Exploring the heterogeneous transcriptional response of the CNS to systemic LPS and Poly(I:C).

Peripheral contact to pathogen-associated molecular patterns (PAMPs) evokes a systemic innate immune response which is rapidly relayed to the central nervous system (CNS). The remarkable cellular heterogeneity of the CNS poses a significant challenge to the study of cell type and stimulus dependent responses of neural cells during acute inflammation. Here we utilized single nuclei RNA sequencing (snRNAseq), serum proteome profiling and primary cell culture methods to systematically compare the acute response of the mammalian brain to the bacterial PAMP lipopolysaccharide (LPS) and the viral PAMP polyinosinic:polycytidylic acid (Poly(I:C)), at single cell resolution. Our study unveiled convergent transcriptional cytokine and cellular stress responses in brain vascular and ependymal cells and a downregulation of several key mediators of directed blood brain barrier (BBB) transport. In contrast the neuronal response to PAMPs was limited in acute neuroinflammation. Moreover, our study highlighted the dominant role of IFN signalling upon Poly(I:C) challenge, particularly in cells of the oligodendrocyte lineage. Collectively our study unveils heterogeneous, shared and distinct cell type and stimulus dependent acute responses of the CNS to bacterial and viral PAMP challenges. Our findings highlight inflammation induced dysregulations of BBB-transporter gene expression, suggesting potential translational implications on drug pharmacokinetics variability during acute neuroinflammation. The pronounced dependency of oligodendrocytes on IFN stimulation during viral PAMP challenges, emphasizes their limited molecular viral response repertoire.

The Secretome of Irradiated Peripheral Mononuclear Cells Attenuates Hypertrophic Skin Scarring.

Hypertrophic scars can cause pain, movement restrictions, and reduction in the quality of life. Despite numerous options to treat hypertrophic scarring, efficient therapies are still scarce, and cellular mechanisms are not well understood. Factors secreted by peripheral blood mononuclear cells (PBMCsec) have been previously described for their beneficial effects on tissue regeneration. In this study, we investigated the effects of PBMCsec on skin scarring in mouse models and human scar explant cultures at single-cell resolution (scRNAseq). Mouse wounds and scars, and human mature scars were treated with PBMCsec intradermally and topically. The topical and intradermal application of PBMCsec regulated the expression of various genes involved in pro-fibrotic processes and tissue remodeling. We identified elastin as a common linchpin of anti-fibrotic action in both mouse and human scars. In vitro, we found that PBMCsec prevents TGFß-mediated myofibroblast differentiation and attenuates abundant elastin expression with non-canonical signaling inhibition. Furthermore, the TGFß-induced breakdown of elastic fibers was strongly inhibited by the addition of PBMCsec. In conclusion, we conducted an extensive study with multiple experimental approaches and ample scRNAseq data demonstrating the anti-fibrotic effect of PBMCsec on cutaneous scars in mouse and human experimental settings. These findings point at PBMCsec as a novel therapeutic option to treat skin scarring.

Single nucleus RNA sequencing reveals glial cell type-specific responses to ischemic stroke.

Reactive neuroglia critically shape the brains response to ischemic stroke. However, their phenotypic heterogeneity impedes a holistic understanding of the cellular composition and microenvironment of the early ischemic lesion. Here we generated a single cell resolution transcriptomics dataset of the injured brain during the acute recovery from permanent middle cerebral artery occlusion. This approach unveiled infarction and subtype specific molecular signatures in oligodendrocyte lineage cells and astrocytes, which ranged among the most transcriptionally perturbed cell types in our dataset. Specifically, we characterized and compared infarction restricted proliferating oligodendrocyte precursor cells (OPCs), mature oligodendrocytes and heterogeneous reactive astrocyte populations. Our analyses unveiled unexpected commonalities in the transcriptional response of oligodendrocyte lineage cells and astrocytes to ischemic injury. Moreover, OPCs and reactive astrocytes were involved in a shared immuno-glial cross talk with stroke specific myeloid cells. In situ, osteopontin positive myeloid cells accumulated in close proximity to proliferating OPCs and reactive astrocytes, which expressed the osteopontin receptor CD44, within the perilesional zone specifically. In vitro, osteopontin increased the migratory capacity of OPCs. Collectively, our study highlights molecular cross talk events which might govern the cellular composition and microenvironment of infarcted brain tissue in the early stages of recovery.

