Salzburger Landeskliniken

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV‐mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd‐sourcing, drawing on the unique EV expertise of academia‐based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

BACKGROUND: Despite consensus on the need for blood cholesterol reductions to prevent coronary heart disease (CHD), available evidence on optimal cholesterol levels or the added predictive value of additional lipids is sparse. METHODS AND RESULTS: After 10 years follow-up of 12 339 middle-aged participants free of CHD in the Atherosclerosis Risk in Communities Study (ARIC), 725 CHD events occurred. The lowest incidence was observed in those at the lowest LDL cholesterol (LDL-C) quintile, with medians of 88 mg/dL in women and 95 mg/dL in men, and risk accelerated at higher levels, with relative risks (RRs) for the highest quintile of 2.7 in women and 2.5 in men. LDL-C, HDL-C, lipoprotein(a) [Lp(a)], and in women but not men, triglycerides (TG) were all independent CHD predictors, providing an RR, together with blood pressure, smoking, and diabetes, of 13.5 in women and 4.9 in men. Lp(a) was less significant in blacks than whites. Prediction was not enhanced by HDL-C density subfractions or apolipoproteins (apo) A-I or B. Despite strong univariate associations, apoB did not contribute to risk prediction in subgroups with elevated TG, with lower LDL-C, or with high apoB relative to LDL-C. CONCLUSIONS: Optimal LDL-C values are <100 mg/dL in both women and men. LDL-C, HDL-C, TG, and Lp(a), without additional apolipoproteins or lipid subfractions, provide substantial CHD prediction, with much higher RR in women than men.

INTRODUCTION: The appropriate strategy for trauma-induced coagulopathy management is under debate. We report the treatment of major trauma using mainly coagulation factor concentrates. METHODS: This retrospective analysis included trauma patients who received >or= 5 units of red blood cell concentrate within 24 hours. Coagulation management was guided by thromboelastometry (ROTEM). Fibrinogen concentrate was given as first-line haemostatic therapy when maximum clot firmness (MCF) measured by FibTEM (fibrin-based test) was <10 mm. Prothrombin complex concentrate (PCC) was given in case of recent coumarin intake or clotting time measured by extrinsic activation test (EXTEM) >1.5 times normal. Lack of improvement in EXTEM MCF after fibrinogen concentrate administration was an indication for platelet concentrate. The observed mortality was compared with the mortality predicted by the trauma injury severity score (TRISS) and by the revised injury severity classification (RISC) score. RESULTS: Of 131 patients included, 128 received fibrinogen concentrate as first-line therapy, 98 additionally received PCC, while 3 patients with recent coumarin intake received only PCC. Twelve patients received FFP and 29 received platelet concentrate. The observed mortality was 24.4%, lower than the TRISS mortality of 33.7% (P = 0.032) and the RISC mortality of 28.7% (P > 0.05). After excluding 17 patients with traumatic brain injury, the difference in mortality was 14% observed versus 27.8% predicted by TRISS (P = 0.0018) and 24.3% predicted by RISC (P = 0.014). CONCLUSIONS: ROTEM-guided haemostatic therapy, with fibrinogen concentrate as first-line haemostatic therapy and additional PCC, was goal-directed and fast. A favourable survival rate was observed. Prospective, randomized trials to investigate this therapeutic alternative further appear warranted.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways.

