Number Of PMN Research Articles

Abstract We063: CD73 EXPRESSION ON NEUTROPHILS PROTECTS AGAINST CARDIOMYOCYTE DAMAGE DURING INVASIVE STREPTOCOCCUS PNEUMONIAE INFECTION

In recent years, Streptococcus pneumoniae (pneumococcus)-induced cardiac events have emerged as one of the most serious and life-threatening infection outcomes of invasive pneumococcal disease (IPD), leading to major adverse cardiac events (MACE) with high mortality rates. S. pneumoniae has the ability to invade myocardium and damage cardiomyocytes through the release of bacterial factors and formation of bacterial-filled cardiac microlesions. We previously found that polymorphonuclear cells (PMNs) or neutrophils are crucial for host defense against S. pneumoniae lung infection and that extracellular adenosine (EAD) production, by the exonucleosidase CD73, controlled the anti-bacterial functions of these cells. The objective of this study was to explore the role of PMNs and CD73 in cardiac damage during invasive pneumococcal infection. Upon intra-peritoneal ( i.p .) injection with TIGR4, an invasive pneumococcal strain, C57BL/6 mice showed an increased influx of PMNs into the cardiac tissue as determined by flow cytometry. However, the increased PMN numbers failed to contain bacterial burden in the heart and showed a positive correlation with serum levels of the cardiac damage marker Troponin-1 as determined through ELISA. Depletion of PMNs prior infection reduced pneumococcal burden in the heart and lowered the Troponin-1 levels suggesting that PMNs induce cardiac damage during infection. While exploring the mechanisms underlying the detrimental PMN response, we found that the late stage of IPD was associated with reduced CD73 expression on PMNs. The role of CD73 in regulating cardiac damage was tested in vivo using CD73 -/- mice which had significantly higher bacterial burden and cardiac damage compared to wild-type mice despite similar PMN numbers. The role of CD73 expression on PMNs was also tested ex vivo using the HL-1 cardiomyocyte cell line which showed increased LDH release upon S. pneumoniae infection in presence of CD73 -/- PMNs, a phenotype reversed by supplementation of exogenous adenosine. Similar iincreased damage in presence of CD73 -/- PMNs was seen with C57BL/6 Mouse Primary Cardiac Microvascular Endothelial Cells which lead to increased migration of beads across endothelium. Our findings suggest that dysregulation of CD73 on PMNs during invasive pneumococcal infection leads to an inability to control bacterial numbers and increased cardiac damage, and that the EAD-pathway can be a potential pharmacological target to limit disease severity.

The healing effects of moderate exercise on acetic acid-induced gastric ulcer in male rats.

This study aimed to evaluate the effect of moderate exercise on the healing of acetic acid-induced gastric ulcers in male rats. Gastric ulcers include benign mucosal and submucosal lesions of the gastric wall. Exercise regulates a wide range of physiological processes. 48 male Wistar rats were randomly divided into three experimental groups (n=16 per group) as follows: control, which was left untreated after causing stomach ulcers; experimental group 1, the rats were first exercised and then received acetic acid; experimental group 2, the rats received acetic acid, and then exercised. The ulcer was caused by injecting 0.12 ml of a 60% acetic acid solution after 24 hours of not eating. The rats had a period of moderate treadmill activity either before or after the development of ulcers, lasting for a duration of 30 days. On the seventh and fourteenth days after the experiment, the rats were sacrificed, their stomach was removed, and the wound healing parameters, and wound depth were determined. Exercise before and after inducing gastric ulcers significantly decreased the depth of gastric ulcers in the experimental groups. The average number of PMN in the control group decreased in comparison to the seventh and fourteenth days following the experiment. Conversely, the number of fibroblasts, epithelialization, and new vessels increased. It seems that exercise before the appearance of ulcers has a greater effect on gastric ulcers compared to exercise after inducing gastric ulcers. Exercise can prepare the gastric mucosa for forthcoming injuries, and heal gastric ulcers. Moderate aerobic exercise has significant restorative effects on gastric ulcers caused by acetic acid and is recommended.