Follistatin-like 1 and Biomarkers of Neutrophil Activation Are Associated with Poor Short-Term Outcome after Lung Transplantation on VA-ECMO.

The investigation of biomarkers associated with undesired outcome following lung transplantation (LuTX) is essential for a better understanding of the underlying pathophysiology, an earlier identification of susceptible recipients and the development of targeted therapeutic options. We therefore determined the longitudinal perioperative course of putative cytokines related to neutrophil activation (chemokine CC motif ligand 4 (CCL-4), interleukin (IL)-23 and Lipocalin 2 (LCN2)) and a cytokine that has been implicated in graft-versus-host disease (Follistatin-like 1 (FSTL1)) in 42 consecutive patients undergoing LuTX. We plotted receiver-operating curves (ROC) to assess the predictive power of the measured cytokines for short-term outcomes namely primary graft dysfunction (PGD), early complications requiring extracorporeal membrane oxygenation (ECMO), and a high postoperative sequential organ failure assessment (SOFA). All cytokines increased immediately after surgery. ROC analyses determined significant associations between CCL4 and a high SOFA score (area under the curve (AUC) 0.74 (95%CI:0.5−0.9; p < 0.05), between LCN2 and postoperative ECMO support (AUC 0.73 (95%CI:0.5−0.9; p < 0.05), and between FSTL1 and PGD (AUC 0.70 (95%CI:0.5−0.9; p < 0.05). The serum concentrations of the neutrophil-derived cytokines LCN2 and CCL4 as well as FSTL1 were all related to poor outcome after LuTX. The specific predictive power, however, still has to be assessed in larger trials. The potential role of FSTL1 as a biomarker in the development of PGD could be of great interest particularly since this protein appears to play a crucial role in allograft tolerance.

Transient perioperative inflammation following lung transplantation and major thoracic surgery with elective extracorporeal support: a prospective observational study.

BACKGROUND: The clinical relevance of inflammation induced by elective perioperative extracorporeal membrane oxygenation (ECMO) usage as an integral part of modern lung transplantation (LUTX) remains elusive. The aim of this study was to determine the perioperative cytokine response accompanying major thoracic surgery employing different extracorporeal devices comprising ECMO, cardiopulmonary bypass (CPB), or no extracorporeal circulation in relation to inflammation, clinically tangible as increased sequential organ failure assessment (SOFA) score, called SOFA. METHODS: In this prospective, observational pilot study 42 consecutive patients with end-stage pulmonary disease undergoing LUTX; 15 patients with chronic thromboembolic pulmonary hypertension (CTEPH) undergoing pulmonary endarterectomy and 15 patients with lung cancer undergoing major lung resections were analysed. Cytokine serum concentrations and SOFA were determined before, at end of surgery and in the following postoperative days. RESULTS: LUTX on ECMO and pulmonary endarterectomy (PEA) on CPB triggered an immediate increase in cytokine serum concentrations at end of surgery: IL-6: 66-fold and 71-fold, IL-10: 3-fold and 2.5-fold, ST2/IL-33R: 5-fold and 4-fold and SOFA: 10.5±2.8 and 10.7±1.7, that decreased sharply to baseline levels from postoperative day 1-5. Despite low perioperative mortality (3 patients, 4.1%) extremely high SOFA ≥13 was associated with mortality after LUTX. Delta-SOFA distinguished survivors from non-survivors: -4.5±3.2 vs. -0.3±1.5 (P=0.001). Increased IL-6 serum concentrations were predictive for increased SOFA (sensitivity: 97%, specificity: 80%). Peak cytokine serum concentrations correlated with ECC duration, maximal lactate, transfusion of red-blood-cells, fresh-frozen-plasma, and catecholamine support. CONCLUSIONS: LUTX and PEA on extracorporeal circulation with an excellent outcome triggered an immediate rise and concomitant fall of inflammation as observed in cytokine serum concentrations and SOFA. High absolute SOFA in the presence of an uncomplicated postoperative course may pertain to specific management strategies rather than organ failure.