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

INTRODUCTION: Thromboelastometry (TEM)-guided haemostatic therapy with fibrinogen concentrate and prothrombin complex concentrate (PCC) in trauma patients may reduce the need for transfusion of red blood cells (RBC) or platelet concentrate, compared with fresh frozen plasma (FFP)-based haemostatic therapy. METHODS: This retrospective analysis compared patients from the Salzburg Trauma Centre (Salzburg, Austria) treated with fibrinogen concentrate and/or PCC, but no FFP (fibrinogen-PCC group, n = 80), and patients from the TraumaRegister DGU receiving ≥ 2 units of FFP, but no fibrinogen concentrate/PCC (FFP group, n = 601). Inclusion criteria were: age 18-70 years, base deficit at admission ≥ 2 mmol/L, injury severity score (ISS) ≥ 16, abbreviated injury scale for thorax and/or abdomen and/or extremity ≥ 3, and for head/neck < 5. RESULTS: For haemostatic therapy in the emergency room and during surgery, the FFP group (ISS 35.5 ± 10.5) received a median of 6 units of FFP (range: 2, 51), while the fibrinogen-PCC group (ISS 35.2 ± 12.5) received medians of 6 g of fibrinogen concentrate (range: 0, 15) and 1200 U of PCC (range: 0, 6600). RBC transfusion was avoided in 29% of patients in the fibrinogen-PCC group compared with only 3% in the FFP group (P< 0.001). Transfusion of platelet concentrate was avoided in 91% of patients in the fibrinogen-PCC group, compared with 56% in the FFP group (P< 0.001). Mortality was comparable between groups: 7.5% in the fibrinogen-PCC group and 10.0% in the FFP group (P = 0.69). CONCLUSIONS: TEM-guided haemostatic therapy with fibrinogen concentrate and PCC reduced the exposure of trauma patients to allogeneic blood products.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.

OBJECTIVE To evaluate whether the sodium–glucose cotransporter 2 inhibitor empagliflozin (EMPA) reduces liver fat content (LFC) in recent-onset and metabolically well-controlled type 2 diabetes (T2D). RESEARCH DESIGN AND METHODS Patients with T2D (n = 84) (HbA1c 6.6 ± 0.5% [49 ± 10 mmol/mol], known disease duration 39 ± 27 months) were randomly assigned to 24 weeks of treatment with 25 mg daily EMPA or placebo. The primary end point was the difference of the change in LFC as measured with magnetic resonance methods from 0 (baseline) to 24 weeks between groups. Tissue-specific insulin sensitivity (secondary outcome) was assessed by two-step clamps using an isotope dilution technique. Exploratory analysis comprised circulating surrogate markers of insulin sensitivity and liver function. Statistical comparison was done by ANCOVA adjusted for respective baseline values, age, sex, and BMI. RESULTS EMPA treatment resulted in a placebo-corrected absolute change of −1.8% (95% CI −3.4, −0.2; P = 0.02) and relative change in LFC of −22% (−36, −7; P = 0.009) from baseline to end of treatment, corresponding to a 2.3-fold greater reduction. Weight loss occurred only with EMPA (placebo-corrected change −2.5 kg [−3.7, −1.4]; P &amp;lt; 0.001), while no placebo-corrected change in tissue-specific insulin sensitivity was observed. EMPA treatment also led to placebo-corrected changes in uric acid (−74 mol/L [−108, −42]; P &amp;lt; 0.001) and high-molecular-weight adiponectin (36% [16, 60]; P &amp;lt; 0.001) levels from 0 to 24 weeks. CONCLUSIONS EMPA effectively reduces hepatic fat in patients with T2D with excellent glycemic control and short known disease duration. Interestingly, EMPA also decreases circulating uric acid and raises adiponectin levels despite unchanged insulin sensitivity. EMPA could therefore contribute to the early treatment of nonalcoholic fatty liver disease in T2D.