Influence of sperm viability on uterine leukocyte chemotaxis

The immune system can differentiate live from dead microorganisms, generating a response as necessary; there has been limited study of equine sperm. We aimed to compare the uterine cellular immune response to artificial insemination (AI) with live versus dead equine sperm. Twenty mares in estrus (follicles ≥ 35 mm, uterine edema), without intrauterine fluid or polymorphonuclear leukocytes (PMNs) were selected from a population of horses sent to an abattoir during the breeding season. Their average age was 10.8 ± 0.9 years (range 6 - 20), body condition score > 3 (scale 1 - 5), and they were clinically healthy. Mares were randomly allocated into two groups. Frozen semen, a pool from two stallions, was inseminated into the uterine body: either LIVE (n = 10), 3.0 mL of 900 × 106 sperm (≥ 35% progressive motility), or DEAD (n = 10), 3.0 mL of 900 × 106 sperm killed by snap freezing in liquid nitrogen. Four hours post AI, mares were slaughtered; ex vivo uteri were subjected to a low-volume lavage (LVL) with 50 mL of PBS and three endometrial tissue samples were collected (both uterine horns and uterine body). The fluid recovered was analyzed in a Neubauer chamber. Endometrial samples were processed for histologicalevaluation using bright-field microscopy. For each mare, the three uterine samples were pooled for analysis. The number of PMNs in the stratum compactum (SC) and spongiosum (SS) was counted in 5 fields per uterine sample at 400x magnification, totaling 15 fields/stratum/mare. Groups were considered independent factors. Leukocyte concentrations in the LVL and PMN presence in both strata were considered dependent factors. Leukocyte concentrations in the LVL were analyzed with a Mann-Whitney test (median ± IQR) and the number of PMNs per field of each stratum using a t-test (mean ± SEM). The significance level was defined as p ≤ 0.05. Mares in the DEAD group presented a lower concentration of leukocytes in the LVL than in the LIVE group (33.2 × 106/mL ± 62.6 versus 63.2 × 106/mL ± 56.5; p = 0.05). Correspondingly, the mean PMN number per field in the SC (50.5 ± 5.2/field versus 69.0 ± 5.3/field; p = 0.02) and SS (32.8 ± 6.2/field versus 54.4 ± 5.7/field; p = 0.02) of mares in the DEAD group were lower than mares in the LIVE group. In conclusion, dead sperm elicited a less intense inflammatory response 4 hours after AI compared to live sperm, suggesting that as with bacteria, the immune system may differentiate sperm according to viability.

Increase in the level of oral neutrophils with gingival inflammation – A population survey

ObjectivesHost responses to oral inflammation include a continuous and substantive response with the influx of polymorphonuclear leukocytes (PMN). PMN, referred to as first responders, migrate rapidly from the circulatory system through the connective tissue to mitigate stimuli and localize in the saliva. This study examined the relationship between the well-established clinical indices of gingivitis and dental plaque and the PMN level. Materials and MethodsThis study enrolled adults aged 18–75 years, who provided voluntary informed consent. Oral rinse samples were collected from 159 participants to estimate the PMN levels prior to the full-mouth assessment for gingivitis and dental plaque using the respective clinical indices. ResultsThe gingival index and dental plaque index scores were in the range of 0.098–2.71 and 0.73–4.78, respectively. Regardless of the age and gender, higher number of PMN was observed with higher gingival index and dental plaque index scores. Our analyses indicated a significant correlation between the oral PMN level and gingival index with a correlation coefficient of 0.66 (p < 0.0001). Similarly, the correlation between the PMN level and dental plaque index was statistically significant with a correlation coefficient of 0.57 (p < 0.0001). Regression analysis identified a significant relationship between the PMN level and clinical indices (p < 0.0005). ConclusionsIncrease in the PMN levels with increasing clinical scores (gingival and dental plaque indices) reflect the oral inflammatory burden, irrespective of age or gender. These observations warrant future studies on participants stratified by health status and research directed toward examining the effects of interventions.

Study on the effect of medicinal leech therapy (Hirudo medicinalis) on full-thickness excisional wound healing in the animal model

The possible impacts of alternative and conventional medicines on wound healing are now of growing interest. This study aimed to evaluate and elucidate the wound healing activity of medicinal leech therapy in wound excision of the rat model. After a round, full-thickness excision was made in the dorsal region of the body, the animals (n = 30) were randomly divided into three equal groups: I) the treatment group (MLT), where the wounds received leech treatment; II) the positive control group (PC), where the wounds received 1% sodium phenytoin treatment; and III) the negative control group (NC), where the wounds did not receive any treatment. On days 6 and 16, wound biopsy specimens were taken, and prepared sections were stained using various methods. The contraction rate differed significantly (P < 0.05) between the NC group and the other groups. The histopathological evaluation revealed that MLT group showed an accelerated healing process and lower inflammatory response compared to other groups. In ML-treated group maturation and remodeling of collagen had occurred, while in 1% sodium phenytoin treated group, proliferation was the prominent feature. Results showed that the fibroblast was significantly lower in the NC group in comparison to other groups. The number of MNC, s, and PMN, s was significantly higher in the NC group compared to other groups (P < 0.0001). In our study, medicinal leech therapy had a higher success rate in healing for the treatment of excisional wounds in animal models.