Potential novel biomarkers for chronic lung allograft dysfunction and azithromycin responsive allograft dysfunction.

Chronic Lung Allograft Dysfunction (CLAD), manifesting as Bronchiolitis Obliterans Syndrome (BOS) or Restrictive Allograft Syndrome (RAS), is the main reason for adverse long-term outcome after Lung Transplantation (LTX). Until now, no specific biomarkers exist to differentiate between CLAD phenotypes. Therefore, we sought to find suitable cytokines to distinguish between BOS, RAS and Azithromycin Responsive Allograft Dysfunction (ARAD); and reveal potential similarities or differences to end-stage fibrotic diseases. We observed significantly increased Lipocalin-2 serum concentrations in RAS compared to BOS patients. In addition, in RAS patients immunohistochemistry revealed Lipocalin-2 expression in bronchial epithelium and alveolar walls. Patients with ARAD showed significantly lower Activin-A serum concentrations compared to Stable-LTX and BOS patients. Further, increased serum concentrations of Lipocalin-2 and Activin-A were predictors of worse freedom-from-CLAD in Stable-LTX patients. These biomarkers serve as promising serum biomarkers for CLAD prediction and seem suitable for implementation in clinical practice.

The secretome of stressed peripheral blood mononuclear cells increases tissue survival in a rodent epigastric flap model.

Reconstructive surgery transfers viable tissue to cover defects and to restore aesthetic and functional properties. Failure rates after free flap surgery range from 3 to 7%. Co-morbidities such as diabetes mellitus or peripheral vascular disease increase the risk of flap failure up to 4.5-fold. Experimental therapeutic concepts commonly use a monocausal approach by applying single growth factors. The secretome of γ-irradiated, stressed peripheral blood mononuclear cells (PBMCsec) resembles the physiological environment necessary for tissue regeneration. Its application led to improved wound healing rates and a two-fold increase in blood vessel counts in previous animal models. We hypothesized that PBMCsec has beneficial effects on the survival of compromised flap tissue by reducing the necrosis rate and increasing angiogenesis. Surgery was performed on 39 male Sprague-Dawley rats (control, N = 13; fibrin sealant, N = 14; PBMCsec, N = 12). PBMCsec was produced according to good manufacturing practices (GMP) guidelines and 2 ml were administered intraoperatively at a concentration of 2.5 × 107 cells/ml using fibrin sealant as carrier substance. Flap perfusion and necrosis (as percentage of the total flap area) were analyzed using Laser Doppler Imaging and digital image planimetry on postoperative days 3 and 7. Immunohistochemical stainings for von Willebrand factor (vWF) and Vascular Endothelial Growth Factor-receptor-3 (Flt-4) were performed on postoperative day 7 to evaluate formation of blood vessels and lymphatic vessels. Seroma formation was quantified using a syringe and flap adhesion and tissue edema were evaluated clinically through a cranial incision by a blinded observer according to previously described criteria on postoperative day 7. We found a significantly reduced tissue necrosis rate (control: 27.8% ± 8.6; fibrin: 22.0% ± 6.2; 20.9% reduction, p = .053 vs. control; PBMCsec: 19.1% ± 7.2; 31.1% reduction, p = .012 vs. control; 12.9% reduction, 0.293 vs. fibrin) together with increased vWF+ vessel counts (control: 70.3 ± 16.3 vessels/4 fields at 200× magnification; fibrin: 67.8 ± 12.1; 3.6% reduction, p = .651, vs. control; PBMCsec: 85.9 ± 20.4; 22.2% increase, p = .045 vs. control; 26.7% increase, p = .010 vs. fibrin) on postoperative day 7 after treatment with PBMCsec. Seroma formation was decreased after treatment with fibrin sealant with or without the addition of PBMCsec. (control: 11.9 ± 9.7 ml; fibrin: 1.7 ± 5.3, 86.0% reduction, 0.004 vs. control; PBMCsec: 0.6 ± 2.0; 94.8% reduction, p = .001 vs. control; 62.8% reduction, p = .523 vs. fibrin). We describe the beneficial effects of a secretome derived from γ-irradiated PBMCs on tissue survival, angiogenesis, and clinical parameters after flap surgery in a rodent epigastric flap model.

