High cysteine concentrations in cell culture media lead to oxidative stress and reduced bioprocess performance of recombinant CHO cells.

Cysteine is considered an essential amino acid in the cultivation of Chinese hamster ovary (CHO) cells. An optimized cysteine supply during fed-batch cultivation supports the protein production capacity of recombinant CHO cell lines. However, we observed that CHO production cell lines seeded at low cell densities in chemically defined media enriched with cysteine greater than 2.5 mm resulted in markedly reduced cell growth during passaging, hampering seed train performance and scale-up. To investigate the underlying mechanism, seeding cell densities and initial cysteine concentrations ranging from low to high cysteine concentrations were varied followed by an analysis of cell culture performance. Additionally, cell cycle analysis, intracellular quantification of reactive oxygen species (ROS) as well as transcriptomic analyses by next-generation sequencing were carried out. Our results demonstrate that CHO cells seeded at low cell densities at high initial cysteine concentrations encountered increased oxidative stress leading to a p21-mediated cell cycle arrest in the G1/S phase. The resulting oxidative stress caused redox imbalance in the endoplasmic reticulum and activation of the unfolded protein response as well as the major antioxidant nuclear factor-like 2 response pathways. Potential signature genes related to oxidative stress and the inhibition of the pentose phosphate pathway were identified in the study. Finally, the study presents that seeding cells at a higher concentration counteract oxidative stress in cysteine-enriched cell culture media.

The systematic use of evidence-based methodologies and technologies enhances shared decision-making in the 2018 International Consensus Conference on Patient Blood Management.

BACKGROUND AND OBJECTIVES: Patient Blood Management (PBM) aims to optimize the care of patients who might need a blood transfusion. The International Consensus Conference on PBM (ICC-PBM) aimed to develop evidence-based recommendations on three topics: preoperative anaemia, red blood cell transfusion thresholds and implementation of PBM programmes. This paper reports how evidence-based methodologies and technologies were used to enhance shared decision-making in formulating recommendations during the ICC-PBM. MATERIALS & METHODS: Systematic reviews on 17 PICO (Population, Intervention, Comparison, Outcomes) questions were conducted by a Scientific Committee (22 international topic experts and one methodologist) according to GRADE (Grades of Recommendation, Assessment, Development and Evaluation) methodology. Evidence-based recommendations were formulated using Consensus Development Conference methodology. RESULTS: We screened 17 607 references and included 145 studies. The overall certainty in the evidence of effect estimates was generally low or very low. During the ICC, plenary sessions (100-200 stakeholders from a range of clinical disciplines and community representatives) were followed by closed sessions where multidisciplinary decision-making panels (>50 experts and patient organizations) formulated recommendations. Two chairs (content-expert and methodologist) moderated each session and two rapporteurs documented the discussions. The Evidence-to-Decision template (GRADEpro software) was used as the central basis in the process of formulating recommendations. CONCLUSION: This ICC-PBM resulted in 10 clinical and 12 research recommendations supported by an international stakeholder group of experts in blood transfusion. Systematic, rigorous and transparent evidence-based methodology in a formal consensus format should be the new standard to evaluate (cost-) effectiveness of medical treatments, such as blood transfusion.

Patient Blood Management: Recommendations From the 2018 Frankfurt Consensus Conference.