Uterine Inflammatory Response After Prostaglandin E1 (Misoprostol) Infusion Prebreeding or Immediately After Embryo Flushing in Commercial Donor Mares

Misoprostol, a synthetic PGE1, is becoming a common therapy for mares with suspected uterine tube obstruction. Recently, there have been concerns that uterine administration of misoprostol induces exacerbated uterine inflammation; however, this has not been critically evaluated. This study aimed to assess the inflammatory response and potential systemic reactions after uterine administration of misoprostol, either during prebreeding or immediately after postembryo flushing. Privately owned embryo donor mares (n = 11) were randomly assigned in a crossover design to receive misoprostol (3 mL +200 µg) or sham (3 mL of lactate Ringer's solution) infusions, bilaterally deposited via deep-horn, at least 72 hours prebreeding (experiment 1) or immediately after embryo flushing (experiment 2). Each mare had one cycle for misoprostol and sham in both experiments and a breeding cycle (no sham or misoprostol) between experiments. Uterine edema, fluid accumulation, and the number of uterine PMN were assessed before each infusion and then daily for 72 hours. Uterine lavage was performed the day after each infusion across groups and experiments. Ovulation was hastened with a GnRH agonist and confirmed at 24 hour-intervals. Mares were bred with semen from one of six stallions per owner's choice. Embryo flushing was performed 8 to 9 days postovulation. In either experiment, misoprostol did not affect uterine edema or fluid accumulation (P > .05). However, both the sham and misoprostol infusions increased the number of PMN up to 48 hours postinfusion in both experiments. Embryo recoveries were similar between sham (45%, 5/11) and misoprostol cycles in experiments 1 (45%, 5/11; P > .05) and 2 (sham, 68%, 7/11; misoprostol, 45%, 5/11; P > .05). In conclusion, misoprostol did not induce exacerbated uterine inflammation in mares or systemic adverse reactions when infused prebreeding or immediately after embryo flushing.

SAFETY OF UTERINE LAVAGE WITH OZONATED SALINE IN MARES

This study aimed to assess the safety of uterine lavage using ozonated saline in mares. Each liter of saline (NaCl 0.9%) was ozonated at 50 µg of ozone per mL for 7 min using a medical ozone generator. Then, uterine lavage was performed during estrus or 4h post-breeding and each liter of saline remained inside the uterus for 10 min before being recovered. In experiment 1, two cycles of six mares were used in a crossover design. Lavage was performed with ozonated (Treated) or non-ozonated (Control) saline 4h after the infusion of dead sperm, and cytology samples and biopsies were collected 6 and 24h later. Experiment 2, endometrial biopsies were collected before lavage (T0) and at 15 (T15), 30 (T30), and 60 (T60) days post-treatment with ozonated (n=11) or non-ozonated (n=3) saline. In experiment 3 (n=10), the pregnancy outcome was evaluated subsequent to lavage with ozonated saline 4h after insemination. In Experiment 1, there were no difference at 6h post-breeding in PMNs count on endometrial cytology between treatment and control groups (P&gt;0.05). However, PMNs tended (P=0.09) to be superior in the experimental treatment compared to the control at 24h post-breeding. In addition, the number of PMNs tended to be lower in the control cycle at 6 h (P=0.06) and 24 h (P=0.08) in endometrial biopsies. In Experiment 2, the endometrial architecture, the numbers of lymphocytes and PMN were not affected by the treatment (P&gt;0.05). In Experiment 3, nine out of 10 mares were confirmed to be pregnant after treatment, and there was not pregnancy loss up to 60 days. In conclusion, uterine lavage with ozonante saline solution may cause a mild endometrial inflammation, however, doesn’t harm the endometrium or impact pregnancy outcomes in healthy mares.

In vivo interferon-gamma induced changes in gene expression dramatically alter neutrophil phenotype.

The cytokine Interferon-γ (IFN-γ) exerts powerful immunoregulatory effects on the adaptive immune system and also enhances functions of the neutrophil (PMN). The clinical use of IFN-γ has been driven by the finding that its administration to patients with chronic granulomatous disease (CGD) results in decreased incidence and severity of infections. However, IFN-γ has no effect on the characteristic defect of CGD, the inability to convert oxygen to microbicidal metabolites including superoxide anion (O2-) during the phagocytosis associated oxidative burst. We administered varying doses of IFN-γ to adult volunteers and studied the effects on plasma drug levels and response molecules and PMNs isolated from blood drawn at intervals over a 96- hour period. Plasma concentrations of IFN-γ, IP-10 and neopterin, and stimulated release of O2- from PMNs exhibited dose- and time-dependent increases after IFN-γ administration. Gene expression in PMNs was altered for 2775 genes; changes occurred rapidly after administration and returned to baseline in 24–36 hours. Several genes involved with neutrophil host defense were upregulated including those for components of the O2- generating NADPH oxidase; innate-immune and Fc receptors; proteins involved in MHCI and II; a regulator of circulating PMN number; guanylate binding proteins; and a key enzyme in synthesis of an essential NOS cofactor. Coordinate changes were detected in protein levels of representative products from several of these genes. Lysates from isolated neutrophils also demonstrated a spike in NO following IFN-γ administration. IFN-γ appears to increase non-oxygen dependent microbicidal functions of PMNs which could provide strategies to compensate for deficiencies, explain its clinical benefit for CGD patients and expand therapeutic applications of IFN-γ to other disorders.Trial registration: Protocol registered in ClinicalTrials.gov, NCT02609932, Effect of IFN-γ on Innate Immune Cells.

