Neutrophil activation and neutrophil extracellular traps (NETs) have been associated with the pathogenesis of venous thromboembolism (VTE). Considering VTE-associated chronic sequelae, which suggest that some pathological mechanisms remain after the acute episode, we investigated whether neutrophil activation is increased in patients with a prior VTE at least one year before this investigation. Thirty-seven patients with prior VTE and 37 individuals with no history of VTE were included. Neutrophil activity was evaluated by the expression of the adhesive molecule activation-specific epitopes LFA-1 (CD11a) and MAC-1 (CD11b), chemotaxis, reactive oxygen species (ROS) and by MPO-DNA complexes as markers of NETs. The adhesive molecules sICAM-1 and sVCAM-1, involved in the cross talk between neutrophil and endothelial cells, were also evaluated. Patient neutrophils presented increased CD11a expression before and after TNF-α stimulus, whereas increased CD11b expression was observed only after TNF-α stimulus, as compared to controls. Neutrophil chemotaxis on both, basal state and after IL-8 stimulus, on circulating levels of sICAM-1 and sVCAM-1, and on MPO-DNA complexes were also increased in VTE patients. ROS release was similar between patients and controls. This is, to our knowledge, the first study to investigate neutrophil inflammatory activity in VTE patients a long period after an acute event (approximately 2 years). The results showed altered neutrophil activation patterns in these patients. While activated neutrophils can cause endothelial activation and injury, the activated endothelium can induce the release of NETs with consequent endothelial cytotoxicity, creating a vicious cycle of activation between neutrophils and endothelium that can lead to thrombosis. Graphical abstract VTE patients (approximately 2 years after the clinical event) present an altered neutrophil activation state evidenced by increased activity of the LFA-1 and Mac-1 adhesive molecules, as well as increased chemotaxis and circulating levels of NETs remnants. Circulating levels of ICAM-1 and VCAM-1, which are endothelial adhesive molecules, are also increased in VTE patients, suggesting not only an exacerbated endothelial activation and dysfunction, but also an interaction of the neutrophil adhesive molecules with their endothelial ligands, favoring the migration process of neutrophil.

Background: Androgenetic alopecia (AGA) is characterized by a pattern hair loss. Currently, treatment with platelet-rich plasma (PRP) has shown promising results due to the growth factors (GFs) released by the platelets. However, the analysis of therapeutic response according to GFs levels and platelet number in PRP has not been established. Objective: Investigate the therapeutic response to treatment of AGA using a standard method of PRP preparation, and the relation with GFs levels and platelet number. Methods: Inclusion criteria comprised diagnosis of AGA-III-vertex profile according to the Norwood-Hamilton scale, age between 18 and 50 years. Exclusion criteria comprised female gender, previous hair transplantation, any disease related to hair loss such as thyroid disease and/or iron deficiency, neoplasia present or past, kidney, liver, infectious, hematologic or rheumatoid disease, use of antiplatelet and/or anti-inflammatory drugs. All patients provided written informed consent approved by the ethic committee from the Faculty of Medical Sciences of the State University of Campinas (UNICAMP). The protocol comprised 20 subcutaneous injections of 100 µL in the scalp totaling 4 applications every fifteen days, with evaluation performed pretreatment (t0), 45 (t1) and 150 (t2) days after the start of the protocol. The endpoints for therapeutic response were hair growth and increase of percentage of anagen hairs evaluated by TrichoScan. For each patient 40 mL of peripheral blood were collected in ACD tubes. L-PRP (PRP with leukocytes) was performed, with double centrifugation (300 g for 5 minutes, and 700 g for 17 minutes). The platelets were counted in the baseline and in the PRP samples. PRP was activated with autologous serum. The platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) were measured by Luminex technique (Millipore®, USA), in two different PRP samples from each patient. Results: During the period of August to December of 2014, 15 male patients were included in the study. The median of platelets in PRP was increased by 5 folds in all four PRP preparations with a minimum of 728.9 and maximum 1.901,90 x 106 cel/uL, and median values of 1.082 x 106 cel/uL (range 608 - 2.023). The baseline number of platelets and PRP preparation showed a significant correlation (r = 0.839, p < 0.0001). The variability of platelet numbers from each individual during the four applications was 19.7% with a minimum of 0.50% and a maximum 56.3%. GF quantification of two different PRP preparations showed a similar intra-individual variation, with a mean of variability coefficient of 18.4% for VEGF, 20.9% for PDGF, and 21.6% for EGF (Table 1). EGF and PDGF concentrations showed a significant correlation to PRP platelets number (r = 0.8287 and P < 0.0001, and r = 0.6925 and P=0.0014, respectively) (Figure 1). Our results showed a significant increase in hair count (P = 0.0018) and anagen hairs (P = 0.0070) in 86.6% and 53.3% of patients, respectively. However, no correlation was found between platelet counts and GFs levels with therapeutic response. The patients who presented high levels of GFs did not show better results for hair growth or anagen hair