Fractional heat shock protein 27 urine excretion as a short-term predictor in acute exacerbation of chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality and is characterized by episodes of acute exacerbations. Finding a systemic biomarker that reliably predicts outcome after an acute exacerbation remains a major challenge. Heat shock protein 27 (HSP27) has been previously studied in COPD, however, urine excretion trajectory and prognostic value after an exacerbation is unknown. METHODS: In this retrospective post hoc analysis of a prospective study that included 253 COPD patients who were hospitalized for acute exacerbation, 207 patients were analyzed. Urine and serum were sampled at admission, discharge, and 180 days after discharge; urine excretion trajectory was analyzed and correlated with clinicopathological and survival data. RESULTS: HSP27 urine excretion increased after an exacerbation episode [1.8% admission, 1.8% discharge, 2.3% 180 days after discharge (P=0.091)]. In severely ill patients (GOLD IV) this course was even more distinct [1.6% admission, 2.1% discharge, 2.8% 180 days after discharge (P=0.007)]. Furthermore, fractional HSP27 urine excretion at discharge was increased in GOLD IV patients (P=0.031). In Kaplan-Meier and univariable Cox proportional hazard models patients with HSP27 urine excretion below 0.845% showed significantly worse survival at 30, 90 and 180 days after discharge. In a multivariable Cox proportional hazard model including established COPD outcome parameters fractional HSP27 urine excretion remained a significant predictor of survival at 30 and 90 days after discharge. Comparing this model to our already published model that includes HSP27 serum concentration we could show that fractional HSP27 urine excretion performs better in short-term survival. CONCLUSIONS: Our findings provide novel information about fractional HSP27 urine excretion trajectory in acute exacerbation of COPD. Fractional HSP27 urine excretion may be significantly reduced during an episode of acute exacerbation in COPD patients and may be used as a predictor of short-term all-cause mortality.

Inflammatory immune response in recipients of transcatheter aortic valves.

Objective: Transcatheter aortic valve implantation (TAVI) is rapidly replacing cardiac surgery due to its minimal invasiveness and practicality. Midterm immunological studies on the biocompatibility of galactose-alpha-1,3-galactose (α-Gal)-carrying bioprosthetic heart valves for TAVI are not available. In this study we investigated whether bioprosthetic heart valves employed for TAVI augment an α-Gal-specific antibody-dependent and antibody-independent immune response 3 months after TAVI implantation. Methods: This prospective observational study included 27 patients with severe aortic valve stenosis undergoing TAVI and 10 patients with severe mitral valve regurgitation treated with a transcatheter MitraClip (Abbott Laboratories, Abbott Park, Ill) procedure. Blood samples were drawn before and 90 days after treatment at a routine checkup. Serum samples were analyzed using enzyme-linked immunosorbent assay. Serum concentrations of α-Gal-specific immunoglobulin (Ig) G, IgG subclasses and IgE, complement factor 3a, NETosis-specific citrullinated H3, and the systemic inflammation markers soluble suppression of tumorigenicity and interleukin 33 were evaluated. Results: Three months after TAVI, we found significantly increased serum concentrations of α-Gal-specific IgG3, complement factor complement factor 3a, citrullinated H3 levels, and soluble suppression of tumorigenicity (P = .002, P = .001, P = .025, and P = .039, respectively). Sensitization of α-Gal-specific IgE antibodies occurred in 55% of all patients after TAVI. Conclusions: Our results indicate that TAVI elicits a midterm, specific humoral immune response against α-Gal and causes an unspecific humoral inflammation compared with patients undergoing MitraClip implantation. This observation will lead to a better understanding of postintervention morbidity and the long-term durability of bioprostheses and indicates that caution is appropriate when designing implantation strategies for younger patients.

TGF-ß in the Secretome of Irradiated Peripheral Blood Mononuclear Cells Supports In Vitro Osteoclastogenesis.