The Determinative Role of Hematopoietic Stem and Progenitor Cell Graft Composition on the Engraftment Kinetics after HSCT

The importance of hematopoietic stem and progenitor cell (HSPC) dose in the outcome of hematopoietic stem cell transplant (HSCT) has been demonstrated by analyses of the threshold dose of CD34+ cells required to achieve donor engraftment, usually defined as endpoints of donor-derived neutrophil (PMN) or platelet (PLT) absolute numbers, and RBC transfusion independence. We recently described the heterogeneous HSPC composition of prospectively obtained umbilical cord blood samples and lack of correlation between CD34+ cell dose and the frequency of hematopoietic stem cells (HSC)(Mantri S et al, Blood Adv 2020).We describe here a pilot analysis of the relationship between HSPC graft composition and engraftment after HSCT. The study population is comprised of 17 children (3.4-22 years, median 13 years) treated with αβT/CD19B - cell depleted mobilized peripheral blood stem cell (PBSC) HSCT (αβhaplo-HSCT). Eight patients had acute lymphoblastic leukemia, seven had acute myeloid leukemia, and two myelodysplastic syndrome. All patients received serotherapy with Thymoglobulin and Rituximab, and a myeloablative conditioning consisting of either TBI 1200 cGy in combination with Fludarabine/Thiotepa (15 patients), Melphalan/Thiotepa (1 patient), or TBI (400 cGy) with Fludarabine/Thiotepa (1 patient receiving a second HSCT). Only 2/17 patients received 1-2 doses of G-CSF before Day+30.The CD34+ Lin- cells in the PBSC products were analyzed for HSPC composition, including HSC (CD38- CD90+ CD45RA-), Multipotent Progenitors (MPP, CD38- CD90- CD45RA-), Common Myeloid Progenitors (CMP, CD38+ CD123+ CD45RA-), Granulocyte-Monocyte Progenitors (GMP, CD38+ CD123+ CD45RA+), Megakaryocyte-Erythroid Progenitors (MEP, CD38+ CD123- CD45RA-) and Common Lymphoid Progenitors (CLP, CD38+ CD127+). Similar HSPC analyses were performed on marrow obtained at Days +30, +60, and +90 post-HSCT. The CD34+ cell dose in the grafts ranged from 8.5-40 x 10e6/kg recipient body weight (mean 15.6). The median time to Absolute Neutrophil Count (ANC) > 500 or 1000/mm 3 were Days +12 and +14, respectively, and for PLT > 50K or 100K/mm 3 Days +14 and +15, respectively. The time to either PMN or PLT engraftment did not correlate with the HSC dose. In contrast, the GMP dose was predictive of PMN engraftment: 7/8 patients receiving the highest GMP dose achieved ANC > 500 at 0-3 days before the median day of engraftment, while PMN engraftment was delayed by 1-5 days beyond the median in 6 of the 9 receiving the lowest GMP dose (χ 2 = 8.14, p=0.004)(Fig A). Of the patients with the highest MEP dose, 7/8 achieved PLT >50K/mm 3 0-4 days before the median, while 5/9 receiving the lowest dose engrafted 1-6 days beyond the median (χ 2 4.9, p=0.026) (Fig B). In the first 100 days post-HSCT, naïve CD4+ T-cells were all CD31+ CD45RA+ Recent Thymic Emigrants (RTE), indicating that they were newly produced T cells and not adoptively transferred from the αβhaplo-HSCT. The HSC dose did not correlate with the number of naïve CD4+ T cells until Day+180 (Fig C). Like PMN and PLT recovery, early T lymphoid recovery after HSCT was mainly derived from infused CLP, and likely switched to HSC-derived lymphopoiesis by Day +180. Consistent with this concept, HSC dose in the PBSC products was unrelated to the numbers of committed progenitors, e.g., CMP, MEP, CLP, in early (Day +30) post-transplant marrow samples.These data are consistent with clonal analyses of patients receiving lentiviral gene therapy and murine experiments demonstrating prolonged steady-state contribution of committed progenitors to peripheral blood cell maintenance (Biasco L et al, Cell Stem Cell 2016; Sun J et al, Nature 2014). While post-HSCT PMN or PLT numbers are frequently equated with “HSC engraftment” and naïve T-cell numbers with HSC-derived immune reconstitution, these early events reflect blood cell production by committed progenitors, which are variably present in the grafts. Although CD34+ cell dose is currently used to predict post-transplant engraftment and to qualify stem cell products for release, more accurate clinical predictions may be determined by the HSPC grafts' composition. Further, engineering of the progenitor composition of clinical HSCT products, e.g., co-transplantation of additional committed progenitors like GMP or CLP, could be strategically used to control post-transplant lymphohematopoietic recovery. [Display omitted] DisclosuresParkman: Jasper Biotech: Consultancy. Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

