AUTOLOGOUS FULL THICKNESS SKIN GRAFT WOUND HEALING ON SPRAGUE DAWLEY RATS Reviewed From TGF-? Expression And Neutrophil Number

Introduction : Skin grafts are now one of treatment option in wound healing process that is always developing. TGF-bexpression and the number of neutrophils have an important role in healing skin graft wounds. Ozone (O3) has disinfecting properties that are effective in wound healing. Objective : Proving the effectiveness of Ozonated VCO for Full Thickness Skin Graft wound healing using parameter of TGF-b expression and neutrophil number. Method : This study is an experimental study with a post-test only design group of 40 Sprague Dawley rats performed autologous skin graft at the same time. Samples were divided randomly into 8 groups (K1 = without Ozonated VCO), (A1 = Ozonated VCO 50 mg / ml), (B1 = Ozonated VCO 100 mg / ml), (C1 = Ozonated VCO 200 mg / ml), ( K2 = without Ozonated VCO) (A2 = Ozonated VCO 50 mg / ml), (B2 = Ozonated VCO 100 mg / ml), (C2 = Ozonated VCO 200 mg / ml). Assessment of TGF-b expression and neutrophil number of tissue was performed by staining hematoxylin & eosin and immunohistochemistry on days 6 and 12 after skin graft. Results : There were significant differences (p <0.05) TGF-b expression and neutrophils number of tissue between the control group and the administration of Ozonated VCO doses of 50 mg / ml, 100 mg / ml and 200 mg / ml on days 6 and 12 post skin graft. Conclusion : The administration of Ozonated VCO effectively improve Full Thickness Skin Graft wound healing seen from macroscopic wounds, increase TGF-b expression and decrease the number of neutrophils.

Osteocytes regulate neutrophil development through IL-19: A potent cytokine for neutropenia treatment

Osteocytes are the most abundant (90-95%) cells in bone and have emerged as an important regulator of hematopoiesis, but their role in neutrophil development and the underlying mechanisms remain unclear. Interleukin (IL)-19 produced predominantly by osteocytes stimulated granulopoiesis and neutrophil formation, which stimulated IL-19 receptor (IL-20Rb)/Stat3 signaling in neutrophil progenitors to promote their expansion and neutrophil formation. Mice with constitutive activation of the mechanistic target of rapamycin complex (mTORC1) signaling in osteocytes (Dmp1-Cre) exhibited a dramatic increase in IL-19 production and promyelocytes/myelocytic expansion, while mTORC1 inactivation in osteocytes reduced IL-19 production and neutrophil number in mice. We showed that IL-19 administration stimulated neutrophil development, while neutralizing endogenous IL-19 or depletion of its receptor inhibited the process. Importantly, low dose IL-19 reversed chemotherapy, irradiation, or chloramphenicol-induced neutropenia in mice more efficiently than G-CSF. These evidence indicated that IL-19 was an essential regulator of neutrophil development and a potent cytokine for neutropenia treatment.

Diabetes-Mediated Toxicity Resulted in the Expression of CD80 and CD86 on Neutrophils after Delayed Wound Healing in Male Rats

Background. Polymorphonuclear neutrophils (PMNs) play an essential role in the innate immune response, and their number increases after prolonged inflammatory diabetic wounds and prolonged wounds in older rats. The expression of CD80 and CD86 on PMNs confirms their participation in acquired immunity, wherein these molecules are involved in antigen presentation. Materials and Methods. We investigated CD80 and CD86 expression on PMNs by flow cytometry and analyzed the mRNA expression of neutrophil chemoattractants macrophage inflammatory protein-2 (MIP-2) and MIP-1α by real-time polymerase chain reaction (PCR) in diabetic wound, which was healed by a camel milk peptide (CMP). The animals were allocated to the following wounded groups: control, diabetic (DM), and diabetic treated with CMP (DM-CMP). Results. Alkaline phosphatase, gamma-glutamyl transpeptidase, and lactate dehydrogenase levels were elevated in DM rats but decreased in peptide-treated rats. The expression of CD80 and CD86 was significantly higher in DM rats with prolonged wounds than in control rats. The expression of both markers was restored to normal levels in diabetic rats treated with CMP. RT-PCR analysis revealed the upregulation in MIP-2 mRNA expression in DM rats. However, neutrophil number at wounded sites of DM rats declined at day 1 after wounding as compared to that in control rats. MIP-2 mRNA expression and neutrophil number were restored in CMP-treated diabetic rats. Conclusion. Prolonged wound stress induced toxicity in DM rats and significantly increased the expression of CD80 and CD86 on PMNs. CMP peptide ameliorated the levels of toxicity markers, CD80 and CD86, and chemoattractant molecules in diabetic rats.

