The Role of Telocytes and Telocyte-Derived Exosomes in the Development of Thoracic Aortic Aneurysm.

A hallmark of thoracic aortic aneurysms (TAA) is the degenerative remodeling of aortic wall, which leads to progressive aortic dilatation and resulting in an increased risk for aortic dissection or rupture. Telocytes (TCs), a distinct type of interstitial cells described in many tissues and organs, were recently observed in the aortic wall, and studies showed the potential regulation of smooth muscle cell (SMC) homeostasis by TC-released shed vesicles. The purpose of the present work was to study the functions of TCs in medial degeneration of TAA. During aneurysmal formation an increase of aortic TCs was identified in human surgical specimens of TAA-patients, compared to healthy thoracic aortic (HTA)-tissue. We found the presence of epithelial progenitor cells in the adventitial layer, which showed increased infiltration in TAA samples. For functional analysis, HTA- and TAA-telocytes were isolated, characterized, and compared by their protein levels, mRNA- and miRNA-expression profiles. We detected TC and TC-released exosomes near SMCs. TAA-TC-exosomes showed a significant increase of the SMC-related dedifferentiation markers KLF-4-, VEGF-A-, and PDGF-A-protein levels, as well as miRNA-expression levels of miR-146a, miR-221 and miR-222. SMCs treated with TAA-TC-exosomes developed a dedifferentiation-phenotype. In conclusion, the study shows for the first time that TCs are involved in development of TAA and could play a crucial role in SMC phenotype switching by release of extracellular vesicles.

Impacts of Telomeric Length, Chronic Hypoxia, Senescence, and Senescence-Associated Secretory Phenotype on the Development of Thoracic Aortic Aneurysm.

Thoracic aortic aneurysm (TAA) is an age-related and life-threatening vascular disease. Telomere shortening is a predictor of age-related diseases, and its progression is associated with premature vascular disease. The aim of the present work was to investigate the impacts of chronic hypoxia and telomeric DNA damage on cellular homeostasis and vascular degeneration of TAA. We analyzed healthy and aortic aneurysm specimens (215 samples) for telomere length (TL), chronic DNA damage, and resulting changes in cellular homeostasis, focusing on senescence and apoptosis. Compared with healthy thoracic aorta (HTA), patients with tricuspid aortic valve (TAV) showed telomere shortening with increasing TAA size, in contrast to genetically predisposed bicuspid aortic valve (BAV). In addition, TL was associated with chronic hypoxia and telomeric DNA damage and with the induction of senescence-associated secretory phenotype (SASP). TAA-TAV specimens showed a significant difference in SASP-marker expression of IL-6, NF-κB, mTOR, and cell-cycle regulators (γH2AX, Rb, p53, p21), compared to HTA and TAA-BAV. Furthermore, we observed an increase in CD163+ macrophages and a correlation between hypoxic DNA damage and the number of aortic telocytes. We conclude that chronic hypoxia is associated with telomeric DNA damage and the induction of SASP in a diseased aortic wall, promising a new therapeutic target.

Mononuclear cell secretome protects from experimental autoimmune myocarditis.

AIMS: Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. METHODS AND RESULTS: BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. CONCLUSION: MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases.

High dose ionizing radiation regulates micro RNA and gene expression changes in human peripheral blood mononuclear cells.