Swine spermatozoa trigger aggregated neutrophil extracellular traps leading to adverse effects on sperm function

In pigs, the number of PMN in uterus lumen increases within few hours after natural or artificial AI resulting in early PMN-derived innate immune reactions. Sperm-NETs formation was recently reported to occur in various mammalian species. Aim of this study was to investigate direct interactions of boar spermatozoa with swine PMN, the release of sperm-mediated NETs, and to assess NET-derived effects on sperm functionality. Sperm-triggered NETs were visualized by SEM- and immunofluorescence analyses. Sperm-mediated NETosis was confirmed by presence of extruded DNA with global histones and NE. Largest sizes of sperm-mediated aggNETs were detected after 5 h thereby resulting in effective massive sperm entrapment. The number of aggNETs increased from 3 h onwards. Kinetic studies of swine sperm-mediated NETosis showed to be a time-dependent cellular process. In addition, number of NETs-entrapped spermatozoa increased at 3 h of exposure whilst few free spermatozoa were detected after 3 h. Anchored NETs also increased from 3 h onwards. The cytotoxicity of NETs was confirmed by diminution of the total motility and the progressive motility. Spermatozoa membrane integrity and function loss exposed to NETs was confirmed from 3 h. Experiments revealed NETs-derived damaging effects on swine spermatozoa in membrane integrity, motility and functionality. We hypothesize that swine sperm-triggered aggNETs might play a critical role in reduced fertility potential in swine reproductive technique. Thus, aggNETs formation needs to be considered in future studies about uterine environment as well as advance of sperm in the porcine female reproductive tract.

Comparison between PRP and PRFM on FTSG healing profile: Macroscopic, microscopic and ELISA evaluation

BackgroundStudies had shown the benefit of PRFM and PRP in wound healing but their use in skin graft healing was rarely studied. This study aims to compare the use of PRP and PRFM in accelerating wound healing process of skin full-thickness skin graft (FTSG). Materials and methodsFive pigs were used to look at the wound healing effect of PRP and PRFM usage prior to FTSG implantation. Subsequent punch biopsies were then conducted on the 1st, 3rd, 7th, 14th, and 30th day to obtain samples for macroscopic (skin color), extracellular matrix (collagen), microscopic (PMN, macrophage, and fibroblast), and ELISA (TGFβ1 and PDGF) analysis to determine the level of wound healing activity. ImageJ software was used to photograph for macroscopic and extracellular matrix analysis. ResultsMacroscopic, extracellular matrix, and ELISA evaluation show no significant difference in FTSG survival rates for all treatment groups. Microscopic examination showed an increase in PMN, macrophage, and fibroblast levels with PRFM application showing higher increases in all observed microscopic variables compared to PRP and control. ConclusionThis study observed that both PRFM and PRP as autologous platelet preparation accelerate wound healing in FTSG, with PRFM being superior due to the higher number of PMN, macrophage, and fibroblast.

Coded Computing for Resilient, Secure, and Privacy-Preserving Distributed Matrix Multiplication

Coded computing is a new framework to address fundamental issues in large scale distributed computing, by injecting structured randomness and redundancy. We first provide an overview of coded computing and summarize some recent advances. Then we focus on distributed matrix multiplication and consider a common scenario where each worker is assigned a fraction of the multiplication task. In particular, by partitioning two input matrices into m-by-p and p-by-n subblocks, a single multiplication task can be viewed as computing linear combinations of pmn submatrix products, which can be assigned to pmn workers. Such block-partitioning-based designs have been widely studied under the topics of secure, private, and batch computation, where the state of the arts all require computing at least “cubic” (pmn) number of submatrix multiplications. Entangled polynomial codes, first presented for straggler mitigation, provides a powerful method for breaking the cubic barrier. It achieves a subcubic recovery threshold, i.e., recovering the final product from any subset of multiplication results with a size order-wise smaller than pmn. We show that entangled polynomial codes can be further extended to also include these three important settings, providing unified frameworks that order-wise reduce the total computational costs by achieving subcubic recovery thresholds.