Osteoclastogenesis required for bone remodeling is also a key pathologic mechanism of inflammatory osteolysis being controlled by paracrine factors released from dying cells. The secretome of irradiated, dying peripheral blood mononuclear cells (PBMCs) has a major impact on the differentiation of myeloid cells into dendritic cells, and macrophage polarization. The impact on osteoclastogenesis, however, has not been reported. For this aim, we used murine bone marrow macrophages exposed to RANKL and M-CSF to initiate osteoclastogenesis, with and without the secretome obtained from γ-irradiated PBMCs. We reported that the secretome significantly enhanced in vitro osteoclastogenesis as determined by means of histochemical staining of the tartrate-resistant acid phosphatase (TRAP), as well as the expression of the respective target genes, including TRAP and cathepsin K. Considering that TGF-ß enhanced osteoclastogenesis, we confirmed the TGF-ß activity in the secretome with a bioassay that was based on the increased expression of IL11 in fibroblasts. Neutralizing TGF-ß by an antibody decreased the ability of the secretome to support osteoclastogenesis. These findings suggested that TGF-ß released by irradiated PBMCs could enhance the process of osteoclastogenesis in vitro.

The inflammatory markers sST2, HSP27 and hsCRP as a prognostic biomarker panel in chronic heart failure patients.

BACKGROUND: The inflammatory markers sST2, HSP27 and hsCRP have already been identified as prognostic markers in chronic heart failure (HF). Though individual biomarkers have proven their value in mortality risk prediction, the role of a multimarker strategy needs further evaluation. MATERIALS AND METHODS: This is an exploratory reanalysis in chronic HF patients. Plasma HSP27, sST2 and hsCRP in outpatients with chronic HF were analysed. Patients were followed for a minimum of twelve months for the endpoint cardiovascular mortality and unplanned HF associated hospitalisation (=event). 15 year overall mortality was assessed retrospectively. The prognostic impact was assessed using a Cox proportional hazard model. RESULTS: 113 chronic HF patients were included. Median follow up time was 614 days and 37 patients (32.7%) experienced an event. A Kaplan-Meier analysis revealed that patients with increased sST2, HSP27 and hsCRP levels have significantly worse prognosis (p < 0.001). The use of a three-biomarker combination was superior in an independent risk prediction of an event (one high vs. two high: HR = 4.5, 95% CI: 1.3-15.5, p = 0.018; and one high vs. all high: HR = 9.8, 95% CI: 2.8-34.3, p < 0.001) as shown in a multivariable cox proportional hazard model. However, the biomarker panel did not predict 15 year overall mortality, in contrast to elevated HSP27 levels (p = 0.012). CONCLUSIONS: The combination of all three markers is an independent predictor of cardiovascular death and unplanned HF associated hospitalisation but not overall mortality. Our findings suggest that adding those markers in combination to well established risk assessment parameters may improve risk stratification.

Quantitative Hybrid Cardiac [18F]FDG-PET-MRI Images for Assessment of Cardiac Repair by Preconditioned Cardiosphere-Derived Cells.

Cardiosphere-derived cells (CDCs) are progenitor cells derived from heart tissue and have shown promising results in preclinical models. APOSEC, the secretome of irradiated peripheral blood mononuclear cells, has decreased infarct size in acute and chronic experimental myocardial infarction (MI). We enhanced the effect of CDCs with APOSEC preconditioning (apoCDC) and investigated the reparative effect in a translational pig model of reperfused MI. Supernatants of CDCs, assessed by proteomic analysis, revealed reduced production of extracellular matrix proteins after in vitro APOSEC preconditioning. In a porcine model of catheter-based reperfused anterior acute MI (AMI), CDCs with (apoCDC, n = 8) or without APOSEC preconditioning (CDC, n = 6) were infused intracoronary, 15 min after the start of reperfusion. Untreated AMI animals (n = 7) and sham procedures (n = 5) functioned as controls. 2-deoxy-2-(18 F)-fluoro-D-glucose-positron emission tomography-magnetic resonance imaging ([18F]FDG-PET-MRI), with late enhancement after 1 month, showed reduced scar volume and lower transmurality of the infarcted area in CDC and apoCDC compared to AMI controls. Segmental quantitative PET images displayed indicated more residual viability in apoCDC. The left-ventricle (LV) ejection fraction was improved nonsignificantly to 45.8% ± 8.6% for apoCDC and 43.5% ± 7.1% for CDCs compared to 38.5% ± 4.4% for untreated AMI. Quantitative hybrid [18F]FDG-PET-MRI demonstrated improved metabolic and functional recovery after CDC administration, whereas apoCDCs induced preservation of viability of the infarcted area.