BACKGROUND: High dose ionizing radiation (IR) induces potent toxic cell effects mediated by either direct DNA damage or the production of reactive oxygen species (ROS). IR-induced modulations in multiple biological processes have been proposed to be partly regulated by radiosensitive microRNA (miRNA). In order to gain new insights into the role of miRNAs in the regulation of biological processes after IR, we have investigated changes in mRNA and miRNA expression after high dose IR. RESULTS: IR induced changes in the mRNA and miRNA profiles of human peripheral blood mononuclear cells (PBMCs). When comparing non-irradiated and irradiated samples, we detected a time-dependent increase in differentially expressed mRNAs and miRNAs, with the highest differences detectable 20 hours after exposure. Gene ontology analysis revealed that very early events (up to 4 hours) after irradiation were specifically associated with p53 signaling and apoptotic pathways, whereas a large number of diverse cellular processes were deregulated after 20 hours. Transcription factor analysis of all up-regulated genes confirmed the importance of p53 in the early post-irradiation phase. When analyzing miRNA expression, we found 177 miRNAs that were significantly regulated in the late post-irradiation phase. Integrating miRNA and target gene expression data, we found a significant negative correlation between miRNA-mRNA and identified hepatic leukemia factor (HLF) as a transcription factor down-regulated in the response to IR. These regulated miRNAs and the HLF target genes were involved in modulating radio-responsive pathways, such as apoptosis, the MAKP signaling pathway, endocytosis, and cytokine-cytokine interactions. CONCLUSION: Using a large dataset of mRNA and miRNA expression profiles, we describe the interplay of mRNAs and miRNAs in the regulation of gene expression in response to IR at a posttranscriptional level and their involvement in the modulation of radiation-induced biological pathways.

Low tidal volume ventilation during cardiopulmonary bypass reduces postoperative chemokine serum concentrations.

BACKGROUND: Open-heart surgery with cardiopulmonary bypass (CPB) is associated with a generalized immune response and postoperative lung dysfunction. Chemokines are involved in the pathogenesis of postoperative lung dysfunction. We investigated whether continued mechanical ventilation during CPB has an impact on chemokine serum concentrations. METHODS: A total of 30 patients undergoing coronary artery bypass graft operation were randomized to either continuous ventilated group (n=15) or nonventilated group (n=15). Blood samples were drawn at the beginning and at the end of surgery and on the 5 consecutive days. Serum CCL2, CCL4, and CCL20 concentrations were measured and given as mean ± standard deviation. RESULTS: Chemokine concentrations were elevated at the end of surgery in both groups. CCL2 and CCL4 levels returned to baseline on postoperative day (POD)-1 in the ventilation group and stayed elevated in the nonventilation group. CCL4 serum levels were significantly lower in ventilated-group patients on POD-1 (10.9 [39.0] vs. 153.2 [168.1]; p=0.005), POD-2 (16.8 [36.8] vs. 147.9 [165.4]; p=0.019), POD-3 (14.2 [24.0] vs. 97.9 [87.1]; p=0.005), and POD-5 (6.5 [25.0] vs. 33.6 [38.4]; p=0.045). CONCLUSION: Continued mechanical ventilation during CPB results in reduced CCL4 concentrations on POD-1 to -5.

Increased serum levels of HSP27 as a marker for incipient chronic obstructive pulmonary disease in young smokers.

BACKGROUND: Although chronic obstructive pulmonary disease (COPD) is amongst the leading causes of morbidity and mortality, no biomarkers for its early detection are known. We have recently demonstrated that COPD is accompanied by elevated serum heat shock protein (HSP) 27 levels as compared to a control population. OBJECTIVES: In an open prospective study, we investigated whether elevated HSP27 levels are associated with the early radiological signs of COPD, i.e., air trapping (AT), emphysema (E) and impaired lung function. METHODS: In total, 120 apparently healthy smokers underwent lung function testing and serum sampling. Serum levels of HSP27, phospho-HSP27, CXCR2 chemokines and proteins related to inflammation, tissue remodeling and apoptosis were evaluated by ELISA. Of these 120 subjects, 94 voluntarily underwent a high-resolution computed tomography scan. RESULTS: AT or AT and E were detected in 57.45%. Subjects with AT and E (n = 23) showed significantly higher HSP27 levels than those without any pathology [i.e., nothing abnormal detected (NAD)] (4,618 +/- 1,677 vs. 3,282 +/- 1,607 pg/ml; p = 0.0081). In a univariate logistic regression model including NAD and AT and E, the area under the curve of HSP27 in the receiver-operating-characteristic curve was 0.724, (0.594­0.854, 95% CI; p = 0.0033). Interestingly, proinflammatory IL-8 was elevated in those subjects with evidence of AT and E compared to those with AT and NAD. Lung function did not correlate with increased HSP27 levels or pathological radiological findings. CONCLUSIONS: HSP27 serum levels correlated with the early radiological signs of COPD, whereas lung function did not match with radiological findings or HSP27 serum levels. Serum HSP27 levels may serve as a potential marker to identify the early signs of COPD independent of lung function in young smokers.

