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OBJECTIVES: Granulocyte transfusions have been used to treat immune cell dysfunction in sepsis. As granulocyte transfusions can trigger tissue injury via local effects of neutrophils, we hypothesized that extracorporeal treatment of plasma using granulocytes would prove beneficial while having less side effects. DESIGN: Prospective controlled three-armed animal study. SETTING: Research laboratory. SUBJECTS: Twenty-one female immature pigs (7.5-12 kg, 7-9 weeks old). INTERVENTIONS: Three groups of spontaneously breathing, sedated pigs (n = 7 each) received an intravenous lethal dose of live Staphylococcus aureus over 1 hour. Although group I had no specific treatment (control), group II and III were subsequently treated for 4 hours with an extracorporeal device containing either no cells (sham control, group II) or human cell line-derived granulocytic cells (group III). Survival time and physiologic, biochemical, and hematologic parameters were monitored for 7 days. MEASUREMENTS AND MAIN RESULTS: All animals of group I died during the observation time (mean survival time: 70 hours). In group II, two of seven and in group III, six of seven animals survived the observation time (mean survival: 75 and 168 hours, respectively). Survival differences were significant between group I and III (p < 0.001) and between group II and III (p < 0.05) but not between group I and II (p = 0.43). Furthermore, group differences in bacterial blood concentrations, differential blood count, blood gases, lactate, and interleukins were observed. The extracorporeal cell treatment was well tolerated by the animals. CONCLUSIONS: Extracorporeal therapy with granulocytic cells significantly improved survival in a pig model of sepsis. Further studies with this approach are encouraged. [ABSTRACT FROM AUTHOR]

Abstract Background Hemorrhagic shock/resuscitation is associated with aberrant neutrophil activation and organ failure. This experimental porcine study was done to evaluate the effects of Fas-directed extracorporeal immune therapy with a leukocyte inhibition module (LIM) on hemodynamics, neutrophil tissue infiltration, and tissue damage after hemorrhagic shock/resuscitation. Methods In a prospective controlled double-armed animal trial 24 Munich Mini Pigs (30.3 ± 3.3 kg) were rapidly haemorrhaged to reach a mean arterial pressure (MAP) of 35 ± 5 mmHg, maintained hypotensive for 45 minutes, and then were resuscitated with Ringer' solution to baseline MAP. With beginning of resuscitation 12 pigs underwent extracorporeal immune therapy for 3 hours (LIM group) and 12 pigs were resuscitated according to standard medical care (SMC). Haemodynamics, haematologic, metabolic, and organ specific damage parameters were monitored. Neutrophil infiltration was analyzed histologically after 48 and 72 hours. Lipid peroxidation and apoptosis were specifically determined in lung, bowel, and liver. Results In the LIM group, neutrophil counts were reduced versus SMC during extracorporeal immune therapy. After 72 hours, the haemodynamic parameters MAP and cardiac output (CO) were significantly better in the LIM group. Histological analyses showed reduction of shock-related neutrophil tissue infiltration in the LIM group, especially in the lungs. Lower amounts of apoptotic cells and lipid peroxidation were found in organs after LIM treatment. Conclusions Transient Fas-directed extracorporeal immune therapy may protect from posthemorrhagic neutrophil tissue infiltration and tissue damage.

Background: Immune cell dysfunction plays a central role in sepsis-induced immunoparalysis. Targeted treatment using healthy donor immune cell transfusions, particularly granulocyte concentrates (GC) potentially induces tissue damage. Initial trials using GC in an extracorporeal immune cell perfusion system provided evidence for beneficial effects with fewer side effects, by separating patient and donor immune cell compartments. A multicenter clinical trial is exploring feasibility and effects of a 6-h treatment (NCT06143137). This ex vivo study examines technical feasibility and cellular effects of an extended treatment interval up to 24 h., Methods: Standard GC were purified to increase the potential storage time and subsequently implemented in the extracorporeal immune cell perfusion system. Parameters assessed included cell viability, phagocytosis activity, oxidative burst, cytokine release, and metabolic parameters of purified. GC during an extended circulation time of up to 24 h., Results: After storage of 72 h granulocytes were viable throughout the study period and exhibited preserved functionality and metabolic activity. The findings highlight a time-dependent nature of cytokine release by neutrophils in the extracorporeal circuit, as cytokine secretion patterns showed IL-8 peaking within 6 h, while MCP-1, IL-6, IL-1β, and TNF-α increased after 24 h of circulation., Conclusion: Purified GC remain functional after 72 h of storage and additional 24 h in the circulating treatment model. Cytokine secretion patterns revealed a significant increase, especially between 10 and 24 h of treatment. Extending treatment time holds promise for enhancing immune response against sepsis-induced immunoparalysis. These findings provide valuable insights for optimizing immune-targeted therapeutic interventions., Competing Interests: Declaration of conflicting interestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: TW, BH, SM, and JA are employees or shareholders of ARTCLINE GmbH. All other authors declare that they have no competing interests.