IMPORTANCE: Blood transfusion is one of the most frequently used therapies worldwide and is associated with benefits, risks, and costs. OBJECTIVE: To develop a set of evidence-based recommendations for patient blood management (PBM) and for research. EVIDENCE REVIEW: The scientific committee developed 17 Population/Intervention/Comparison/Outcome (PICO) questions for red blood cell (RBC) transfusion in adult patients in 3 areas: preoperative anemia (3 questions), RBC transfusion thresholds (11 questions), and implementation of PBM programs (3 questions). These questions guided the literature search in 4 biomedical databases (MEDLINE, EMBASE, Cochrane Library, Transfusion Evidence Library), searched from inception to January 2018. Meta-analyses were conducted with the GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) methodology and the Evidence-to-Decision framework by 3 panels including clinical and scientific experts, nurses, patient representatives, and methodologists, to develop clinical recommendations during a consensus conference in Frankfurt/Main, Germany, in April 2018. FINDINGS: From 17 607 literature citations associated with the 17 PICO questions, 145 studies, including 63 randomized clinical trials with 23 143 patients and 82 observational studies with more than 4 million patients, were analyzed. For preoperative anemia, 4 clinical and 3 research recommendations were developed, including the strong recommendation to detect and manage anemia sufficiently early before major elective surgery. For RBC transfusion thresholds, 4 clinical and 6 research recommendations were developed, including 2 strong clinical recommendations for critically ill but clinically stable intensive care patients with or without septic shock (recommended threshold for RBC transfusion, hemoglobin concentration <7 g/dL) as well as for patients undergoing cardiac surgery (recommended threshold for RBC transfusion, hemoglobin concentration <7.5 g/dL). For implementation of PBM programs, 2 clinical and 3 research recommendations were developed, including recommendations to implement comprehensive PBM programs and to use electronic decision support systems (both conditional recommendations) to improve appropriate RBC utilization. CONCLUSIONS AND RELEVANCE: The 2018 PBM International Consensus Conference defined the current status of the PBM evidence base for practice and research purposes and established 10 clinical recommendations and 12 research recommendations for preoperative anemia, RBC transfusion thresholds for adults, and implementation of PBM programs. The relative paucity of strong evidence to answer many of the PICO questions supports the need for additional research and an international consensus for accepted definitions and hemoglobin thresholds, as well as clinically meaningful end points for multicenter trials.

Patient blood management: recommendations rrom the 2018 Frankfurt consensus conference

What is the current evidence base for patient blood management (PBM) in adults, and what international clinical recommendations can be derived for preoperative anemia, red blood cell transfusion thresholds, and PBM implementation strategies? Diagnosis and management of preoperative anemia is crucial, and iron-deficient anemia should be treated with iron supplementation. Red blood cell transfusion thresholds for critically ill, clinically stable patients (hemoglobin concentration <7 g/dL), patients undergoing cardiac surgery (hemoglobin concentration <7.5 g/dL), patients with hip fractures and cardiovascular disease or risk factors (hemoglobin concentration <8 g/dL), and hemodynamically stable patients with acute gastrointestinal bleeding (hemoglobin concentration 7-8 g/dL) are relatively well defined, although the quality of evidence is moderate to low. Further high-quality research to support PBM is required for a range of clinical scenarios and implementation of PBM programs.

Liberal transfusion strategy to prevent mortality and anaemia-associated, ischaemic events in elderly non-cardiac surgical patients - the study design of the LIBERAL-Trial.

BACKGROUND: Perioperative anaemia leads to impaired oxygen supply with a risk of vital organ ischaemia. In healthy and fit individuals, anaemia can be compensated by several mechanisms. Elderly patients, however, have less compensatory mechanisms because of multiple co-morbidities and age-related decline of functional reserves. The purpose of the study is to evaluate whether elderly surgical patients may benefit from a liberal red blood cell (RBC) transfusion strategy compared to a restrictive transfusion strategy. METHODS: The LIBERAL Trial is a prospective, randomized, multicentre, controlled clinical phase IV trial randomising 2470 elderly (≥ 70 years) patients undergoing intermediate- or high-risk non-cardiac surgery. Registered patients will be randomised only if Haemoglobin (Hb) reaches ≤9 g/dl during surgery or within 3 days after surgery either to the LIBERAL group (transfusion of a single RBC unit when Hb ≤ 9 g/dl with a target range for the post-transfusion Hb level of 9-10.5 g/dl) or the RESTRICTIVE group (transfusion of a single RBC unit when Hb ≤ 7.5 g/dl with a target range for the post-transfusion Hb level of 7.5-9 g/dl). The intervention per patient will be followed until hospital discharge or up to 30 days after surgery, whichever occurs first. The primary efficacy outcome is defined as a composite of all-cause mortality, acute myocardial infarction, acute ischaemic stroke, acute kidney injury (stage III), acute mesenteric ischaemia and acute peripheral vascular ischaemia within 90 days after surgery. Infections requiring iv antibiotics with re-hospitalisation are assessed as important secondary endpoint. The primary endpoint will be analysed by logistic regression adjusting for age, cancer surgery (y/n), type of surgery (intermediate- or high-risk), and incorporating centres as random effect. DISCUSSION: The LIBERAL-Trial will evaluate whether a liberal transfusion strategy reduces the occurrence of major adverse events after non-cardiac surgery in the geriatric population compared to a restrictive strategy within 90 days after surgery. TRIAL REGISTRATION: ClinicalTrials.gov (identifier: NCT03369210 ).