Anti-alpha-Gal antibody titres remain unaffected by the consumption of fermented milk containing Lactobacillus casei in healthy adults.

Alpha-Gal is a glycoconjugate present on cell membranes of non-primate mammals and bacteria, but not in humans, who display anti-Gal antibodies (ABs) in high titres. Probiotics contain bacterial strains which colonize the intestinal tract. In the present study, we investigated whether intake of fermented milk containing Lactobacillus casei (FML) affects anti-Gal AB titres. Serum was drawn from healthy probands (n = 19) for 6 weeks. After the second week, the probands consumed 125 ml of FML per day. Anti-Gal ABs of all isotypes and cytokines were measured. Bacterial cultures were bred from FML and bacteria were stained for alpha-Gal. Concentration of bacteria in FML was manifold higher than in conventional yoghurt (2 × 10(5)/g yoghurt vs. 1.1 × 10(7)/g FML). Both stained highly positive for Alpha-Gal. Alpha-Gal-specific ABs and cytokines remained unaffected by FML intake. Our results indicated that the consumption of FML does not elicit a humoral immune response in healthy adults.

Electrical Stimulation of the Greater Auricular Nerve to Reduce Postoperative Atrial Fibrillation.

BACKGROUND: Postoperative atrial fibrillation (POAF) occurs in up to 40% of patients undergoing cardiac surgery. Invasive stimulation of the vagal nerve previously demonstrated a reduced risk of POAF. Therefore, we examined the antiarrhythmic and anti-inflammatory effects of noninvasive low-level transcutaneous electrical stimulation (LLTS) of the greater auricular nerve in a pilot trial including patients undergoing cardiac surgery. METHODS: Patients were randomized into a sham (n=20) or a treatment group (n=20) for LLTS. After cardiac surgery, electrodes were applied in the triangular fossa of the ear. Stimulation (amplitude 1 mA, frequency 1 Hz for 40 minutes, followed by a 20 minutes break) was performed for up to 2 weeks after cardiac surgery. Heart rhythm was recorded continuously using an ECG during the observation period. CRP (C-reactive protein) and IL (interleukin)-6 plasma concentrations were measured immediately after surgery as well as on day 2 and 7 postsurgery. RESULTS: Patients receiving LLTS had a significantly reduced occurrence of POAF (4 of 20) when compared with controls (11 of 20, P=0.022) during a similar mean Holter recording period. The median duration of POAF was comparable between the treatment and the control group (878 [249; 1660] minutes versus 489 [148; 1775] minutes; P=0.661). No effect of LLTS on CRP or IL-6 levels was detectable. CONCLUSIONS: LLTS of the greater auricular nerve may be a potential therapy for POAF. We demonstrated the feasibility to conduct a randomized trial of neurostimulation as an outlay for a multisite clinical trial.

Publisher Correction: Exercise-induced bronchoconstriction, temperature regulation and the role of heat shock proteins in non-asthmatic recreational marathon and half-marathon runners.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exercise-induced bronchoconstriction, temperature regulation and the role of heat shock proteins in non-asthmatic recreational marathon and half-marathon runners.