Background: Immune cell dysfunction is a central part of immune paralysis in sepsis. Granulocyte concentrate (GC) transfusions can induce tissue damage via local effects of neutrophils. The hypothesis of an extracorporeal plasma treatment with granulocytes is to show beneficial effects with fewer side effects. Clinical trials with standard GC have supported this approach. This ex vivo study investigated the functional properties of purified granulocyte preparations during the extracorporeal plasma treatment., Methods: Purified GC were stored for up to 3 days and compared with standard GC in an immune cell perfusion therapy model. The therapy consists of a plasma separation device and an extracorporeal circuit. Plasma is perfused through the tubing system with donor immune cells of the GC, and only the treated plasma is filtered for re-transfusion. The donor immune cells are retained in the extracorporeal system and discarded after treatment. Efficacy of granulocytes regarding phagocytosis, oxidative burst as well as cell viability and metabolic parameters were assessed., Results: In pGC, the metabolic surrogate parameters of cell functionality showed comparable courses even after a storage period of 72 h. In particular, glucose and oxygen consumption were lower after extended storage. The course of lactate dehydrogenase concentration yields no indication of cell impairment in the extracorporeal circulation. The cells were viable throughout the entire study period and exhibited preserved phagocytosis and oxidative burst functionality., Conclusion: The granulocytes demonstrated full functionality in the 6 h extracorporeal circuits after 3 days storage and in septic shock plasma. This is demonstrating the functionality of the system and encourages further clinical studies., (© 2023 The Authors. Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

Sepsis is a leading cause of morbidity and mortality worldwide. Dysregulated immune response to infection is a hallmark of sepsis, leading to life-threatening organ dysfunction or even death. Advancing knowledge of the complex pathophysiological mechanisms has been a strong impetus for the development of therapeutic strategies aimed at rebalancing the immune response by modulating the excess of both pro- and anti-inflammatory mediators. There is a wealth of preclinical data suggesting clinical benefits of various extracorporeal techniques in an attempt to modulate the exaggerated host inflammatory response. However, the evidence base is often weak. Owing to both an advancing comprehension of the pathophysiology and the increased quality of clinical trials, progress has been made in establishing extracorporeal therapies as part of the general therapeutic canon in sepsis. We aim for a comprehensive overview of the technical aspects and clinical applications in the context of the latest evidence concerning these techniques., (© 2022 The Authors. Therapeutic Apheresis and Dialysis published by John Wiley & Sons Australia, Ltd on behalf of International Society for Apheresis and Japanese Society for Apheresis.)

Background & Aims: Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach., Methods: We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice., Results: In vitro analysis proved the vaccine's stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40 °C for 3 months and at 25 °C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4 °C/-80 °C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8 + T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice., Conclusion: SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide., Impact and Implications: Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world., Competing Interests: UP is a co-founder and shareholder of SCG Cell Therapy, and an ad hoc scientific advisor to Abbvie, Arbutus, Biontech, Gilead, GSK, J&J, Roche, Sanofi, Sobi, Vaccitech and VIR Biotechnology. UP and AK are named as inventors on a patent application describing the therapeutic vaccination scheme of TherVacB (PCT/EP2017/050553). KK, MS, JA are or were employees of LEUKOCARE AG. UP is member of the advisory board of LEUKOCARE AG. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Authors.)

Background: Immune cell dysfunction plays a central role in sepsis-associated immune paralysis. The transfusion of healthy donor immune cells, i.e., granulocyte concentrates (GC) potentially induces tissue damage via local effects of neutrophils. Initial clinical trials using standard donor GC in a strictly extracorporeal bioreactor system for treatment of septic shock patients already provided evidence for beneficial effects with fewer side effects, by separating patient and donor immune cells using plasma filters. In this ex vivo study, we demonstrate the functional characteristics of a simplified extracorporeal therapy system using purified granulocyte preparations., Methods: Purified GC were used in an immune cell perfusion model prefilled with human donor plasma simulating a 6-h treatment. The extracorporeal circuit consisted of a blood circuit and a plasma circuit with 3 plasma filters (PF). PF1 is separating the plasma from the patient's blood. Plasma is then perfused through PF2 containing donor immune cells and used in a dead-end mode. The filtrated plasma is finally retransfused to the blood circuit. PF3 is included in the plasma backflow as a redundant safety measure. The donor immune cells are retained in the extracorporeal system and discarded after treatment. Phagocytosis activity, oxidative burst and cell viability as well as cytokine release and metabolic parameters of purified GCs were assessed., Results: Cells were viable throughout the study period and exhibited well-preserved functionality and efficient metabolic activity. Course of lactate dehydrogenase and free hemoglobin concentration yielded no indication of cell impairment. The capability of the cells to secret various cytokines was preserved. Of particular interest is equivalence in performance of the cells on day 1 and day 3, demonstrating the sustained shelf life and performance of the immune cells in the purified GCs., Conclusion: Results demonstrate the suitability of a simplified extracorporeal system. Furthermore, granulocytes remain viable and highly active during a 6-h treatment even after storage for 3 days supporting the treatment of septic patients with this system in advanced clinical trials., (© 2022. The Author(s).)