NADPH Oxidase Controls Neutrophil Infiltration in the Lung in Early Response to Fungal Cell Walls Via LTB4

The leukocyte NADPH oxidase generates superoxide, the precursor to reactive oxygen species important for host defense and immunoregulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by serious bacterial and fungal infections and aberrant inflammation. Aspergillus pneumonia is frequent, and associated with pyogranulomatous inflammation. Of note, even sterile fungal cell walls elicit increased neutrophilic inflammation in CGD mice, demonstrating that that NADPH oxidase can limit inflammation independent of its antimicrobial effects. Leukotriene B4 (LTB4) is a potent inflammatory mediator, acting as a chemoattractant and activating other polymorphonuclear leukocyte (PMN) functions. PMN themselves are prominent source of LTB4, and sense LTB4 through its receptor BLT1, which stimulates additional LTB4 synthesis by autocrine and paracrine routes. This leads to feed forward amplification of LTB4 production locally and formation of PMN clusters. Ca2+ acts as an important second messenger in PMN, including regulation of LTB4 synthesis. Activated human CGD PMN have increased intracellular Ca2+ levels compared to PMN from healthy donors due to electrogenic effects of the NADPH oxidase (Geiszt M. et al JBC 1997). Intracellular Ca2+ overload in PMN is speculated to contribute to increased PMN pro-inflammatory activity, but specific pathways and in vivo relevance are not well defined. Therefore, we therefore investigated the production of LTB4 by murine CGD PMN and the role of LTB4 in PMN recruitment and lung hyperinflammation following pulmonary challenge with zymosan, a sterile fungal cell wall preparation. Zymosan-stimulated PMN from Cybb-null mice, a model for X-linked CGD, produced higher LTB4 in vitro compared to wild type (WT), even in the presence of SOD and catalase to counteract oxidative degradation. Higher LTB4 production correlated with higher levels of intracellular Ca2+ in stimulated X-CGD PMN. The increased LTB4 produced by X-CGD PMN was dependent on cell density and the LTB4 receptor BLT1, consistent with a feed-forward loop that amplified LTB4 production. Zymosan-stimulated X-CGD PMN also formed larger and more numerous clusters in vitro compared to WT PMN. Cluster formation was abrogated by inhibiting LTB4 synthesis with the 5-lipoxygenase (5-LO) inhibitor zileuton or by the BLT1 receptor antagonist U75302, demonstrating that cluster formation was LTB4 and BLT1 dependent. We next examined whether LTB4 regulated the response to zymosan in the lung. Following intranasal administration of zymosan, X-CGD mice had higher PMN numbers in bronchoalveolar lavage (BAL), larger PMN foci by lung histology, and increased lung LTB4 as compared to WT mice. These differences were evident by 8 hr and progressed over the first 24 hr post-challenge, in contrast to WT mice where inflammation plateaued at 8 hr. Treatment with zileuton or U75302 just prior to zymosan significantly reduced lung PMN numbers for both WT and X-CGD mice at 8 hr post challenge, and lung PMN remained substantially reduced in X-CGD mice at 24 hrs. Moreover, delaying administration of zileuton to 8 hr after zymosan challenge also significantly decreased PMN inflammation in X-CGD but not WT mice at 24 hrs, indicating that ongoing synthesis of LTB4 otherwise continued to promote PMN recruitment in CGD mice. These results demonstrate that PMN production of LTB4 in response to fungal cell walls is limited by NADPH oxidase via its effects on intracellular Ca2+. In oxidase-deficient CGD mice, LTB4 plays a major role in driving excessive PMN recruitment to the lung in the early response to zymosan. This study is the first to implicate LTB4 in promoting neutrophilic lung inflammation in response to fungal cell walls in CGD, likely by an amplified feed-forward loop involving increased production of LTB4 by CGD PMN. LTB4 could be a potential therapeutic target to ameliorate CGD hyperinflammation. Disclosures No relevant conflicts of interest to declare.

Wound healing acceleration in inflammation phase of post-tooth extraction after aerobic and anaerobic exercise

Summary Background Tooth extraction leads a wound healing that is expected to be physiologically healed in a short time. Both aerobic and anaerobic physical exercises are scientifically proven to enhance various biological functions as well as to accelerate the wound healing in oral mucosa. Aerobic exercise increases tissue oxygenation, accelerates the wound healing and this is different to anaerobic exercise which is considered to produce more free radicals and interferes the wound healing process. Purpose To observe the differences of wound healing effectiveness in the inflammatory phase after tooth extraction based on the number of PMN and macrophage cells in Wistar rats (Rattus norvegicus) that received aerobic and anaerobic exercise. Methods Forty-two rats were divided into three groups: control group (K), aerobic exercise group (P1), and anaerobic exercise group (P2). In each group the number of PMN cells was calculated one day after tooth extraction and the counting of the macrophage cells were performed on the other groups two days later. Data was analyzed statistically using Kruskal Wallis, Levene's test, and proceed with Oneway Anova. Results The highest number of PMN and macrophage cells was obtained in P1 group and the lowest results were in K group. The data between groups demonstrated significant differences. Conclusion This study represents significant differences in the number of PMN and macrophage cells between aerobic and anaerobic exercises in wound healing after tooth extraction which can be a strong indicator of wound healing acceleration in the inflammatory phase.