Exercise is the most common trigger of bronchospasm. Heat shock protein (HSP) expression was linked to asthmatic patients. The prevalence and pathophysiology of exercise-induced bronchoconstriction (EIB) in non-professional non-asthmatic runners is unknown. We sought to investigate the frequency of EIB and cytokine changes in non-professional non-asthmatic marathon and half marathoners with and without EIB. Testing was performed before the marathon (baseline), immediately post-marathon at the finish area (peak), and 2-7 days after the marathon (recovery): immunosorbent assays for measurement of HSP70, blood count analysis, spirometry and temperature measurements. We experienced a decline in FEV1 of ≥10% in 35.29% of marathon and 22.22% of half marathon runners. Runners with EIB had significantly higher HSP70 serum concentrations at baseline than those without EIB (987.4 ± 1486.7 vs. 655.6 ± 1073.9; p = 0.014). Marathoners with EIB had significantly increased WBC before participating in the competition (7.4 ± 1.7 vs. 6.0 ± 1.5; p = 0.021). After recovery we found increased HSP70 serum concentrations in marathoners with EIB compared to those without (2539.2 ± 1692.5 vs. 1237.2 ± 835.2; p = 0.032), WBC (7.6 ± 1.8 vs. 6.4 ± 1.6; p = 0.048) and PLT (273.0 ± 43.0 vs 237.2 ± 48.3; p = 0.040). At all measured skin sites skin temperatures in runners were significantly lower immediately after participating in the competition when compared to temperature before the race (skin temperature baseline vs. peak: abdominal: 33.1 ± 0.2 vs. 30.0 ± 0.4; p < 0.001; upper arm: 31.6 ± 0.2 vs. 29.4 ± 0.3; p < 0.001; upper leg: 30.7 ± 0.3 vs. 29.4 ± 0.2; p = 0.014; lower leg: 30.6 ± 1.0 vs. 30.2 ± 1.5; p = 0.007). We found a higher than expected number of non-professional athletes with EIB. HSP70 serum concentrations and elevated WBC could indicate a predisposition to EIB.

Effect of thymoquinone on head and neck squamous cell carcinoma cells in vitro: Synergism with radiation.

Thymoquinone (TQ) is the main bioactive constituent present in black seed oil (Nigella sativa); it has shown anti-inflammatory and anti-neoplastic effects in various cancer cell types. The aim of the present study was to investigate the effects of TQ on head and neck squamous cell carcinoma (HNSCC) cell lines, on its own and in combination with radiation and cisplatin, respectively. The SCC25 and CAL27 HNSCC cell lines were treated with TQ alone and in combination with cisplatin or radiation, respectively. Proliferation assays and clonogenic assays were performed. Apoptosis was detected by flow cytometry. TQ exhibited dose-dependent cytotoxicity via apoptosis in the investigated cell lines. In combination with cisplatin, TQ resulted in no significant increase in cytotoxicity. Combined with radiation, TQ significantly reduced clonogenic survival compared with each treatment method alone. TQ is a promising agent in the treatment of head and neck cancer due to its anti-proliferative and radiosensitizing properties. However, the combination of TQ with cisplatin showed no therapeutic benefit in vitro.

Standard donor lung procurement with normothermic ex vivo lung perfusion: A prospective randomized clinical trial.

BACKGROUND: Ex vivo lung perfusion (EVLP) was primarily developed for evaluation of impaired donor lungs. The good clinical results raise the question for its possible impact on lungs meeting standard criteria. Before application of EVLP on such lungs enters routine clinical practice, it must be demonstrated whether EVLP would affect or improve outcome when used in standard donor lungs. We performed a prospective randomized trial to investigate the role of EVLP in standard lung transplantation (Tx). METHODS: This prospective randomized clinical trial compared patients who underwent Tx with ex vivo evaluated donor lungs with an equivalent patient population without previous EVLP. RESULTS: From October 2013 to May 2015, 193 lung Tx were performed at the Medical University of Vienna. During this period, 80 recipient/donor pairs that met the inclusion criteria were included in this trial, 41 pairs in the control group, and 39 in the EVLP group. In the EVLP group, 4 lungs (10.2%) ultimately did not qualify for Tx and were rejected for lung Tx owing to technical reasons (n = 2) and quality criteria (n = 2). Donor and recipient characteristics were comparable in both groups. Total cold ischemic time in the EVLP group was significantly longer for both implanted lungs (first side, 372 minutes vs 291 minutes, p < 0.001; second side, 437 minutes vs 370 minutes, p = 0.001); median duration of surgery showed no differences (277 minutes vs 275 minutes). Median oxygen partial pressure/fraction of inspired oxygen ratio at 24 hours after Tx was 516 (range, 280-557) in the EVLP group and 491 (range, 352-575) in the control group (p = 0.63). Incidence of primary graft dysfunction >1 was lower in the EVLP group at all time points compared with the control group (24 hours, 5.7% vs 19.5%, p = 0.10), and need for post-operative prolonged extracorporeal membrane oxygenation was lower in the EVLP group (5.7% vs 12.2%, p = 0.44). Short-term clinical outcomes did not differ between recipients in the 2 groups. Patients remained intubated (1.6 days vs 1.6 days, p = 0.67), in the intensive care unit (6 days vs 6 days, p = 0.76), and in the hospital (23 days vs 19 days, p = 0.42) for a comparable period of time. The 30-day survival was 97.1% vs 100% (p = 0.46). CONCLUSIONS: This study provides evidence that EVLP can safely be used in standard donor lungs. Functional results and perioperative outcome are comparable to those achieved with standard donor lung preservation techniques. As an evaluation tool, EVLP allows clinicians to identify and to possibly exclude lungs with functional impairment. Finally, EVLP can safely extend total preservation time.