Background: Use of donor granulocyte concentrate (GC) has been limited due to its short storage time of 6-24 h, which is partially due to residual red blood cells (RBCs) and platelets and the resulting lactate production leading to an acidotic milieu. To increase this storage time, we developed a closed system procedure compatible with standard blood bank technologies to remove RBC and platelets and to enrich the GC., Methods: Standard GCs (sGCs) were sedimented, washed twice with 0.9% sodium chloride (NaCl), and resuspended in blood group-identical fresh frozen plasma. The resulting purified GCs (pGCs) were then stored in platelet bags at a cell concentration of about 5 × 10 7  ± 1.8 × 10 7 leukocytes/ml without agitation at room temperature for up to 72 h. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst were monitored daily., Results: A significant reduction in RBC (98%) through sedimentation, and platelets (96%) by washing, purified the white blood cell (WBC) population and enriched the granulocytes to 96% of the WBC in the pGC. After 72 h of storage, over 90% of the initial WBC count of pGC remained, was viable (≥97%), and the granulocytes exhibited a high phagocytosis and oxidative burst functionality, comparable to sGC after 24 h., Conclusion: Purification extends the maximum storage period of GC from 24 to 72 h and may therefore improve the availability of GC and its clinical use., (© 2021 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Specifically tailored amino acid-based formulations were previously shown to have a high potential to avoid stress-mediated degradation of complex molecules such as monoclonal antibodies and viral vectors. By using adenovirus 5 (Ad5) as a model, we studied whether such formulations may also efficiently protect viral vectors in thermal stress experiments and during long-term liquid storage. Algorithm-based amino acid preselection using an excipient database and subsequent application of design of experiments (DoE) in combination with a 37°C challenging model enabled the prediction of long-term storage stability of Ad5. By statistical analysis of the Ad5 infectivity, amino acids with significant influence on Ad5 stability were detected after 2 and 3 weeks of liquid storage at 37°C. Ad5 formulations comprising positively selected amino acids did not reveal any loss of infectivity after 24 months in liquid storage at 5°C. By contrast, a 2 log reduction after 3 months and complete loss of infectivity after 18 months was observed with a standard viral vector formulation. By an optimization round, we designed a simple and well-balanced formulation avoiding MgCl 2 , previously considered essential in Ad5 formulations. This work demonstrates the efficacy of an algorithm-based development approach in the formulation development for viral vectors., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

A granulocyte bioreactor for the extracorporeal treatment was developed to enhance the immune cell function in patients with severe sepsis. The influence of oxygenation on the used cells was tested in a prospective clinical study. Ten patients with severe sepsis were treated twice with the granulocyte bioreactor. The used cells were screened for functionality; values of blood gases, glucose and lactate were obtained from the recirculating bioreactor circuit. Five patients were treated with an oxygenator setup (Oxy group), five without oxygenator (Non-Oxy group). The overall in-hospital mortality was 50%. Significantly lower values of oxygen saturation, partial oxygen pressure, lactate, oxyburst and phagocytosis were seen in the Non-Oxy group compared with the Oxy group in the bioreactor circuit. Further studies with this approach are encouraged and should focus on the influence of oxygenation on production of reactive oxygen species and cytokines of used cells., (© 2018 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.)

To develop highly concentrated therapeutic antibodies enabling convenient subcutaneous application, well stabilizing pharmaceutical formulations with low viscosities are considered to be key. The purpose of this study is to select specific amino acid combinations that reduce and balance aggregation, fragmentation and chemical degradation, and also lower viscosity of highly concentrated liquid antibodies. As a model, the therapeutically well-established antibody trastuzumab (25->200 mg mL -1 ) in liquid formulation is used. Pre-testing of formulations based on a stabilizing and protecting solutions (SPS®) platform is conducted in a thermal unfolding model using differential scanning fluorimetry (DSF) and accelerated aging at 37 and 45 °C. Pre-selected amino acid combinations are further iteratively adjusted to obtain stable highly concentrated antibody formulations with low viscosity. Size exclusion chromatography (SE-HPLC) reveals significantly lower aggregation and fragmentation at specific amino acid:sugar and protein:excipient ratios. Dynamic viscosities <20 mPa * s of highly concentrated trastuzumab (≥200 mg mL -1 ) are measured by falling ball viscosimetry. Moreover, less chemical degradation is found by cationic exchange chromatography (CEX-HPLC) even after 6 months liquid storage at 25 °C. In conclusion, specifically tailored and advanced amino acid-based liquid formulations avoid aggregation and enable the development of stable and low viscous highly concentrated biopharmaceuticals., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Liver dysfunction (LD) and liver failure are associated with poor outcome in critically ill patients. In patients with severe sepsis or septic shock, LD occurred in nearly 19% of patients. An early diagnosis of LD at time of initial damage of the liver can lead to a better prognosis of these patients because an early start of therapy is possible. We performed a second prospective study with septic patients to test a new cell-based cytotoxicity device (biosensor) to evaluate clinical relevance for early diagnosis of LD and prognostic capacity. In the clinical study, 99 intensive care unit patients were included in two groups. From the patients of the septic group ( n  = 51, SG), and the control (non-septic) group [ n  = 49, control group (CG)] were drawn 20 ml blood at inclusion, after 3, and 7 days for testing with the biosensor. Patients' data were recorded for hospital survival, organ function, and demographic data, illness severity [acute physiology and chronic health evaluation (APACHE) II-, sepsis-related organ failure assessment (SOFA) scores], cytokines, circulating-free deoxyribonucleic acid/neutrophil-derived extracellular traps (cf-DNA/NETs), microbiological results, and pre-morbidity. For the developed cytotoxicity test, the human liver cell line HepG2/C3A was used. Patients' plasma was incubated in a microtiter plate assay with the test cells and after 6 days incubation the viability (trypan blue staining, XTT-test) and functionality (synthesis of albumin, cytochrome 1A2 activity) was analyzed. An impairment of viability and functionality of test cells was only seen in the SG compared with the CG. The plasma of non-survivors in the SG led to a more pronounced impairment of test cells than the plasma of survivors at inclusion. In addition, the levels of cf-DNA/NETs were significantly higher in the SG at inclusion, after 3, and after 7 days compared with the CG. The SG showed an in-hospital mortality of 24% and the values of bilirubin, APACHE II-, and SOFA scores were markedly higher at inclusion than in the CG. Hepatotoxicity of septic plasma was already detected with the liver cell-based biosensor at inclusion and also in the course of disease. The biosensor may be a tool for early diagnosis of LD in septic patients and may have prognostic relevance.