Role for Lipids Secreted by Irradiated Peripheral Blood Mononuclear Cells in Inflammatory Resolution in Vitro.

Periodontal inflammation is associated with dying cells that potentially release metabolites helping to promote inflammatory resolution. We had shown earlier that the secretome of irradiated, dying peripheral blood mononuclear cells support in vitro angiogenesis. However, the ability of the secretome to promote inflammatory resolution remains unknown. Here, we determined the expression changes of inflammatory cytokines in murine bone marrow macrophages, RAW264.7 cells, and gingival fibroblasts exposed to the secretome obtained from γ-irradiated peripheral blood mononuclear cells in vitro by RT-PCR and immunoassays. Nuclear translocation of p65 was detected by immunofluorescence staining. Phosphorylation of p65 and degradation of IκB was determined by Western blot. The secretome of irradiated peripheral blood mononuclear cells significantly decreased the expression of IL1 and IL6 in primary macrophages and RAW264.7 cells when exposed to LPS or saliva, and of IL1, IL6, and IL8 in gingival fibroblasts when exposed to IL-1ß and TNFα. These changes were associated with decreased phosphorylation and nuclear translocation of p65 but not degradation of IκB in macrophages. We also show that the lipid fraction of the secretome lowered the inflammatory response of macrophages exposed to the inflammatory cues. These results demonstrate that the secretome of irradiated peripheral blood mononuclear cells can lower an in vitro simulated inflammatory response, supporting the overall concept that the secretome of dying cells promotes inflammatory resolution.

Therapeutic potential of lipids obtained from γ-irradiated PBMCs in dendritic cell-mediated skin inflammation.

BACKGROUND: Since numerous pathological conditions are evoked by unwanted dendritic cell (DC) activity, therapeutic agents modulating DC functions are of great medical interest. In regenerative medicine, cellular secretomes have gained increasing attention and valuable immunomodulatory properties have been attributed to the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs). Potential effects of the PBMC secretome (PBMCsec) on key DC functions have not been elucidated so far. METHODS: We used a hapten-mediated murine model of contact hypersensitivity (CH) to study the effects of PBMCsec on DCs in vivo. Effects of PBMCsec on human DCs were investigated in monocyte-derived DCs (MoDC) and ex vivo skin cultures. DCs were phenotypically characterised by transcriptomics analyses and flow cytometry. DC function was evaluated by cytokine secretion, antigen uptake, PBMC proliferation and T-cell priming. FINDINGS: PBMCsec significantly alleviated tissue inflammation and cellular infiltration in hapten-sensitized mice. We found that PBMCsec abrogated differentiation of MoDCs, indicated by lower expression of classical DC markers CD1a, CD11c and MHC class II molecules. Furthermore, PBMCsec reduced DC maturation, antigen uptake, lipopolysaccharides-induced cytokine secretion, and DC-mediated immune cell proliferation. Moreover, MoDCs differentiated with PBMCsec displayed diminished ability to prime naïve CD4+T-cells into TH1 and TH2 cells. Furthermore, PBMCsec modulated the phenotype of DCs present in the skin in situ. Mechanistically, we identified lipids as the main biomolecule accountable for the observed immunomodulatory effects. INTERPRETATION: Together, our data describe DC-modulatory actions of lipids secreted by stressed PBMCs and suggest PBMCsec as a therapeutic option for treatment of DC-mediated inflammatory skin conditions. FUNDING: This research project was supported by the Austrian Research Promotion Agency (Vienna, Austria; grant "APOSEC" 862068; 2015-2019) and the Vienna Business Agency (Vienna, Austria; grant "APOSEC to clinic" 2343727).

