophages without inducing cytotoxicity. Moreover, HCE decreased the TNF-α-induced adhesion of leukocytes to human endothelial cells (ECs) and the surface expression of EC adhesion molecules (ICAM-1, VCAM-1, E-selectin) without affecting EC viability. Extract fractions containing basic alkaloids did not display any activity, whereas the non-basic main alkaloid narciclasine (1 nM to 10µM) clearly reduced adhesion molecule expression. HCE as well as narciclasine attenuated the TNF-α-triggered expression of NF-κB-dependent genes (reporter gene assay) without influencing NF-κB DNA-binding activity (gel shift assay), IκB-α degradation (Western blot), or p65 nuclear translocation (microscopy). In vivo, HCE (450 mg/kg, orally) was found to clearly reduce edema formation and leukocyte infiltration in a dermal ear edema model by croton oil or arachidonic acid (AA). Also in a renal injury model we could demonstrate that HCE (50 µg/animal, i.p.) strongly attenuates leukocyte infiltration and cytokine expression. In conclusion, our study highlights that the use of HCE/narciclasine could represent a novel interesting anti-inflammatory approach.

Anti-Inflammatory and Analgesic Effects of the Marine-Derived Compound Comaparvin Isolated from the Crinoid Comanthus bennetti

To date, no study has been conducted to explore the bioactivity of the crinoid Comanthus bennetti. Here we report the anti-inflammatory properties of comaparvin (5,8-dihydroxy-10-methoxy-2-propylbenzo[h]chromen-4-one) based on in vivo experiments. Our preliminary screening for anti-inflammatory activity revealed that the crude extract of Comanthus bennetti significantly inhibited the expression of pro-inflammatory proteins in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophage cells. Comaparvin isolated from crinoids significantly decreased the expression of inducible nitric oxide synthase (iNOS) protein and mRNA in LPS-stimulated macrophage cells. Moreover, our results showed that post-treatment with comaparvin significantly inhibited mechanical allodynia, thermal hyperalgesia and weight-bearing deficits in rats with carrageenan-induced inflammation. Comaparvin also attenuated leukocyte infiltration and iNOS protein expression in carrageenan-induced inflamed paws. These results suggest that comaparvin is a potential anti-inflammatory therapeutic agent against inflammatory pain.

The Heme Oxygenase 1 Inducer (CoPP) Protects Human Cardiac Stem Cells against Apoptosis through Activation of the Extracellular Signal-regulated Kinase (ERK)/NRF2 Signaling Pathway and Cytokine Release

Intracoronary delivery of c-kit-positive human cardiac stem cells (hCSCs) is a promising approach to repair the infarcted heart, but it is severely limited by the poor survival of donor cells. Cobalt protoporphyrin (CoPP), a well known heme oxygenase 1 inducer, has been used to promote endogenous CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning hCSCs with CoPP promotes CSC survival. c-kit-positive, lineage-negative hCSCs were isolated from human heart biopsies. Lactate dehydrogenase release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress induced by H(2)O(2), concomitant with up-regulation of heme oxygenase 1, COX-2, and anti-apoptotic proteins (BCL2, BCL2-A1, and MCL-1) and increased phosphorylation of NRF2. Apoptotic cytometric assays showed that pretreatment of CSCs with CoPP enhanced the cells' resistance to apoptosis induced by oxidative stress. Conversely, knocking down HO-1, COX-2, or NRF2 by shRNA gene silencing abrogated the cytoprotective effects of CoPP. Further, preconditioning CSCs with CoPP led to a global increase in release of cytokines, such as EGF, FGFs, colony-stimulating factors, and chemokine ligand. Conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the anti-apoptotic effects of CoPP. Preconditioning CSCs with CoPP also induced an increase in the phosphorylation of Erk1/2, which are known to modulate multiple pro-survival genes. These results potentially provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.

Acute administration of recombinant Angiopoietin-1 ameliorates multiple-organ dysfunction syndrome and improves survival in murine sepsis