Patient Blood Management is Associated With a Substantial Reduction of Red Blood Cell Utilization and Safe for Patient's Outcome: A Prospective, Multicenter Cohort Study With a Noninferiority Design.

OBJECTIVE: To determine whether the implementation of patient blood management (PBM) is effective to decrease the use of red blood cell without impairment of patient's safety. BACKGROUND: The World Health Organization encouraged all member states to implement PBM programs employing multiple combined strategies to increase and preserve autologous erythrocyte volume to minimize red blood cell transfusions. Data regarding safety issues are not sufficiently available. METHODS: In this prospective, multicenter study, surgical inpatients from four German University Hospitals were analyzed before (pre-PBM) and after the implementation of PBM. PBM program included multiple measures (ie, preoperative optimization of hemoglobin levels, blood-sparing techniques, and standardization of transfusion practice). Primary aim was to show noninferiority of the PBM cohort with a margin of 0.5%. Secondary endpoints included red blood cell utilization. RESULTS: A total of 129,719 patients discharged between July 2012 and June 2015 with different inclusion periods for pre-PBM (54,513 patients) and PBM (75,206 patients) were analyzed. The primary endpoint was 6.53% in the pre-PBM versus 6.34% in the PBM cohort. The noninferiority aim was achieved (P < 0.001). Incidence of acute renal failure decreased in the PBM cohort (2.39% vs 1.67%; P < 0.001, regression model). The mean number of red blood cell transfused per patient was reduced from 1.21 ±â€Š0.05 to 1.00 ±â€Š0.05 (relative change by 17%, P < 0.001). CONCLUSIONS: The data presented show that implementation of PBM with a more conscious handling of transfusion practice can be achieved even in large hospitals without impairment of patient's safety. Further studies should elucidate which PBM measures are most clinically and cost effective. TRIAL REGISTRATION: PBM-Study ClinicalTrials.gov, NCT01820949.

A randomized placebo-controlled phase Ia malaria vaccine trial of two virosome-formulated synthetic peptides in healthy adult volunteers.

BACKGROUND AND OBJECTIVES: Influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. The aim of the study was to proof the concept that virosomes can also be used to elicit high titers of antibodies against synthetic peptides. The specific objective was to demonstrate the safety and immunogenicity of two virosome-formulated P. falciparum protein derived synthetic peptide antigens given in two different doses alone or in combination. METHODOLOGY/PRINCIPAL FINDINGS: The design was a single blind, randomized, placebo controlled, dose-escalating study involving 46 healthy Caucasian volunteers aged 18-45 years. Five groups of 8 subjects received virosomal formulations containing 10 microg or 50 microg of AMA 49-CPE, an apical membrane antigen-1 (AMA-1) derived synthetic phospatidylethanolamine (PE)-peptide conjugate or 10 ug or 50 ug of UK39, a circumsporozoite protein (CSP) derived synthetic PE-peptide conjugate or 50 ug of both antigens each. A control group of 6 subjects received unmodified virosomes. Virosomal formulations of the antigens (designated PEV301 and PEV302 for the AMA-1 and the CSP virosomal vaccine, respectively) or unmodified virosomes were injected i. m. on days 0, 60 and 180. In terms of safety, no serious or severe adverse events (AEs) related to the vaccine were observed. 11/46 study participants reported 16 vaccine related local AEs. Of these 16 events, all being pain, 4 occurred after the 1(st), 7 after the 2(nd) and 5 after the 3(rd) vaccination. 6 systemic AEs probably related to the study vaccine were reported after the 1(st) injection, 10 after the 2(nd) and 6 after the 3(rd). Generally, no difference in the distribution of the systemic AEs between either the doses applied (10 respectively 50 microg) or the synthetic antigen vaccines (PEV301 and PEV302) used for immunization was found. In terms of immunogenicity, both PEV301 and PEV302 elicited already after two injections a synthetic peptide-specific antibody response in all volunteers immunized with the appropriate dose. In the case of PEV301 the 50 microg antigen dose was associated with a higher mean antibody titer and seroconversion rate than the 10 microg dose. In contrast, for PEV302 mean titer and seroconversion rate were higher with the lower dose. Combined delivery of PEV301 and PEV302 did not interfere with the development of an antibody response to either of the two antigens. No relevant antibody responses against the two malaria antigens were observed in the control group receiving unmodified virosomes. CONCLUSIONS: The present study demonstrates that three immunizations with the virosomal malaria vaccine components PEV301 or/and PEV302 (containing 10 microg or 50 microg of antigen) are safe and well tolerated. At appropriate antigen doses seroconversion rates of 100% were achieved. Two injections may be sufficient for eliciting an appropriate immune response, at least in individuals with pre-existing anti-malarial immunity. These results justify further development of a final multi-stage virosomal vaccine formulation incorporating additional malaria antigens. TRIAL REGISTRATION: ClinicalTrials.gov NCT00400101.