than who presented lower levels. Conclusion: Our results corroborate previous studies that showed PRP as a quite promising therapeutic option for AGA, up to 3 months after the injections. However, there was a lack of correlation between the therapeutic responses and platelet numbers or GFs levels. Although, the GFs were not considered biomarker for PRP, it may play an important role in the PRP therapeutic effect. In addition, our results suggest that the PRP effects depend on an orchestration between many mechanisms involved in the increase of number of hairs and its growth. Furthermore, local receptors might present a central role in this response. Graphs of correlation between the platelet mean in PRP and the mean of the growth factor concentrations Graphs of correlation between the platelet mean in PRP and the mean of the growth factor concentrations Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Increasing prevalence of diabetes and obesity has registered a simultaneous increase in chronic wounds and defects in tissue repair. Recently PRP was described to be used in the treatment of chronic ulcers. PRP contains cytokines, chemokines and growth factors (GF) derived from platelets which induce molecular and cell mechanisms of natural wound healing. The possible use of GF derived from platelets, and the potential to prolong the shelf life of platelet concentrates, freeze-drying becomes one particularly suitable method to improve wound healing treatment. The use of lyophilized PRP appears to be interesting for greater convenience: only one blood collection, after hydration its soon ready for use, and also due to the highest concentration of bioactive molecules. This study aims to compare intra-lesional freeze-dried and fresh PRP for treatment of acute wound in a pre-clinic model. Methods: Fresh and freeze-dried PRP was prepared from two human platelet concentrate bags using double spin method. PRP characterization included platelet number and quantification of PDGF-AA, EGF, VEGF and TGF-β1 by Luminex (Millipore). Animal wound model was performed after shaving the dorsum of the rat, and a full-thickness excisional wound (1 cm2) was made to the level of the panniculus carnosus muscle. Thirty animals were divided according to the type o treatment in 3 groups: Fresh PRP (n=10), Freeze-dried PRP (FD-PRP) (n=10) and control saline (n=10). The animals received one perilesional application of 500 µL in the day of wound induction. The monitoring of wound closure was made through macroscopical analysis of wound size in days 3, 7 and 10 after wounding. Animals were sacrificed in the tenth day and the skin was removed for histology with hematoxilin-eosin and Masson´s Trichrome, and immunohistochemistry with α-actin smooth muscle for myofibroblasts and blood vessels, and quantified through image J. Results: The platelet number of PRP was 5714 x 103 cells / µL, presenting a high platelet recovery. The FD-PRP presented higher level of all GF when compared to fresh PRP from 1.64 to 3.72 folds. The application of PRP or FD-PRP did not induce significantly changes in wound healing kinetics compared to control during all evaluated days. The mean and standard deviation (SD) of area in D3 was 112.9 ± 16.6%, 107.9 ± 21.3 and 100.8 ± 30.2 for control, PRP and FD-PRP, respectively. In D7 it was 54.3 ± 28.9, 63.9 ± 22.8 and 61.6 ± 20.6 for control, PRP and FD-PRP, respectively. In D10 it was 9.9 ± 6.1, 13.9 ± 11.2, 7.7 ± 6.0 for control, PRP and FD-PRP, respectively. In immunohistochemistry of deep epidermis FD-PRP presented a significantly higher concentration of myofibroblasts in comparison with fresh PRP (16.61 ± 9.04 vs. 13.99 ± 14.07, p=0.0095). A significantly higher number of blood vessels was observed in the group treated with FD-PRP (in percentage of area) in comparison to the controls, both in superficial and deep regions of epidermis (0.43 ± 0.5 vs. 0.21 ± 0.22, P=0.01; 0.38 ± 0.44 vs. 0.24 ± 0.25, P=0.03, respectively). Discussion and conclusion: The most interesting result of this study was the increased number of blood vessels. VEGF plays a central role in promoting angiogenesis during wound repair. Previous studies demonstrated accelerated wound closure with collagen-binding VEGF topical treatment. However, the use of one growth factor has limited success and heterogeneity of clinical results, probably due to the need of multiple bioactive molecules that are necessary in the cascade of complexes events of healing. The use of multiples factors necessary to healing can orchestrated the physiological events resulting in chemotaxis, proliferation and differentiation of cells and the angiogenic process. The use of FD-PRP in this study of acute model of wounds showed positive results, especially in angiogenesis. The practicality and the improve in the shelf life of PRP have a great value in clinical practice and becomes very attractive, showing low cost, the possibility to increase the concentration of GF, various samples ready-to-use and a single blood collection. The investigation of FD-PRP in other clinical situations in which fresh PRP have been used is an interesting approach to be to evaluated to determine its effectiveness. Figure Quantification of blood vessels through imunohistochemistry using smooth α-actin and treated by Image J software; A) Superficial epidermis; B) Deep epidermis. Figure. Quantification of blood vessels through imunohistochemistry using smooth α-actin and treated by Image J software; A) Superficial epidermis; B) Deep epidermis. Disclosures No relevant conflicts of interest to declare.