IntroductionEndothelial activation leading to vascular barrier breakdown plays an essential role in the pathophysiology of multiple-organ dysfunction syndrome (MODS) in sepsis. Increasing evidence suggests that the function of the vessel-protective factor Angiopoietin-1 (Ang-1), a ligand of the endothelial-specific Tie2 receptor, is inhibited by its antagonist Angiopoietin-2 (Ang-2) during sepsis. In order to reverse the effects of the sepsis-induced suppression of Ang-1 and elevation of Ang-2 we aimed to investigate whether an intravenous injection of recombinant human (rh) Ang-1 protects against MODS in murine sepsis. MethodsPolymicrobiological abdominal sepsis was induced by cecal ligation and puncture (CLP). Mice were treated with either 1μg of intravenous rhAng-1 or control buffer immediately after CLP induction and every 8h thereafter. Sham-operated animals served as time-matched controls. ResultsCompared to buffer-treated controls, rhAng-1 treated septic mice showed significant improvements in several hematologic and biochemical indicators of MODS. Moreover, rhAng-1 stabilized endothelial barrier function, as evidenced by inhibition of protein leakage from lung capillaries into the alveolar compartment. Histological analysis revealed that rhAng-1 treatment attenuated leukocyte infiltration in lungs and kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression in rhAng-1 treated mice. Finally, the protective effects of rhAng-1 treatment were reflected by an improved survival time in a lethal CLP model. ConclusionsIn a clinically relevant murine sepsis model, intravenous rhAng-1 treatment alone is sufficient to significantly improve a variety of sepsis-associated organ dysfunctions and survival time, most likely by preserving endothelial barrier function. Further studies are needed to pave the road for clinical application of this therapy concept.

A Critical Role of Gβγ in Tumorigenesis and Metastasis of Breast Cancer

A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Here we evaluated the role of Gβγ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking Gβγ signaling with Gα(t) or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of Gα(t) in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking Gβγ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that Gβγ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of Gβγ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of Gβγ signaling in promoting breast tumor growth and metastasis and suggest that targeting Gβγ may represent a novel therapeutic approach for breast cancer.

Timing and duration of anti–α4β1 integrin treatment after spinal cord injury: effect on therapeutic efficacy

After spinal cord injury (SCI) leukocytes infiltrate the injured cord, causing significant damage and further impairment of functional recovery. The leukocyte integrin alpha4beta1 is crucial for their entry. The authors previously demonstrated that an anti-alpha4 monoclonal antibody (mAb) treatment attenuates leukocyte infiltration, improves motor and autonomic function, and reduces neuropathic pain when administered at 2 hours and 24 hours after SCI. The authors conducted 2 preclinical studies: the first determined effects of treatment commencing at 6 hours, a clinically relevant time after injury, and the second examined effects of long-lasting treatment (28 days) on neurological recovery after SCI, as current clinically used anti-inflammatory monoclonal antibodies have such longevity. In the first study (timing study), rats were treated with anti-alpha4 or control mAb (intravenously) at 6 hours and 48 hours after moderate (35 g) thoracic compression SCI. Effects on intraspinal inflammation and oxidative injury were assessed at 3 and 7 days after SCI; motor function and pain were examined for 6 weeks. In the second study (duration study), anti-alpha4 mAb was administered starting 2 hours after SCI and subsequently every 3 days for 4 weeks (total of 8 doses), using a schedule of decreasing doses to resemble the pharmacodynamics of long-lasting antibodies used clinically. Motor function and pain were examined for 6 weeks. Lesions were assessed for tissue sparing and inflammation at 6 weeks by histological examination and MR imaging. Anti-alpha4 mAb treatment at 6 hours and 48 hours after SCI (timing study) significantly decreased neutrophil and monocyte/macrophage influx at 3 days by 36% and 20%, respectively, but had no effect by at 7 days after SCI. Antibody treatment significantly reduced intraspinal myeloperoxidase activity by 48% and lipid peroxidation by 27% at 3 days post-injury. The treatment did not improve locomotor function but reduced mechanical allodynia elicited from the trunk and hind paw by ~50% at 3-6 weeks after SCI. In contrast, long-term mAb treatment commencing at 2 hours after SCI (duration study) significantly improved locomotor function at 2-6 weeks after SCI, (mean BBB scores +/- SE: treated rats, 8.3 +/- 0.16; controls, 7.3 +/- 0.2 at 6 weeks). At 3-6 weeks, mAb treatment decreased mechanical allodynia elicited from the trunk and hind paw by ~55%. This recovery correlated with 30% more myelin-containing white matter in treated rats than controls at 6 weeks. The lesion cavity was smaller in the treated rats when assessed by both histological (-37%) and imaging (-50%) methods. The accumulation of ED1-immunoreactive microglia/macrophages at the lesion was similar in treated and control rats. Although delayed treatment reduced intraspinal inflammation and pain, motor function was not improved, revealing decreased efficacy at the more clinically feasibly treatment onset. Long-term anti-alpha4 mAb treatment starting 2 hours after SCI improved neurological outcomes, with tissue sparing near the lesion and no impairment of the late immune response to injury. These findings reveal no disadvantage of long-lasting immunosuppression by the treatment but show that efficacy depends upon very early delivery.