Purpose. Granulocyte transfusions have been used to treat immune cell dysfunction in sepsis. A granulocyte bioreactor for the extracorporeal treatment of sepsis was tested in a prospective clinical study focusing on the dosage of norepinephrine in patients and influence on dynamic and cell based liver tests during extracorporeal therapies. Methods and Patients. Ten patients with severe sepsis were treated twice within 72 h with the system containing granulocytes from healthy donors. Survival, physiologic parameters, extended hemodynamic measurement, and the indocyanine green plasma disappearance rate (PDR) were monitored. Plasma of patients before and after extracorporeal treatments were tested with a cell based biosensor for analysis of hepatotoxicity. Results. The observed mortality rate was 50% during stay in hospital. During the treatments, the norepinephrine-dosage could be significantly reduced while mean arterial pressure was stable. In the cell based analysis of hepatotoxicity, the viability and function of sensor-cells increased significantly during extracorporeal treatment in all patients and the PDR-values increased significantly between day 1 and day 7 only in survivors. Conclusion. The extracorporeal treatment with donor granulocytes showed promising effects on dosage of norepinephrine in patients, liver cell function, and viability in a cell based biosensor. Further studies with this approach are encouraged.

Currently, the need for cooled storage and the impossibility of terminal sterilisation are major drawbacks in vaccine manufacturing and distribution. To overcome current restrictions a preclinical safety and efficacy study was conducted to evaluate new influenza A vaccine formulations regarding thermal resistance, resistance against irradiation-mediated damage and storage stability. We evaluated the efficacy of novel antigen stabilizing and protecting solutions (SPS) to protect influenza A(H1N1)pdm09 split virus antigen under experimental conditions in vitro and in vivo. Original or SPS re-buffered vaccine (Pandemrix) was spray-dried and terminally sterilised by irradiation with 25 kGy (e-beam). Antigen integrity was monitored by SDS-PAGE, dynamic light scattering, size exclusion chromatography and functional haemagglutination assays. In vitro screening experiments revealed a number of highly stable compositions containing glycyrrhizinic acid (GA) and/or chitosan. The most stable composition was selected for storage tests and in vivo assessment of seroconversion in non-human primates (Macaca fascicularis) using a prime-boost strategy. Redispersed formulations with original adjuvant were administered intramuscularly. Storage data revealed high stability of protected vaccines at 4°C and 25°C, 60% relative humidity, for at least three months. Animals receiving original Pandemrix exhibited expected levels of seroconversion after 21 days (prime) and 48 days (boost) as assessed by haemagglutination inhibition and microneutralisation assays. Animals vaccinated with spray-dried and irradiated Pandemrix failed to exhibit seroconversion after 21 days whereas spray-dried and irradiated, SPS-protected vaccines elicited similar seroconversion levels to those vaccinated with original Pandemrix. Boost immunisation with SPS-protected vaccine resulted in a strong increase in seroconversion but had only minor effects in animals treated with non SPS-protected vaccine. In conclusion, utilising the SPS formulation technology, spray-drying and terminal sterilisation of influenza A(H1N1)pdm09 split virus vaccine is feasible. Findings indicate the potential utility of such formulated vaccines e.g. for needle-free vaccination routes and delivery to countries with uncertain cold chain facilities., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In an extracorporeal combination therapy, the impact of different replacement fluids on survival was tested in a bacterial sepsis model in pigs. In an animal study 19 pigs, weighing 7.5-11.1 kg, were included. All groups received an intravenous lethal dose of live Staphylococcus aureus over 1 h. The animals were treated by an extracorporeal circuit consisting of online centrifugation and subsequent plasma filtration for 4 h. The extracorporeal circuit was pre-filled with 400 mL replacement fluid. In the P0 group 100% hydroxyethyl starch 130/0.4 was used as replacement fluid; in the P30 group 30% pig plasma and 70% hydroxyethyl starch; and in the P100 group 100% pig plasma. The observation time was 7 days. All animals of the group P100 survived, while all animals of group P0 and five out of seven animals of the P30 group died during the observation time. Extracorporeal therapy consisting of online centrifugation and plasma filtration with 100% pig plasma as replacement fluid significantly improved survival in a pig model of sepsis. Further studies with this approach are encouraged., (© 2012 The Authors. Therapeutic Apheresis and Dialysis © 2012 International Society for Apheresis.)