Obesity and associated metabolic disorders have become highly prevalent diseases worldwide, and the human gut microbiota, due to its influence on host energy metabolism, has been attributed an important role therein. This pilot study explores host-microbiota relationships in men and women affected by various types of glucose metabolism disorder. Among 20 individuals aged 58 to 71 years with either normal glucose tolerance, prediabetes, or type 2 diabetes mellitus the gut bacterial communities were compared based on barcoded 454 sequencing of 16S rRNA genes amplified from stool samples. We found that specific microbiota groups were relatively enriched or reduced in different metabolic states. Further, positive or negative associations with clinical manifestations of metabolic disease suggest that these organisms indicate and possibly contribute to metabolic impairment or health. For instance, a higher prevalence of Erysipelotrichaceae and Lachnospiraceae was found associated with metabolic disorders, and the Holdemania and Blautia genera correlated with clinical indicators of an impaired lipid and glucose metabolism. The Bacteroidetes and groups therein, by contrast, displayed inverse relationships with metabolic disease parameters and were found relatively enriched in participants not diagnosed with metabolic syndrome or obesity. Further, the prevalence of specific Clostridia and Rikenellaceae members also pointed towards a healthier metabolic state. Links with diet as an intermediate factor included positive and negative associations of Lachnospiraceae with relative consumption rates of fat and carbohydrates, respectively, and positive associations of Turicibacteraceae with the consumption of protein. Identifying critical roles of major gut microbiota components in metabolic disorders has important translational implications regarding the prevention and treatment of metabolic diseases by means of preventing or reversing dysbiosis and by controlling exacerbating diet and life style factors particularly in sensitive population groups.

OBJECTIVE: To assess the potential role of FoxP3-expressing regulatory T cells (Tregs) in reversing obesity-linked insulin resistance and diabetic nephropathy in rodent models and humans. RESEARCH DESIGN AND METHODS: To characterize the role of Tregs in insulin resistance, human visceral adipose tissue was first evaluated for Treg infiltration and second, the db/db mouse model was evaluated. RESULTS: Obese patients with insulin resistance displayed significantly decreased natural Tregs but an increase in adaptive Tregs in their visceral adipose tissue as compared with lean control subjects. To further evaluate the pathogenic role of Tregs in insulin resistance, the db/db mouse model was used. Treg depletion using an anti-CD25 monoclonal antibody enhanced insulin resistance as shown by increased fasting blood glucose levels as well as an impaired insulin sensitivity. Moreover, Treg-depleted db/db mice developed increased signs of diabetic nephropathy, such as albuminuria and glomerular hyperfiltration. This was paralleled by a proinflammatory milieu in both murine visceral adipose tissue and the kidney. Conversely, adoptive transfer of CD4(+)FoxP3(+) Tregs significantly improved insulin sensitivity and diabetic nephropathy. Accordingly, there was increased mRNA expression of FoxP3 as well as less abundant proinflammatory CD8(+)CD69(+) T cells in visceral adipose tissue and kidneys of Treg-treated animals. CONCLUSIONS: Data suggest a potential therapeutic value of Tregs to improve insulin resistance and end organ damage in type 2 diabetes by limiting the proinflammatory milieu.

A common 936 C/T polymorphism in the gene for the vascular endothelial growth factor (VEGF) has been associated with VEGF plasma levels. In our case-control study, we investigated the role of this polymorphism for breast cancer risk. VEGF genotype was determined in 500 women with breast cancer and 500 sex- and age-matched healthy control subjects. Carriers of a 936T-allele were more frequent among controls (29.4%) than among patients (17.6%; p = 0.000014). The odds ratio for carriers of a 936T-allele for breast cancer was 0.51 (95% confidence interval 0.38-0.70). Additionally, VEGF plasma levels were determined in 21 nonsmoking post-menopausal controls; carriers of a 936T allele had significantly lower levels (median 23 pg/ml; range 6-50 pg/ml) than noncarriers (37; 21-387; p = 0.034). We conclude that carriers of a VEGF 936T-allele are at decreased risk for breast cancer, this, however, requiring further confirmation in a larger study.

