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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: Granulocyte concentrates (GCs) are usually prepared by single-donor apheresis after G-CSF pretreatment and have to be transfused within 24 h after cell collection because of the rapid decrease in pH and cell survival due to high lactate production by red blood cell contamination. GCs pooled from buffy coats of whole blood donations could improve the availability of these products. Methods to reduce red blood cell and platelet contamination may improve storability. We developed a manufacturing process for pooled GCs and investigated cell viability and functionality over time. Methods: Six ABO blood group-identical buffy coats were pooled. Subsequently, the red blood cells spontaneously sedimented after the addition of hydroxyethyl starch. The resulting leukocyte-enriched supernatant was washed twice with saline to reduce platelets and was resuspended in ABO-identical donor plasma. The leukocyte concentrate was transferred to a platelet storage bag and stored up to 72 h at 20–24°C w/o agitation. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst activity were monitored. Results: The number of red blood cells and platelets was reduced to 0.4% and 6.1% of the baseline levels. About 50% of the original present leukocytes could be extracted (n = 76). In the course of 72 h of storage, there were no significant changes in white blood cell counts (p = 0.12). The viability exceeded 98% during the entire period. The rate of granulocytes performing phagocytosis and oxidative burst remained above 95% anytime. Conclusion: GCs prepared from pooled buffy coats provide a precious alternative to granulocytes obtained from apheresis. Reduction of red blood cells and platelets by more than 90% extends the maximum shelf life of GCs from 24 h to 72 h. For a therapeutic dose of at least 1 × 1010 granulocytes, 15–20 buffy coats are required. [ABSTRACT FROM AUTHOR]

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: 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.)

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.)