Monocyte-derived extracellular trap (MET) formation induces aggregation and affects motility of human spermatozoa in vitro

ABSTRACTThe presence of bacteria and/or leukocytes can alter semen quality resulting in low sperm quality and infertility. Inflammation or infection increases the numbers of PMN or macrophages/monocytes in male genital tract. Release of extracellular traps (ETs) by leukocytes has been recognized as a novel mechanism of early host innate immunity, in response to invasive pathogens. This is the first work that evaluated the mechanism of triggered ETs in monocytes co-incubated with spermatozoa or bacteria and the effect on sperm function. Selected spermatozoa and human monocytes isolated from peripheral blood were obtained by healthy donors. Two experimental models were developed, one aseptic (non-infectious) incubating spermatozoa and monocytes, and septic models (infectious) incubating spermatozoa with monocytes and uropathogenic Escherichia coli (E. coli). ETs of monocytes (METs) (DNA, global histone and citrullinated histones) were visualized by scanning electron microscopy (SEM) and immunofluorescence analyses. Progressive motility was performed at 0, 10, 30, 60, and 180 min after co-incubation with CASA system. SEM- and immunofluorescence-analyses revealed human spermatozoa alone or in the presence of E. coli as strong inducers METs. In aseptic model, the motility decreased to 65.2 ± 3.5% at 10 min of incubation and 29.3 ± 3.3% at 30 min (p < 0.001). In septic model, motility decreased to 44.5 ± 5.9% (10 min) and 12.7 ± 2.2% (30 min) (p < 0.001). MET-derived small spermatozoa aggregations were observed in both models. METs might physically block spermatozoa and decrease motility after a brief contact. This may impair male fertility, especially in patients with genital tract infections or chronic inflammation.Abbreviations: PMN: polymorphonuclear; ETs: extracellular traps; E. coli: Escherichia coli; METs: ETs of monocytes; SEM: scanning electron microscopy; NE: neutrophil elastase; MPO: myeloperoxidase; MAGI: male accessory gland infection; PBMC: peripheral blood mononuclear cells; RT: room temperature; CFU: colony forming units; CASA: computer-aided sperm analysis; H4Cit3: histone H4 citrullinated 3

Extract effect of Tintir (Jathropa Multifida Linn) to Total PMN and Selt Mast in Wound Healing Model Rats with Acute Injury

Wound is considered as a serious health problem, because it can interfere with quality of life due to pain, weakness, and decreased mobility and quality of life. This study aims to determine the effect ofj jathropha multifida, linn to the number of PMN and selt mast in the process of wound healing in rats with acute injury models. This study is an experimental research laboratory using the design of post test control group design Wistar rats were used as research subjects. Based on Tukey HSD test results, the average number of PMN leukocytes third day the negative control group had no significant difference in the positive control group (0.037) and treatment group (0004). On the seventh day the number of PMN leukocytes negative control group had no significant difference in the positive control group (0.069) and had a significant difference to the treatment group (0.00). Meanwhile, on the fourteenth day the number of PMN leukocytes negative control group had no significant difference in the positive control group (0.000) and had a significant difference to the treatment group (0.00). Based on the results of data analysis Annova one direction on the third day obtained a significant level of 0:50 means there is no significant difference in the number of mast cells in each group on the seventh day. While on day three significant values ​​of 0:08 was also no significant difference, as well as on the fourteenth day found no significant difference between the group with a value of 0.68.&#x0D; Conclusions there was a decrease in the number of Leukocyte PMN in rat wounds given a 10% stem extract ointment, and there was a decrease in the number of mast cells in the wounds of rodents given a 10% stem extract ointment. Suggestions need to be investigated regarding the use of distance stem extract in various concentrations to obtain effective results for wound healing and the need to examine other factors related to wound healing.

12/15-lipoxygenase supports inflammatory cytokine production and PMN recruitment during invasive aspergillosis

Abstract Eicosanoids are a family of inflammatory lipid mediators produced via the metabolism of arachidonic acid by a network of biosynthetic enzymes. We and others have recently shown that pathways within this network, specifically the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) pathways, participate in induction of protective immune responses during invasive aspergillosis (IA). Based on the high degree of cooperation and interconnection within the eicosanoid network, we hypothesized that a third core enzyme, 12/15-lipoxygenase (12/15-LOX), also may support innate antifungal responses. We report here that mice deficient in the gene encoding 12/15-LOX, Alox15, exhibit decreased survival and increased fungal burden in a model of IA. These defects were associated with decreased PMN numbers in the lung and decreased levels of PMN chemokines (CXCL1, CXCL2, CCL3) in Alox15−/− mice, while fungal killing by PMNs in vitro was unaffected. Alox15−/− mice produced lower levels of the inflammatory cytokines IL-17A, IL-22, IL-1α and IL-6 during infection. iNKT cells, which we have reported to be a major cell source of IL-22 during IA, were present in decreased numbers in Alox15−/− mice. This reduction was associated with lower levels of IL-7, a cytokine we have recently shown to be required for optimal numbers of iNKT cells during IA. Decreased IL-22 levels were further associated with decreased levels of IL-23. Together, these data indicate that 12/15-LOX is a critical player in induction of multiple aspects of the innate immune response to A. fumigatus.