BACKGROUND: Serum anti-Müllerian hormone (AMH) levels provide a powerful means for predicting ovarian response, which is reflected not only by the size of the primordial follicle pool but also by the quality of the oocytes. Considering a mutual interdependence between AMH-expressing somatic cells and gametes, this prospective morphological study was set up to evaluate whether extreme AMH levels represent diminished oocyte quality and developmental incompetence. METHODS: A total of 141 consecutive ICSI patients were subdivided into three groups using the 25th and 75th percentiles of the serum AMH levels (cycle day 3). In these three groups, morphology of all oocytes and fertilization rate, embryo quality and blastocyst formation were evaluated, and FSH, LH and estradiol (E(2)) levels were also measured. RESULTS: Cycle cancellation rate was correlated with AMH levels (P < 0.05). AMH groups 1 (<1.66 ng/ml) and 3 (>4.52 ng/ml) showed oocytes of lower quality [dark central granulation, aggregation of smooth endoplasmic reticulum (sER)] compared with the median group 2 (1.66-4.52 ng/ml). Basal serum FSH did not allow for adequate prognosis in terms of gamete appearance. Fertilization and further cleavage up to blastocyst stage was not affected by AMH levels. CONCLUSIONS: AMH seems to be superior to FSH in predicting both oocyte number and quality.

An elevated concentration of lipoprotein (a) [Lp(a)] in the serum has been considered a risk factor for coronary heart disease by various investigators. In the present study, the turnover of Lp(a) was investigated in nine individuals with serum Lp(a) levels ranging from 1 to 68 mg/100 ml. After intravenous injection of radioiodinated Lp(a), the radioactivity time-curve of the serum and the specific activitity time-curves of the isolated Lp(a) and Lp(a) apolipoproteins were measured for 14 d. More than 97% of the label was found in the protein moiety of Lp(a). During the entire study period, the serum radioactivity remained with Lp(a), only insignificant amounts of radioactivity were detectable in other lipoprotein fractions. The serum radioactivity time-curves and the specific activity time-curves of the isolated Lp(a) and Lp(a) apolipoproteins were identical. The kinetic parameters of Lp(a) turnover were calculated in terms of a two-compartment model. 76.5+/-5.1% (mean+/-1 SD) of total Lp(a) was contained in the intravascular space. The biological half-life of Lp(a) was 3.32+/-0.52 d, the fractional catabolic rate (FCR) was 0.306+/-0.054/d, and the rate of synthesis was 5.00+/-3.37 mg/kg/d. A positive correlation was found between serum concentration and synthetic rate of Lp(a) apoprotein. No relationship could be demonstrated between serum level and FCR of Lp(a). The results of this study indicate that Lp(a) is not converted to other serum lipoproteins. From the correlations between serum concentration and kinetic parameters of Lp(a) it is concluded that an elevated Lp(a) level is the consequence of an increased Lp(a) apoprotein synthesis.

In Brief Objective: The aim of this study was to investigate the prognostic relevance of lymphangiogenesis and lymphovascular invasion in a large cohort of breast cancer patients. Introduction: Invasion of tumor cells into blood and lymphatic vessels is one of the critical steps for metastasis. The presence or absence of lymph node metastasis is one of the main decision criteria for further therapy. One shortcoming of previous morphologic studies was the lack of specific markers that could exact discriminate between blood and lymphatic vessels. The aim of this study was to evaluate the prognostic relevance of lymphangiogenesis and lymphovascular invasion in breast cancer patients. Methods: We investigated 374 tissue specimens of patients suffering from invasive breast cancer by immunostaining for the lymphatic endothelial specific marker podoplanin. Lymphangiogenesis, quantified by evaluating the lymphatic microvessels density (LMVD), and lymphovascular invasion (LVI) were correlated with various clinical parameters and prognostic relevance. Results: LMVD correlated significantly with LVI (P = 0.001). LVI was associated significantly with a higher risk for developing lymph-node metastasis (P = 0.004). Calculating the prognostic relevance, LVI presented as an independent prognostic parameter for disease free as well as overall survival (P = 0.001, and P = 0.001, respectively). Conclusion: Our data provide evidence that the biologic system of lymphangiogenesis constitutes a potential new target for development of anti-breast cancer therapeutic concepts. Our results further suggest that young, premenopausal patients with low differentiated breast tumors and high LMVD and LVI would, in particular, benefit from lymphangiogenesis-associated therapeutic strategies. The prognostic relevance of lymphangiogenesis and lymphovascular invasion in breast cancer has not been investigated using a specific lymph-endothelial marker. Here we investigated the clinical relevance of lymphangiogenesis and lymphovascular invasion in 374 breast cancer patients using the lymph-endothelial specific marker podoplanin. Our data provide evidence that the system of lymphangiogenesis constitutes a potential new target for the development of anti-breast cancer therapeutic concepts.