The impact on survival of a combination of plasma separation by centrifugation and subsequent plasma filtration was tested in a bacterial sepsis model in pigs. In this animal study 19 pigs were included. Groups II and III received an intravenous lethal dose of live Staphylococcus aureus over 1 h; group I received saline (non-septic control--NC). Groups I and II were treated by an extracorporeal circuit consisting of online centrifugation and subsequent plasma filtration (group II: treated group--TG) for 4 h; group III had no specific treatment (septic control, SC). The observation time was 7 days. All animals of group I (NC) and group II (TG) survived, while all animals of group III (SC) died during the observation time. Extracorporeal therapy with online centrifugation and plasma filtration significantly improved survival in a pig model of sepsis. Further studies with this approach are encouraged., (© 2012 The Authors. Therapeutic Apheresis and Dialysis © 2012 International Society for Apheresis.)

Objective and Design: The development of liver failure is a major problem in septic patients. In this prospective clinical experimental study the hepatotoxicity of plasma from septic and non-septic patients was tested., Methods and Subjects: The basic test components consist of human liver cells (HepG2/C3A) used in a standardized microtiter plate assay. After incubation with patient's plasma viability of cells (XTT-test), the cytochrome 1A2 activity and synthesis of micro albumin were measured. Subjects (28) enrolled comprise the septic shock group (SSG, n=10), the non-septic group (NSG, n=5) and the healthy volunteers group (HVG, n=13)., Results: The 28-day mortality was 30% in the SSG. The APACHE II-, SOFA-, and SAPS-scores and the values of bilirubin and prothrombin time as INR were significantly higher in the SSG than in the NSG. The cytochrome 1A2 activity and the release of albumin were significantly reduced in HepG2/C3A cells incubated with plasma of the SSG (p<0.05). The cytochrome 1A2 activities were higher in survivors compared to non-survivors at the time point 0 and were increasing in survivors and decreasing in non-survivors within 54 h in the SSG. In the SSG there was a significant decrease in IL-10 and IL-8 between inclusion and 54 h. Values of IL-6, TNF alpha and IL-10 were significantly lower in the NSG compared with the values of the SSG at inclusion and after 54 h., Conclusion: The plasma of patients with septic shock impaired cellular functions of HepG2/C3A cells.

The pathophysiology leading to delayed wound healing is complex and efficient therapeutic approaches for accelerated wound healing currently do not exist. We developed a novel drug-eluting platform for the potential use in wound dressings. Here, we report on the potential of eluting ascorbic acid-2-phosphate (ASC-2P), a highly stable variant of ascorbic acid, to induce angiogenesis and to promote collagen synthesis by fibroblasts. The drug-eluting platform device (DEPD) consists of biocompatible polymeric layers comprising polyethylene terephtalate, polyvinyl alcohol (PVA), and polyurethane with PVA as the solvent for ASC-2P. The angiogenic potential of ASC-2P was evaluated in the endothelial cell tube formation assay (TFA) and in the chorion allantoic membrane (CAM) model. Collagen synthesis by ASC-2P-stimulated fibroblasts was determined by Sirius Red staining. ASC-2P significantly induced angiogenesis in five independent TFA and CAM assays and induced collagen synthesis in two different fibroblast cell lines. The eluting kinetics of ASC-2P was determined by the ultraviolet NanoDrop method and the functional 2,2'-Azinobis-(3-ethylbenzthiazolin-6-sulfonic acid) method. Eluting profiles showed a continuous release in the range of biologically effective concentrations >10 days. This is the first report showing the proangiogenic- and collagen-promoting features of ASC-2P. DEPD loaded with ASC-2P ought to be further evaluated as wound dressings or as supplementary pads for topical treatment of delayed wound healing in preclinical studies., (© 2011 by the Wound Healing Society.)