Neutrophils have a complex migrating process out of the vascular lumen, that consists of chemoattraction and rolling, followed by firm attachment and migration to extravascular tissues. In these sites, neutrophils are capable of promoting phagocytosis, secreting proteases, generating reactive oxygen species (ROS), and probably releasing neutrophil extracellular traps (NETs). Furthermore, neutrophil activation also induces major tissue injury associated with acute and chronic inflammatory disorders, such as the venous thromboembolism (VTE). Recently, animal models and clinical studies in acute VTE have explored the participation of neutrophils in the pathophysiology of VTE. However, VTE has been associated with a chronic inflammatory condition, and it remains unclear whether the activation of neutrophils is persistent after the acute phase of the disease. Furthermore, there are clinical evidences supporting that simvastatin may prevent VTE, since the drug has pleiotropic anti-inflammatory effects. The aim of this study is to evaluate the occurrence of neutrophil activation in patients with VTE compared to health controls and determines the effect of simvastatin in these adhesive properties of the neutrophils. Neutrophils were separated from blood collected over Ficoll-Paque densities. Neutrophils activation was determined by the expression of activated adhesive molecules (LFA-1/CD11a and MAC-1/CD11b) and ROS generation, detected by flow cytometry. Chemotaxis assays (chemoTx, Neuro Probe, Inc) and serum nucleosome, a marker of NETs, were quantified by optical density (OD) (Cell Death Detection ELISAPLUS Kit, Roche). Serum high sensitive CRP (hs-CRP) levels were measured in BN ProSpec System (Siemens) by nephelometry. For CD11a and CD11b integrins expression, cells were evaluated under basal conditions and after TNFα inflammatory stimulus, pretreated, or not, with simvastatin. For the migration assays, neutrophils were treated, or not, with IL-8, a neutrophil chemotactic factor. The results were displayed as mean and standard deviation (±SD). The study group consisted of thirty-seven patients with personal history of VTE, the median time since VTE occurred was 25 months (range 13 - 42 months), the event was spontaneous in 51.35% of the cases and 23 patients presented proximal VTE. Thirty-seven controls, matched with patients according to age, gender and ethnicity, were also included. The mean fluorescence intensity (MFI) of CD11a was higher in VTE patient neutrophils, both in basal conditions (30.84 ± 6.82 vs. 38.72 ± 22.75, P= 0.04) and after TNFα stimulus (34.09 ± 9.64 vs. 45.65 ± 33.06, P= 0.01). Higher MFI of CD11b was observed in patient neutrophils, compared with controls, only after TNFα stimulus (149.10 ± 52.74 vs. 200.0 ± 100.5, P= 0.02), and the stimulus was reverted by pre-treatment with simvastatin (200.8 ± 100.50 vs. 174.60 ± 80.63, P= 0.001). The amount of ROS (MFI) was similar in patients and controls (908.30 ± 423.7 vs. 844.0 ± 312.0, P= 0.83). Neutrophils from VTE patients also presented increased basal chemotaxis (17.55% ± 9.79 vs. 12.64% ± 4.78, P=0.02) and IL-8-stimulated chemotaxis (63.48% ± 29.73 vs. 49.88% ± 19.48%, P=0.06). Serum levels of nucleosomes were similar in patients and controls (1.05 ± 0.81 vs. 0.88 ± 0.62, P= 0.64), however higher levels of circulating nucleosomes were observed in patients with severe post-thrombotic syndrome (PTS), compared to patients with non-severe PTS, without PTS and controls (1.49 ± 0.81 vs. 1.06 ± 0.64 vs. 0.64 ± 0.60 vs. 0.88 ± 0.62, P=0.04). Furthermore, serum levels of hs-CRP were significantly higher in VTE patients when compared with controls (0.59 mg/dl ± 0.58 vs. 0.17 mg/dl ± 0.12, P=0.00). We demonstrated that patients with VTE presented patterns of neutrophil activation long time after the acute thrombotic episode. In particular, the stimuli for neutrophil adhesion and chemotaxis were higher in patients, as detected by the increased activation of adhesive molecules and cell migration. Furthermore, we observed that simvastatin may abrogate the expression of CD11b in inflamed neutrophils. Neutrophil activities associated with ROS generation and the releases of nucleosomes were not increased in these patients. The results may support the hypothesis that increased neutrophils activation is part of the chronic inflammatory condition associated with VTE and may be downregulated by the effects of simvastatin. Disclosures No relevant conflicts of interest to declare.