Viral safety of APOSECTM: a novel peripheral blood mononuclear cell derived-biological for regenerative medicine.

BACKGROUND: Viral reduction and inactivation of cell-derived biologicals is paramount for patients' safety and so viral reduction needs to be demonstrated to regulatory bodies in order to obtain marketing authorisation. Allogeneic human blood-derived biological medicinal products require special attention. APOSECTM, the secretome harvested from selected human blood cells, is a new biological with promising regenerative capabilities. We evaluated the effectiveness of inactivation of model viruses by methylene blue/light treatment, lyophilisation, and gamma irradiation during the manufacturing process of APOSECTM. MATERIALS AND METHODS: Samples of intermediates of APOSECTM were acquired during the manufacturing process and spiked with bovine viral diarrhoea virus (BVDV), human immunodeficiency virus type 1 (HIV-1), pseudorabies virus (PRV), hepatitis A virus (HAV), and porcine parvovirus (PPV). Viral titres were assessed with suitable cell lines. RESULTS: Methylene blue-assisted viral reduction is mainly effective against enveloped viruses: the minimum log10 reduction factors for BVDV, HIV-1, and PRV were ≥6.42, ≥6.88, and ≥6.18, respectively, with no observed residual infectivity. Viral titres of both HAV and PPV were not significantly reduced, indicating minor inactivation of non-enveloped viruses. Lyophilisation had minor effects on the viability of several enveloped model viruses. Gamma irradiation contributes to the viral safety by reduction of enveloped viruses (BVDV: ≥2.42; HIV-1: 4.53; PRV: ≥4.61) and to some degree of non-enveloped viruses as seen for HAV with a minimum log10 reduction factor of 2.92. No significant reduction could be measured for the non-enveloped virus PPV (2.60). DISCUSSION: Three manufacturing steps of APOSECTM were evaluated under Good Laboratory Practice conditions for their efficacy at reducing and inactivating potentially present viruses. It could be demonstrated that all three steps contribute to the viral safety of APOSECTM.

Heat shock protein 27 as a predictor of prognosis in patients admitted to hospital with acute COPD exacerbation.

Episodes of acute exacerbations are major drivers of hospitalisation and death from COPD. To date, there are no objective biomarkers of disease activity or biomarkers to predict patient outcome. In this study, 211 patients hospitalised for an acute exacerbation of COPD have been included. At the time of admission, routine blood tests have been performed including complete blood count, C-reactive protein, cardiac troponin T and NT-proBNP. Heat shock protein 27 (HSP27) serum concentrations were determined at time of admission, discharge and 180 days after discharge by ELISA. We were able to demonstrate significantly increased HSP27 serum concentrations in COPD patients at time of admission to hospital as compared to HSP27 concentrations obtained 180 days after discharge. In univariable Cox regression analyses, a HSP27 serum concentration ≥ 3098 pg/mL determined at admission was a predictor of all-cause mortality at 90 days, 180 days, 1 year and 3 years. In multivariable analyses, an increased HSP27 serum concentration at admission retained its prognostic ability with respect to all-cause mortality for up to 1-year follow-up. However, an increased HSP27 serum concentration at admission was not an independent predictor of long-term all-cause mortality at 3 years. Elevated serum HSP27 concentrations significantly predicted short-term mortality in patients admitted to hospital with acute exacerbation of COPD and could help to improve outcomes by identifying high-risk patients.

Systemic release of heat-shock protein 27 and 70 following severe trauma.

Trauma represents a major cause of morbidity and mortality worldwide. The endogenous inflammatory response to trauma remains not fully elucidated. Pro-inflammation in the early phase is followed by immunosuppression leading to infections, multi-organ failure and mortality. Heat-shock proteins (HSPs) act as intracellular chaperons but exert also extracellular functions. However, their role in acute trauma remains unknown. The aim of this study was to evaluate serum concentrations of HSP 27 and HSP 70 in severely injured patients. We included severely injured patients with an injury severity score of at least 16 and measured serum concentration of both markers at admission and on day two. We found significantly increased serum concentrations of both HSP 27 and HSP 70 in severely injured patients. Concomitant thoracic trauma lead to a further increase of both HSPs. Also, elevated concentrations of HSP 27 and HSP 70 were associated with poor outcome in these patients. Standard laboratory parameters did not correlate with neither HSP 27, nor with HSP 70. Our findings demonstrate involvement of systemic release of HSP 27 and HSP 70 after severe trauma and their potential as biomarker in polytraumatized patients.