Effective inhibition of experimental metastasis and prolongation of survival in mice by a potent factor Xa-specific synthetic serine protease inhibitor with weak anticoagulant activity.

Clinical and experimental evidence suggests that the blood coagulation system is involved in the dissemination of malignant tumors. Consequently, anticoagulant agents have been tested as metastasis suppressors in experimental models. Recently, we have found a close correlation between factor Xa (FXa)-specificity of a series of synthetic serine protease inhibitors and their anti-metastatic potential in a murine T-cell lymphoma metastasis model. Interference of such inhibitors with blood-coagulation may represent a major experimental and clinical obstacle. Here, we test anti-metastatic effects of a recently developed, highly specific 3-amidinophenylalanine-type FXa inhibitor, WX-FX4, with weaker anticoagulant activity when compared to well-established FXa inhibitors, such as DX-9065a, as measured by the activated partial thromboplastin time, prothrombin time, prothrombinase complex activity, and coagulation time. Treatment of mice with WX-FX4 (1.5 mg/kg twice daily) led to significant reduction of experimental liver metastasis of a syngeneic T-cell lymphoma in DBA/2 mice (> 90%), and of experimental lung metastasis of a human fibrosarcoma in CD1 nu/nu mice (> 60%). Due to its relatively low anticoagulant activity, daily treatment over 100 days was possible, leading to significant survival benefits without inducing bleeding anomalities. FXa-inhibitors with highly efficient anti-metastatic potential without coagulation-related side effects may represent important new tools as anticancer agents.

Fresh frozen plasma in patients with disseminated intravascular coagulation or in patients with liver diseases.

Disseminated intravascular coagulation (DIC) and liver diseases are complex clinical conditions. Both disorders frequently disturb the finely tuned coagulation and fibrinolysis equilibrium. In DIC, a wide range of underlying disorders can induce a systemic activation of the coagulation system with generation of soluble fibrin, possible deposition of platelet-rich fibrin clots in the microvasculature and subsequent micro- or macroembolism, impaired organ perfusion and organ failure. Such coagulation activation depletes platelets, coagulation factors, and inhibitors and clinically can result in severe, sometimes untreatable bleeding, especially when bone marrow or liver function is diminished or invasive procedures are performed. In addition, a secondary counterbalancing activation of the fibrinolytic system to dissolve microcirculatory clots adds to the bleeding tendency. In conjunction with other options based on prompt and rigorous treatment of the underlying cause of DIC, fresh frozen plasma plays an important role in therapeutic management when overt bleeding is present or anticipated in DIC patients with disturbed coagulation or when an invasive procedure is being planned. In liver disease, factor and inhibitor synthesis in both the coagulation and fibrinolytic system is impaired, both quantitatively and qualitatively. This destabilizes the balance between the two systems. In addition, the clearance of activated coagulation factors and fibrin(ogen) degradation products (FDP) from the systemic circulation is impaired. In patients with liver diseases and acute or imminent bleeding, or before invasive procedures, fresh frozen plasma (FFP) offers advantages over clotting factor concentrates. However, hypervolemia following the required doses of FFP might pose a problem in some liver disease patients.The complex pathophysiology both in DIC and in liver disease requires early diagnosis and adequate management including plasma and platelet substitution after treatment of the underlying disease. Due to the heterogeneity of DIC and liver disease, prospective randomized trials are difficult to perform. Therefore, treatment recommendations are mostly empirical and less evidence-based. Therapy must be accompanied by close and repeated clinical and laboratory monitoring.