Posttrauma apoptosis resistance of neutrophils (PMN) is related to overshooting immune responses, systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Recently, we have shown that the apoptosis resistance in circulating PMN from severely injured patients which is known to be mediated by high serum levels of pro-inflammatory cytokines can be overcome by the activation of Fas death receptor. Here, we aimed to study whether stimulation of surface Fas leads to the inactivation of hyperactivated PMN from critically ill patients with SIRS. PMN from 23 multiple trauma patients (mean injury severity score (ISS) 34±1.9) were isolated at day 1 after admission to the trauma center. PMN from 17 volunteer blood donors served as controls. Neutrophil activity has been determined after ex vivo short (1 h) and long-term (4 h) stimulation of freshly isolated PMN with immobilized agonistic anti-Fas antibodies. We found neutrophil chemotactic migration in response to IL-8, phagocytosis and oxidative burst to be significantly inhibited in control cells already after short-term (1 h) Fas stimulation. In contrast, inactivation of trauma PMN by agonistic anti-Fas antibodies was found to be efficient only after long-term (4 h) incubation of cells with agonistic antibodies. Thus, in trauma PMN down-regulation of neutrophil activity seems to be delayed when compared to cells isolated from healthy controls, suggesting impaired susceptibility for Fas stimulation in these cells. Interestingly, whereas Fas-mediated inhibition of phagocytosis and oxidative burst could be prevented by the broad range caspase inhibitor t-butoxycarbonyl-aspartyl(O-methyl)-fluoromethyl ketone (BocD-fmk), the chemotactic activity in response to IL-8 was unaffected. In conclusion, we demonstrate that stimulation of neutrophil Fas does not only initiate apoptosis but also induces inhibition of neutrophil functions, partially by non-apoptotic signaling., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Introduction: Neutrophil granulocytes are the first defense line in bacterial infections. However, granulocytes are also responsible for severe local tissue impairment. In order to use donor granulocytes, but at the same time to avoid local side effects, we developed an extracorporeal immune support system. This first-in-man study investigated whether an extracorporeal plasma treatment with a granulocyte bioreactor is tolerable in patients with septic shock. A further intention was to find suitable efficacy end-points for subsequent controlled trials., Methods: The trial was conducted as a prospective uncontrolled clinical phase I/II study with 28-day follow-up at three university hospital intensive care units. Ten consecutive patients (five men, five women, mean age 60.3 ± 13.9 standard deviation (SD) years) with septic shock with mean ICU entrance scores of Acute Physiology and Chronic Health Evaluation (APACHE) II of 29.9 ± 7.2 and of Simplified Acute Physiology Score (SAPS) II of 66.2 ± 19.5 were treated twice within 72 hours for a mean of 342 ± 64 minutes/treatment with an extracorporeal bioreactor containing 1.41 ± 0.43 × 10E10 granulocytes from healthy donors. On average, 9.8 ± 2.3 liters separated plasma were treated by the therapeutic donor cells. Patients were followed up for 28 days., Results: Tolerance and technical safety during treatment, single organ functions pre/post treatment, and hospital survival were monitored. The extracorporeal treatments were well tolerated. During the treatments, the bacterial endotoxin concentration showed significant reduction. Furthermore, noradrenaline dosage could be significantly reduced while mean arterial pressure was stable. Also, C-reactive protein, procalcitonin, and human leukocyte antigen DR (HLA-DR) showed significant improvement. Four patients died in the hospital on days 6, 9, 18 and 40. Six patients could be discharged., Conclusions: The extracorporeal treatment with donor granulocytes appeared to be well tolerated and showed promising efficacy results, encouraging further studies., Trial Registration: ClinicalTrials.gov Identifier: NCT00818597.

The predictive value of circulating free DNA/neutrophil extracellular traps (cf-DNA/NETs) has recently been shown in patients with major trauma for sepsis, multiple organ failure, and mortality. Here we report on the predictive potential of cf-DNA/NETs for mortality in patients with severe burn injury. In a prospective study 32 patients with severe burn injury were included. Blood samples were sequentially obtained on day 1, 3, 5, and 7 after admission. cf-DNA/NETs was directly quantified from plasma by means of rapid fluorescence assay. Time kinetics of cf-DNA/NETs were correlated with clinical data, C-reactive protein (CRP), procalcitonin (PCT), and interleukin (IL)-6. Furthermore sensitivity, specificity, and positive and negative predictive value, as well as receiver operation characteristic (ROC) curves were calculated. Seven patients died within the first month after burn injury. cf-DNA/NETs values from these patients were significantly increased already on day 1 and 3 after admission compared with patients who survived (p < 0.01). In contrast, PCT levels of nonsurvivors were significantly elevated on day 3 and 5 (p < 0.01), while CRP and IL-6 did not show any significant difference between survivors and nonsurvivors. At a cutoff of 255 ng/ml, cf-DNA/NETs had sensitivity of 0.8 and specificity of 0.74. ROC revealed largest areas under the curve (AUC) for cf-DNA/NETs on day 1 (0.851) and 3 (0.883) after admission. For all values between day 1 and 7, AUC was 0.815. cf-DNA/NETs seems to be a rapid, valuable marker for prediction of mortality in burn patients. A larger confirmation trial ought to be carried out.