Treatment of primary graft dysfunction after lung transplantation with orally inhaled AP301: A prospective, randomized pilot study.

BACKGROUND: Primary graft dysfunction (PGD) after lung transplantation (LTx) carries significant morbidity and mortality in the early post-operative period and is associated with the development of chronic lung allograft dysfunction. AP301, an activator of epithelial sodium channel-mediated Na+ uptake represents a new concept for prevention and treatment of pulmonary edema and has shown promising results in the pre-clinical setting. This pilot study investigated the clinical effect of inhaled AP301 on patients with development of PGD > 1 according to International Society of Heart and Lung Transplantation criteria after primary LTx in a high-volume center and was conducted as a randomized, placebo-controlled, single-center pilot-study including 20 patients. All consecutive patients fulfilling inclusion criteria were screened for PGD at arrival on the intensive care unit (ICU) after LTx. After randomization, inhaled AP301 or placebo was administered by nebulizer twice daily for 7 days or until extubation. Otherwise, patients were treated according to routine clinical protocol. Partial pressure of arterial oxygen (Pao2)/fraction of inspired oxygen (Fio2) values were obtained until extubation and assessed as a primary outcome parameter. Patients were monitored for 30 days within the study protocol. RESULTS: From July 2013 to August 2014, 20 patients were randomized 1:1 to AP301 (Group 1) or placebo (Group 2). Both groups were comparable with regard to sex (40% women/60% men vs 50% women/50% men), mean age (55 ± 13 vs 54 ± 6 years), comorbidities, and diagnosis leading to LTx. The Pao2/Fio2 ratio at the time of inclusion was comparable in both groups, with a mean 235.65 ± 90.78 vs 214.2 ± 95.84 (p = 0.405), and there was no significant difference in the extravascular lung water index (13.88 ± 5.28 vs 16 ± 6.29 ml/kg, p = 0.476). The primary end point was mean Pao2/Fio2 ratio values between baseline and Day 3. In the AP301 group, only 1 patient was ventilated at Day 4 and no patients were ventilated after Day 4. In the placebo group, 5 patients were ventilated on Day 4 and 2 patients on Days 5, 6, and 7. The mean increase in the Pao2/Fio2 ratio was significantly higher in Group 1 patients, and the mean between baseline and at 72 hours was 365.6 ± 90.4 in Group 1 vs 335.2 ± 42.3 in Group 2 (p = 0.049). The duration of intubation was shorter in Group 1 than in Group 2 patients (2 ± 0.82 vs 3.7 ± 1.95 days; p = 0.02). ICU stay was 7.5 ± 3.13 days in Group 1 vs 10.8 ± 8.65 days in group 2 (p = 0.57). Survival at 30 days was 100%. No severe adverse events were recorded. CONCLUSIONS: This study was designed as a proof-of-concept pilot study. Although it was not powered to achieve statistical significances, the study demonstrated relevant clinical effects of inhaled AP301 on patients with PGD after primary LTx. The improved gas exchange led to a significantly shorter duration of mechanical ventilation and a trend towards a shorter ICU stay. Further investigation of AP301 for treatment of PGD in larger studies is warranted. TRIAL REGISTRATION: The trial is registered at https://www.clinicaltrialsregister.eu/ctr-search/trial/2013-000716-21/AT.