Mesenchymal Stem Cells Promote the Resolution of Cardiac Inflammation After Ischemia Reperfusion Via Enhancing Efferocytosis of Neutrophils

Background Neutrophils play a major role in inflammation after myocardial ischemia‐reperfusion (I/R) injury. The effects of mesenchymal stem cells (MSCs) on neutrophils in I/R are complex and not fully understood. This study was designed to investigate the effects and mechanism of MSCs on alleviating myocardial I/R injury in rats. Methods and Results MSCs induced M2 macrophages polarization in vitro and enhanced macrophage efferocytosis of apoptotic neutrophils, measured by fluorescence‐activated cell sorting analysis and immunofluorescence staining. Rats myocardial I/R were induced by transient ligation of left anterior descending coronary. Adipose‐derived MSCs or vehicle were infused at initiation (immediate after reperfusion) or peak of inflammation (24 hours after I/R). Hematoxylin and eosin, 2,3,5‐triphenyltetrazolium chloride/Evans Blue staining and immunofluorescence staining were applied within 72 hours after cell infusion. Cardiac function was assessed by echocardiography and left cardiac catheterization analysis at 28 days post‐operation. MSCs infused immediately and 24 hours later both markedly ameliorated myocardial I/R injury, and immediate infusion had more significant outcome. These improvements were associated with neutrophils infiltration, measured by fluorescence‐activated cell sorting analysis and immunofluorescence staining. When infused immediately, MSCs did not significantly change neutrophil number at 24 hours but CD11b expression was significantly higher. When infused at 24 hours, MSCs markedly decreased neutrophil number by enhanced M2 macrophage infiltration and macrophage efferocytosis of neutrophils within 72 hours. Conclusions Efferocytosis is pivotal to relieve neutrophil‐mediated I/R injury and initial the immune response for healing. MSCs infusion improves cardiac function in rats after myocardial I/R via the possible mechanism of enhancing M2 macrophages‐induced efferocytosis of apoptotic neutrophils.

Efficacy of antiinflammatory therapy in patients with severe asthma and cold air-provoked bronchial hyperresponsiveness

The aim of this study was to assess effects of antiinflammatory therapy with leukotriene receptor antagonists (LTA) and/or combination of an inhaled corticosteroid (ICS) and a long-acting β2-agonist (LABA) on the clinical course and airway inflammatory patterns in patients with severe asthma and cold air-provoked bronchial hyperresponsiveness. Methods. Asthma symptoms, lung function, and spontaneous sputum cytology were assessed at baseline and after 24 weeks of the therapy. Subgroup analysis was performed for patients with sputum eosinophils < 61% and sputum neutrophils < 61%. Eosinophilic patients were treated with fluticasone propionate/salmeterol, neutrophilic patients with treated with fluticasone propionate/salmeterol plus montelukast during 24 weeks. The control of the disease was assessed using Asthma Control Test (ACT). Results. After 24-wk treatment, eosinophilic patients improves asthma control from 10.9 ± 0.5 to 19.6 ± 1.3 according to ACT questionnaire (р < 0.001), FEV1 improved from 45.9 ± 3.7% pred. to 79.2 ± 2.2% pred. (р < 0.001). Sputum eosinophil number decreased from 27.9 ± 2.1% to 7.1 ± 1.9% (р < 0.001); sputum neutrophil number decreased from 21.1 ± 2.1% to 8.7 ± 2.3% (р < 0.001). In neutrophilic patients, ACT score improved from 8.9 ± 0.6 to 15.9 ± 1.2 (р < 0.001), FEV1 improved from 42.9 ± 2.6% pred. to 72.3 ± 2.5% pred. (р < 0.001). Sputum neutrophil number decreased from 76.8 ± 3.7 to 52.2 ± 4.3 % (р < 0.001); Sputum eosinophil number decreased from 8.1 ± 0.7% to 6.2 ± 0.4% (р < 0.05). After 24 weeks of the treatment, partial control of asthma (ACT 20 -24) was achieved in 63% and 29% of patients in eosinophilic and neutrophilic groups, respectively (χ2 = 1.81; р > 0.05) after treatment. Conclusion. Adding montelukast to the combined therapy with fluticasone propionate/salmeterol in patients with severe asthma, cold air-provoked bronchial hyperresponsiveness and increased sputum neutrophils did not resulted in better control of the disease. The analysis of airway inflammatory pattern could be used as an additional marker to predict treatment efficiency.

Automated High-Throughput Light-Sheet Fluorescence Microscopy of Larval Zebrafish

ABSTRACTLight sheet fluorescence microscopy enables fast, minimally phototoxic, three-dimensional imaging of live specimens, but is currently limited by low throughput and tedious sample preparation. Here, we describe an automated high-throughput light sheet fluorescence microscope in which specimens are positioned by and imaged within a fluidic system integrated with the sheet excitation and detection optics. We demonstrate the ability of the instrument to rapidly examine live specimens with minimal manual intervention by imaging fluorescent neutrophils over a nearly 0.3 mm3 volume in dozens of larval zebrafish. In addition to revealing considerable inter-individual variability in neutrophil number, known previously from labor-intensive methods, three-dimensional imaging allows assessment of the correlation between the bulk measure of total cellular fluorescence and the spatially resolved measure of actual neutrophil number per animal. We suggest that our simple experimental design should considerably expand the scope and impact of light sheet imaging in the life sciences.

NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival

Key Points We demonstrate an important role for NR4A receptors in regulating neutrophil lifespan and homeostasis in vitro and in vivo. These findings may define targets for therapies for diseases driven by defects in neutrophil number and/or survival.