Kynurenine, the major degradation product of tryptophan has been shown to directly damage various tissues. Its potential contribution to septic arthritis is unknown. In this study, we analyzed the putative diagnostic value of kynurenine for bacterial joint infection and its potential harmful effects on cartilage. In a prospective study 41 patients with a joint effusion who had undergone arthrocentesis were included. Tryptophan and kynurenine levels from synovial fluid were quantified by HPLC. Diagnostic value of kynurenine was evaluated and its effects on the proliferation of the chondrocyte cell line ATDC5 were determined. Synovial fluid kynurenine values from patients with septic arthritis (4.1 ± 0.8 µmol/L, n = 9) were significantly increased compared to patients with non-infectious inflammatory arthropathy (1.8 ± 0.2 µmol/L, n = 17) or osteoarthritis (1.2 ± 0.1 µmol/L, n = 15, p < 0.01). At a cut-off value of 2.28 µmol/L kynurenine had a sensitivity of 0.89 and a specificity of 0.87. Further, kynurenine inhibited chondrocyte (ATDC5) cell proliferation in a dose-dependent manner. Septic arthritis is associated with significantly increased values of synovial kynurenine. Furthermore kynurenine inhibits proliferation of chondrocytes, which strongly suggests a pathophysiological effect of kynurenine on cartilage in inflammatory arthropathies., (© 2010 Orthopaedic Research Society.)

Conventional treatment of severe viral disease is limited by the narrow choice as well as the often-significant side effects or lack of clear efficacy of antiviral chemotherapy. At the same time, however, it is known that a reduction in viral load leads to significant clinical improvement in a number of important viral diseases. In this paper we discuss the possibility of using preconditioned human phagocytes in an extracorporeal biohybrid system for adsorption of viral pathogens. We present data from in vitro experiments testing adsorption of an enterovirus and of hepatitis B virus (HBV) by a preconditioned human promyelocytic cell line. While no clearance of HBV could be detected, the results revealed a near elimination of enterovirus with the cell line displaying robust viability. Enterovirus titers of 1000 (reciprocal) were reduced to a mean titer of 10(0.6) CCID(50) with no virus detectable after adsorption in two out of five samples. Titers of 10000 (reciprocal) were in turn reduced to a mean of 10(1.4) CCID(50). The kinetics of the process was remarkable with this near elimination of the pathogen occurring within only 15min. Extracorporeal viral adsorption by a cellular biohybrid system appears feasible. Pairing target pathogens with suitable cell lines may offer a versatile antiviral technology., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Hemorrhagic shock/resuscitation is associated with aberrant neutrophil activation and organ failure. This experimental porcine study was done to evaluate the effects of Fas-directed extracorporeal immune therapy with a leukocyte inhibition module (LIM) on hemodynamics, neutrophil tissue infiltration, and tissue damage after hemorrhagic shock/resuscitation., Methods: In a prospective controlled double-armed animal trial 24 Munich Mini Pigs (30.3 +/- 3.3 kg) were rapidly haemorrhaged to reach a mean arterial pressure (MAP) of 35 +/- 5 mmHg, maintained hypotensive for 45 minutes, and then were resuscitated with Ringer' solution to baseline MAP. With beginning of resuscitation 12 pigs underwent extracorporeal immune therapy for 3 hours (LIM group) and 12 pigs were resuscitated according to standard medical care (SMC). Haemodynamics, haematologic, metabolic, and organ specific damage parameters were monitored. Neutrophil infiltration was analyzed histologically after 48 and 72 hours. Lipid peroxidation and apoptosis were specifically determined in lung, bowel, and liver., Results: In the LIM group, neutrophil counts were reduced versus SMC during extracorporeal immune therapy. After 72 hours, the haemodynamic parameters MAP and cardiac output (CO) were significantly better in the LIM group. Histological analyses showed reduction of shock-related neutrophil tissue infiltration in the LIM group, especially in the lungs. Lower amounts of apoptotic cells and lipid peroxidation were found in organs after LIM treatment., Conclusions: Transient Fas-directed extracorporeal immune therapy may protect from posthemorrhagic neutrophil tissue infiltration and tissue damage.

The systemic inflammatory response syndrome and subsequent organ failure are mainly driven by activated neutrophils with prolonged life span, which is believed to be due to apoptosis resistance. However, detailed underlying mechanisms leading to neutrophil apoptosis resistance are largely unknown, and possible therapeutic options to overcome this resistance do not exist. Here we report that activated neutrophils from severely injured patients exhibit cell death resistance due to impaired activation of the intrinsic apoptosis pathway, as evidenced by limited staurosporine-induced mitochondrial membrane depolarization and decreased caspase-9 activity. Moreover, we found that these neutrophils express high levels of antiapoptotic Mcl-1 and low levels of proapoptotic Bax protein. Mcl-1 up-regulation was dependent on elevated concentrations of GM-CSF in patient serum. Accordingly, increased Mcl-1 protein stability and GM-CSF serum concentrations were shown to correlate with staurosporine-induced apoptosis resistance. However, cross-linking of neutrophil Fas by immobilized agonistic anti-Fas IgM resulted in caspase-dependent mitochondrial membrane depolarization and apoptosis induction. In conclusion, the observed impairment of the intrinsic pathway and the resulting apoptosis resistance may be overcome by immobilized agonistic anti-Fas IgM. Targeting of neutrophil Fas by immobilized agonistic effector molecules may represent a new therapeutic tool to limit neutrophil hyperactivation and its sequelae in patients with severe immune disorders.