Non-professional marathon running: RAGE axis and ST2 family changes in relation to open-window effect, inflammation and renal function.

Conflicting data exist on the relevance of marathon (M) and half marathon (HM) running for health. The number of non-professional athletes finishing M and HM events is steadily growing. In order to investigate molecular changes occurring in amateur athletes, we enrolled 70 non-professional runners finishing a single M (34) or HM (36) event at baseline, the finish line and during recovery, and 30 controls. The measurement of the Receptor for Advanced Glycation Endproducts, Interleukin 1 receptor antagonist, ST2 and cytokeratin 18 was combined with molecules measured during clinical routine. Results were analyzed in the light of blood cell analysis, lactate measurements, correction for changes in plasma volume and body composition assessments. There were intrinsic differences in body mass index, abdominal body fat percentage and training time between M and HM runners. C-reactive protein changes in M and HM runners. While soluble RAGE, AGEs and ST2 increased immediately after the race in HM runners, HMGB1 increased in HM and M after the race and declined to baseline after a recovery period. We give insights into the regulation of various molecules involved in physical stress reactions and their possible implications for the cardiovascular system or renal function.

Paracrine Factors from Irradiated Peripheral Blood Mononuclear Cells Improve Skin Regeneration and Angiogenesis in a Porcine Burn Model.

Burn wounds pose a serious threat to patients and often require surgical treatment. Skin grafting aims to achieve wound closure but requires a well-vascularized wound bed. The secretome of peripheral blood mononuclear cells (PBMCs) has been shown to improve wound healing and angiogenesis. We hypothesized that topical application of the PBMC secretome would improve the quality of regenerating skin, increase angiogenesis, and reduce scar formation after burn injury and skin grafting in a porcine model. Full-thickness burn injuries were created on the back of female pigs. Necrotic areas were excised and the wounds were covered with split-thickness mesh skin grafts. Wounds were treated repeatedly with either the secretome of cultured PBMCs (Sec(PBMC)), apoptotic PBMCs (Apo-Sec(PBMC)), or controls. The wounds treated with Apo-Sec(PBMC) had an increased epidermal thickness, higher number of rete ridges, and more advanced epidermal differentiation than controls. The samples treated with Apo-Sec(PBMC) had a two-fold increase in CD31+ cells, indicating more angiogenesis. These data suggest that the repeated application of Apo-Sec(PBMC) significantly improves epidermal thickness, angiogenesis, and skin quality in a porcine model of burn injury and skin grafting.

Clinical-radiological, histological and genetic analyses in a lung transplant recipient with Mounier-Kuhn syndrome and end-stage chronic obstructive pulmonary disease.

BACKGROUND AND AIMS: The Mounier-Kuhn syndrome (MKS) is a rare disease characterized by a pathological dilation of the trachea and the bronchial system. The etiology of the disorder remains elusive, but genetic alterations and degradation of elastic fibers are thought to be involved in the pathogenesis. No causative treatment is available although transplantation is an option for end-stage disease. Here, we describe a patient suffering from MKS who received a double lung transplant at our department. METHODS: Since a familial clustering of MKS is discussed in the literature, we performed a chromosomal analysis and an array-comparative genomic hybridization (CGH) to search for genetic abnormalities. At the time of transplantation, we collected samples from the bronchi and performed hematoxylin and eosin (HE), Elastic von-Gieson (EVG) and immunohistochemical stains of the explanted MKS bronchus, a control bronchus and of the inflammatory infiltrates. Specimens of main bronchi from the donor lung harvested for transplant served as control. Bronchial smears were taken from both main bronchi of the recipient for microbiological cultures. RESULTS: No genetic alterations could be found in chromosomal analysis and in array-CGH. Histological analysis revealed a strong reduction of elastic fibers in the submucosal connective tissue and a diffuse inflammatory infiltrate, mainly comprised of CD4+ cells. In addition, immunohistochemistry showed increased matrix metalloproteinases (MMPs) protein expression of MMP-1, 2, 3 and 9. CONCLUSIONS: Based on our findings, we hypothesize that MKS is a chronic inflammatory disease characterized by an MMP-mediated degradation of submucosal elastic fibers.