Extracorporeal shock wave treatment modulates skin fibroblast recruitment and leukocyte infiltration for enhancing extended skin‐flap survival

Extracorporeal shock wave (ESW) treatment has a positive effect of rescuing ischemic skin flaps. This study assessed whether ESW treatment rescues the compromised flap tissue by suppressing the apoptosis of ischemic tissue and recruiting tissue remodeling. We used a random-pattern extended dorsal-skin-flap (10 x 3 cm) rodent model. Thirty-six male Sprague-Dawley rats were divided into three groups. Group I, the control group, received no treatment. Group II received one session of ESW treatment (500 impulses at 0.15 mJ/mm(2)) immediately after surgery. Group III received two sessions of ESW treatment, immediately and the day after the surgery. Results indicated that the necrotic area in the flaps in group II was significantly smaller than that of the flaps in group I (p<0.01). Transferase dUTP-nick end labeling (TUNEL) analysis revealed a significant decrease in the number of apoptotic cells in group II. Hydrogen peroxide (H(2)O(2)) expression in circulation blood was significantly decreased in group II on the day after ESW treatment. Immunohistochemical staining indicated that compared with no treatment, ESW treatment could substantially increase proliferating cell nuclear antigen (PCNA), endothelial nitric oxide synthase, and prolyl 4-hydroxylase (rPH) expression, reduce CD45 expression, and suppress 8-hydroxyguanosine (8-OG) expression in the ischemic zone of the flap tissue. In conclusion, ESW treatment administered at an optimal dosage exerts a positive effect of rescuing ischemic extended skin flaps. The mechanisms of action of ESWs involve modulation of oxygen radicals, attenuation of leukocyte infiltration, decrease in tissue apoptosis, and recruitment of skin fibroblasts, which results in increased flap tissue survival.

The effect of C1 inhibitor on intestinal ischemia and reperfusion injury

Complement activation and neutrophil stimulation are two major components in events leading to ischemia and reperfusion (IR) injury. C1 inhibitor (C1INH) inhibits activation of each of the three pathways of complement activation and of the contact system. It is also endowed with anti-inflammatory properties that are independent of protease inhibition. The goal of these studies was to investigate the role and mechanism of C1INH in alleviating IR-induced intestinal injury. C57BL/6, C1INH-deficient (C1INH(-/-)), bradykinin type 2 receptor-deficient (Bk2R(-/-)), and C3-deficient mice (C3(-/-)) were randomized into three groups: sham operated control, IR, and IR + C1INH-treated groups. Ischemia was generated by occlusion of the superior mesenteric artery followed by reperfusion. C1INH or reactive center-cleaved inactive C1INH (iC1INH) was injected intravenously before reperfusion. IR resulted in intestinal injury in C57BL/6, C1INH(-/-), Bk2R(-/-), and C3(-/-) mice with significantly increased neutrophil infiltration into intestinal tissue. In each mouse strain, C1INH treatment reduced intestinal tissue injury and attenuated leukocyte infiltration compared with the untreated IR group. C1INH inhibited leukocyte rolling in the mesenteric veins of both C57BL/6 and C3-deficient mice subjected to IR. C1INH treatment also improved the survival rate of C57BL/6 and C1INH(-/-) mice following IR. Similar findings were observed in the IR animals treated with iC1INH. These studies emphasize the therapeutic benefit of C1INH in preventing intestinal injury caused by IR. In addition to the protective activities mediated via inhibition of the complement system, these studies indicate that C1INH also plays a direct role in suppression of leukocyte transmigration into reperfused tissue.

Cyclic Compressive Loading Facilitates Recovery after Eccentric Exercise

To assess the biologic basis of massage therapies, we developed an experimental approach to mimic Swedish massage and evaluate this approach on recovery from eccentric exercise-induced muscle damage using a well-controlled animal model. Tibialis anterior muscles of six New Zealand White rabbits were subjected to one bout of damaging, eccentric contractions. One muscle was immediately subjected to cyclic compressive loads, and the contralateral served as the exercised control. We found that commencing 30 min of cyclic compressive loading to the muscle, immediately after a bout of eccentric exercise, facilitated recovery of function and attenuated leukocyte infiltration. In addition, fiber necrosis and wet weight of the tissue were also reduced by compressive loading. We conclude that subjecting muscle to compressive loads immediately after exercise leads to an enhanced recovery of muscle function and attenuation of the damaging effects of inflammation in the rabbit model. Although these observations suggest that skeletal muscle responds to cyclic compressive forces similar to those generated by clinical approaches, such as therapeutic massage, further research is needed to assess the translational efficacy of these findings.