Toxicological testing of allogeneic secretome derived from peripheral mononuclear cells (APOSEC): a novel cell-free therapeutic agent in skin disease.

A cell-free approach using secretomes derived from stem cells or peripheral blood mononuclear cells is an active area of regenerative medicine that holds promise for therapies. Regulatory authorities classify these secretomes as biological medicinal products, and non- clinical safety assessment thus falls under the scope of ICH S6. A secretome of stressed peripheral blood mononuclear cells (APOSEC) was successfully tested in a toxicology program, supporting clinical use of the new drug candidate. Here, to allow for topical, dermal treatment of patients with diabetic foot ulcer, several non-clinical safety studies were performed. Acute toxicity (single dose) and neuropharmacological screening were tested intravenously in a rat model. Risk for skin sensitisation was tested in mice. A 4-week intravenous toxicity study in mice and a 4-week subcutaneous toxicity study in minipigs were conducted to cover the clinical setting and application in a rodent and a non-rodent model. Acute and repeated-dose toxicity studies show that APOSEC administered intravenously and subcutaneously does not involve major toxicities or signs of local intolerance at levels above the intended total human maximal dose of 3.3 U/kg/treatment, 200 U/wound/treatment, and 100 U/cm2/treatment. The non-clinical data support the safe topical use of APOSEC in skin diseases related to deficient wound healing.

Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions.

Secretomes from various cell sources exert strong regenerative activities on numerous organs, including the skin. Although secretomes consist of many diverse components, a growing body of evidence suggests that small extracellular vesicles (EVs) account for their regenerative capacity. We previously demonstrated that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs) exhibits wound healing capacity. Therefore, we sought to dissect the molecular composition of EVs present in the secretome and compared wound healing-related activities of these EVs to other subfractions of the secretome and the fully supplemented secretome (MNCaposec). Compared to EVs derived from non-irradiated PBMCs, γ-irradiation significantly increased the size and number and changed the composition of released EVs. Detailed characterization of the molecular components of EVs, i.e. miRNA, proteins, and lipids, derived from irradiated PBMCs revealed a strong association with regenerative processes. Reporter gene assays and aortic ring sprouting assays revealed diminished activity of the subfractions compared to MNCaposec. In addition, we showed that MNCaposec accelerated wound closure in a diabetic mouse model. Taken together, our results suggest that secretome-based wound healing represents a promising new therapeutic avenue, and strongly recommend using the complete secretome instead of purified subfractions, such as EVs, to exploit its full regenerative capacity.

Increased serum concentrations of soluble ST2 predict mortality after burn injury.

BACKGROUND: Large burn injuries induce a systemic response in affected patients. Soluble ST2 (sST2) acts as a decoy receptor for interleukin-33 (IL-33) and has immunosuppressive effects. sST2 has been described previously as a prognostic serum marker. Our aim was to evaluate serum concentrations of sST2 and IL-33 after thermal injury and elucidate whether sST2 is associated with mortality in these patients. METHODS: We included 32 burn patients (total body surface area [TBSA] >10%) admitted to our burn intensive care unit and compared them to eight healthy probands. Serum concentrations of sST2 and IL-33 were measured serially using an enzyme-linked immunosorbent assay (ELISA) technique. RESULTS: The mean TBSA was 32.5%±19.6%. Six patients (18.8%) died during the hospital stay. Serum analyses showed significantly increased concentrations of sST2 and reduced concentrations of IL-33 in burn patients compared to healthy controls. In our study cohort, higher serum concentrations of sST2 were a strong independent predictor of mortality. CONCLUSIONS: Burn injuries cause an increment of sST2 serum concentrations with a concomitant reduction of IL-33. Higher concentrations of sST2 are associated with increased in-hospital mortality in burn patients.