Neutrophil extracellular traps (NETs) have recently been discovered as a central part of antimicrobial innate immunity. In the meanwhile, evidence accumulated that NETs are also generated upon non-infectious stimuli in various clinical settings. In acute or chronic inflammatory disorders aberrantly enhanced NET formation and/or decreased NET degradation seems to correlate with disease outcome. This review summarizes current knowledge about the relation of NETs in a broad spectrum of clinical settings. Specifically, we focus on the importance of NETs as a predictive marker in severely ill patients and further, we speculate about the potential pathophysiology of NETs.

The release of "neutrophil extracellular traps" (NETs) has been identified as a novel immune response in innate immunity. NETs are composed of neutrophil-derived circulating free DNA (cf-DNA) and neutrophil cytoplasm-derived proteins such as proteases. In this study, we analyzed the putative diagnostic value of synovial cf-DNA/NETs for identification of septic arthritis. Forty-two patients with a joint effusion who had undergone arthrocentesis were included. From synovial fluid, cf-DNA/NETs (j-cf-DNA) levels were directly quantified. Diagnostic value of j-cf-DNA was compared with white blood cells (WBC), synovial white blood cells (j-WBC), C-reactive protein (CRP), j-IL-6, j-TNF alpha, j-IL-1 beta, and myeloperoxidase (j-MPO). Sensitivity, specificity, positive and negative predictive value, as well as ROC-curves for each parameter were calculated. Synovial fluid cf-DNA/NETs values from patients with septic arthritis (3,286 +/- 386 ng/ml, n = 9) were significantly increased compared to patients with noninfectious joint inflammation (1,040 +/- 208 ng/ml, n = 17) or osteoarthritis (278 +/- 34 ng/ml, n = 16, p < 0.01). In conjunction with j-cf-DNA, j-IL-6 and j-IL-1 beta were significantly elevated (p < 0.01), but WBC, CRP, and j-WBC were not. At a cut-off of 300 ng/ml, j-cf-DNA had a sensitivity of 0.89, a specificity of 1.0, a positive predictive value of 1.0, and a negative predictive value of 0.97. Receiver operation curves revealed largest areas under the curve for cf-DNA/NETs (0.933) and j-IL-6 (0.951). cf-DNA/NETs seem to be a valuable additional marker for the diagnosis of septic arthritis or periprosthetic infections. However, this result should be confirmed in a large clinical trial., ((c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Granulocyte dysfunction is a central component of immunodeficiency in septic patients. Granulocyte transfusions appear to be pathophysiologically useful; however, they cause unwanted side-effects in the lungs and other organs. This study evaluates the safety of an extracorporeal immune support system with granulocytic cells in a rat model of Gram-negative sepsis. Three groups of male CD rats received either saline (control group, I), a dose of Escherichia coli O7:K1 lethal to 90% of the animals (LD90) (septic group, II), or an LD90 dose of E. coli that was incubated with the human promyelocytic leukemia cell line (HL-60) (differentiated into the granulocytic direction) for 20 min prior to infusion (second septic group, III). The animals were observed for seven days. Pre-treatment with HL-60 cells resulted in no adverse effects in the group III animals. Significantly lower bacterial counts and endotoxin levels in the plasma were detected after 24 h as compared to group II (P < 0.05). Group III animals had better weight gain and more stable hemodynamics than group II animals (P < 0.01). Seven day survival was 0/8 in group II, 6/8 in group III, and 8/9 in group I (log-rank test: II-III: P < 0.001). The data suggest that extracorporeal use of granulocytes allows the therapeutic use of these cells while avoiding unwanted effects resulting from direct contact to internal organs.

Kynurenine, the major degradation product of tryptophan has been shown to directly damage tissues, but its possible contribution to posttraumatic morbidity is unknown. Here, we studied the kinetics of kynurenine in patients after major trauma and whether this correlates with the development of posttraumatic sepsis. Kynurenine and tryptophan levels of 60 multiple-injured patients with Injury Severity Score of more than 16 were quantified prospectively by high-performance liquid chromatography. Blood samples were obtained daily from admission until day 10 after admission. Significantly increased kynurenine values were detectable already at day 1 after admission in blood from patients who later developed sepsis, regardless of injury pattern (P < 0.01). In contrast, kynurenine values of nonsepsis patients remained low throughout the observation period. However, all patients exhibited significantly decreased tryptophan values versus healthy controls (P < 0.01). Moreover, significantly increased kynurenine-tryptophan ratios rapidly predicted subsequent sepsis, multiple organ failure, and death (P < 0.01). Both increased kynurenine values and kynurenine-tryptophan ratios predicted posttraumatic development of sepsis and organ failure. This ought to be validated in subsequent studies.