Analysis of the Secretome of Apoptotic Peripheral Blood Mononuclear Cells: Impact of Released Proteins and Exosomes for Tissue Regeneration.

We previously showed that, when peripheral blood mononuclear cells (PBMCs) were stressed with ionizing radiation, they released paracrine factors that showed regenerative capacity in vitro and in vivo. This study aimed to characterize the secretome of PBMCs and to investigate its biologically active components in vitro and vivo. Bioinformatics analysis revealed that irradiated PBMCs differentially expressed genes that encoded secreted proteins. These genes were primarily involved in (a) pro-angiogenic and regenerative pathways and (b) the generation of oxidized phospholipids with known pro-angiogenic and inflammation-modulating properties. Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects. We tested a viral-cleared PBMC secretome, prepared according to good manufacturing practice (GMP), in a porcine model of closed chest, acute myocardial infarction. We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes. Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs. In addition our findings implicate the use of secretome fractions as valuable material for the development of cell-free therapies in regenerative medicine.

The secretome of apoptotic human peripheral blood mononuclear cells attenuates secondary damage following spinal cord injury in rats.

After spinal cord injury (SCI), secondary damage caused by oxidative stress, inflammation, and ischemia leads to neurological deterioration. In recent years, therapeutic approaches to trauma have focused on modulating this secondary cascade. There is increasing evidence that the success of cell-based SCI therapy is due mainly to secreted factors rather than to cell implantation per se. This study investigated peripheral blood mononuclear cells as a source of factors for secretome- (MNC-secretome-) based therapy. Specifically, we investigated whether MNC-secretome had therapeutic effects in a rat SCI contusion model and its possible underlying mechanisms. Rats treated with MNC-secretome showed substantially improved functional recovery, attenuated cavity formation, and reduced acute axonal injury compared to control animals. Histological evaluation revealed higher vascular density in the spinal cords of treated animals. Immunohistochemistry showed that MNC-secretome treatment increased the recruitment of CD68(+) cells with concomitant reduction of oxidative stress as reflected by lower expression of inducible nitric oxide synthase. Notably, MNC-secretome showed angiogenic properties ex vivo in aortic rings and spinal cord tissue, and experiments showed that the angiogenic potential of MNC-secretome may be regulated by CXCL-1 upregulation in vivo. Moreover, systemic application of MNC-secretome activated the ERK1/2 pathway in the spinal cord. Taken together, these results indicate that factors in MNC-secretome can mitigate the pathophysiological processes of secondary damage after SCI and improve functional outcomes in rats.

Antimicrobial peptides are highly abundant and active in postoperative pleural drainage fluids.

BACKGROUND: The human lung is considered a nonsterile organ, and surgical interventions therefore take place in a more or less contaminated operating field. Nevertheless, infectious complications of the pleural cavity are low after major lung resections. Antimicrobial peptides (AMPs) are part of the innate immunity and display a broad capacity to kill pathogens. We hypothesized that the pleural space must have a high natural antimicrobial barrier and that AMPs might effectively protect the pleural cavity. METHODS: Pleural effusions were collected after lung operations. Antimicrobial activity of the fluids against gram-positive and gram-negative pathogens was analyzed by microdilution assays. AMPs were determined by enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and immunohistochemical analysis. The impact of proinflammatory triggers on AMP release from pleural mesothelial cells was evaluated. RESULTS: Antimicrobial activity assays revealed high bactericidal properties of postoperative pleural drainage fluids. They effectively killed gram-negative pathogens (Escherichia coli, Pseudomonas aeruginosa) as well as gram-positive pathogens (Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes). A variety of AMPs was detected at constantly high concentrations in the pleural fluids. They mainly derived from leukocytes and pleural epithelium. Although proinflammatory cytokine levels were elevated in the postoperative pleural fluids, AMP expression could not be augmented by Toll-like receptor (TLR) triggering or by the proinflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)α. CONCLUSIONS: We provide the first evidence of a high abundance of AMPs in postoperative pleural fluids. Our findings might explain the broad protection against infectious complications of the pleural space after major lung operations.