514 Effect of Topical Platelet Rich Plasma on Burn Healing After Partial Thickness Burn Injury

Burn injury has direct impact on local and systemic changes. Many modalities for wound bed preparation was not satisfied. One of the most progressive dressing nowadays is PRP. PRP induces the synthesis of collagen with fibroblast. PRP accelerates wound healing and decrease the use of skin graft. Sixty Wistar albino rats were divided into 3 groups of 20 rats each. Group 1 (platelet-rich plasma group) was exposed to burn injury and topical platelet-rich plasma was applied. Group 2 (control group) was exposed to burn injury and normal saline was applied. Group 3 (blood donor group) was used as blood donors for platelet-rich plasma. Wound was excised on the fourteenth day then fixated in 10% formaldehyde. Specimen was then embedded in parafin block and stained with HE to observe the number of PMN and Masson’s Trichrome to observe collagen deposition. Collagen deposition was found significantly higher in Group 1 (platelet-rich plasma group) (P<0.05). The number of PMN is significantly less in Group 1 (P<0.05) meaning topical PRP also has anti-inflammatory effect. PRP is proven to accelerate burn healing after partial thickness burn injury by increasing the number of collagen deposition also having anti-inflammatory effect shown by the significantly less number of PMN in PRP group. PRP as a new method of burn wound dressing may be able to accelerate wound healing. In large burn area, PRP might help in decreasing the use of skin graft.

Neutrophil isolation from feline blood using discontinuous Percoll dilutions.

Some studies have performed in vitro neutrophil isolation from feline blood. The major limiting factor for these studies is the small volume of blood that can be collected without development of potentially life-threatening complications. In the present study we attempted neutrophil isolation from feline venous blood samples using discontinuous Percoll gradients. Blood was collected from the cephalic vein of clinically healthy adult cats. The blood samples were layered on Percoll dilutions (72 %, 63 %, 54 % and 45 %). After centrifugation, the feline polymorphonuclear leukocytes (PMN) accumulated as a band between 72-63 % Percoll dilutions. The total cell count was calculated using light microscopy counts. The percentage of the neutrophils was determined microscopically after staining with Diff-Quik stain. Neutrophil viability was evaluated with a 0.01 % Trypan blue assay. The activation was determined based on intact cell morphology in the isolated neutrophils. The mean PMN number was 22 x 105 per ml (minimum - maximum: 20-26 x 105/ml). Neutrophil homogeneity was > 95 % in the cell suspensions. The viability of isolated neutrophils was > 98 %. The technique did not result in neutrophil activation. Discontinuous Percoll gradients (72 %, 63 %, 54 % and 45 %) can be used to isolate neutrophils from blood samples of cats. The technique was simple to perform and neutrophil activation was minimal.

Pro-resolving mediators promote resolution in a human skin model of UV-killed Escherichia coli-driven acute inflammation.

While the treatment of inflammatory disorders is generally based on inhibiting factors that drive onset of inflammation, these therapies can compromise healing (NSAIDs) or dampen immunity against infections (biologics). In search of new antiinflammatories, efforts have focused on harnessing endogenous pathways that drive resolution of inflammation for therapeutic gain. Identification of specialized pro-resolving mediators (SPMs) (lipoxins, resolvins, protectins, maresins) as effector molecules of resolution has shown promise in this regard. However, their action on inflammatory resolution in humans is unknown. Here, we demonstrate using a model of UV-killed Escherichia coli–triggered skin inflammation that SPMs are biosynthesized at the local site at the start of resolution, coinciding with the expression of receptors that transduce their actions. These include receptors for lipoxin A4 (ALX/FPR2), resolvin E1 (ChemR23), resolvin D2 (GPR18), and resolvin D1 (GPR32) that were differentially expressed on the endothelium and infiltrating leukocytes. Administering SPMs into the inflamed site 4 hours after bacterial injection caused a reduction in PMN numbers over the ensuing 6 hours, the phase of active resolution in this model. These results indicate that in humans, the appearance of SPMs and their receptors is associated with the beginning of inflammatory resolution and that their therapeutic supplementation enhanced the resolution response.