CONCLUSIONS: Hearing may be conserved in adults after implantation with the Nucleus Contour Advance perimodiolar electrode array. The degree of hearing preservation and the maximum insertion depth of the electrode array can vary considerably despite a defined surgical protocol. Residual hearing combined with electrical stimulation in the same ear can provide additional benefits even for conventional candidates for cochlear implantation. OBJECTIVES: We present preliminary results from a prospective multicentre study investigating the conservation of residual hearing after implantation with a standard-length Nucleus Contour Advance perimodiolar electrode array and the benefits of combined electrical and acoustic stimulation. MATERIAL AND METHODS: The subjects were 12 adult candidates for cochlear implantation recruited according to national selection criteria. A "soft" surgery protocol was defined, as follows: 1-1.2-mm cochleostomy hole anterior and inferior to the round window; Nucleus Contour Advance electrode array inserted using the "Advance-off-stylet" technique; and insertion depth controlled by means of three square marker ribs left outside the cochleostomy hole. These procedures had been shown to reduce insertion forces in temporal bone preparations. Variations in surgical techniques were monitored using a questionnaire. Pure-tone thresholds were measured pre- and postoperatively. Patients who still retained thresholds <90 dB HL for frequencies up to 500 Hz were re-fitted with an in-the-ear (ITE) hearing aid. Word recognition was tested in quiet and sentence perception in noise for the cochlear implant alone and in combination with an ipsilateral hearing aid. RESULTS: Hearing threshold level data were available for 12 patients recruited from 6 of the centres. Median increases in hearing threshold levels were 23, 27 and 33 dB for the frequencies 125, 250 and 500 Hz, respectively. These median increases include the data for two patients who had total loss of residual hearing due to difficulties encountered during surgery. "Cochlear view" X-ray images indicated that the depth of insertion varied between 300 and 430 degrees, despite modest variations in the length of the electrode inserted (17-19 mm). The insertion angle had some influence on the preservation of residual hearing at frequencies of 250-500 Hz. Six of the 12 patients retained sufficient hearing for effective use of an ipsilateral ITE hearing aid (< or = 80 dB HL at 125 and 250 Hz; < or = 90 dB HL at 500 Hz). Word recognition scores in quiet were improved from 10% to 30% with the cochlear implant plus ipsilateral hearing aid in 3 patients who had at least 3 months postoperative experience. Signal:noise ratio thresholds for sentence recognition were improved by up to 3 dB. Patients reported that they experienced greatly improved sound quality and preferred to use the two devices together.

Major bleeding during complex surgery increases the need for blood transfusions, prolongs the patient’s stay in the intensive care unit (ICU), and is associated with increased morbidity and mortality.1,2The standard treatment of perioperative bleeding involves the transfusion of allogeneic blood components (erythrocytes, fresh frozen plasma [FFP], platelet concentrate, or cryoprecipitate). Although these components have become safer, there is evidence that the transfusion of allogeneic blood components may be associated with a risk of serious adverse events.3–5Thus, treatment approaches that can reduce transfusion appear desirable.Clot strength increases with increasing concentrations of fibrinogen, and fibrinogen levels higher than 200 mg/dl have been suggested for optimal clot formation.6–8Immediately after cardiopulmonary bypass (CPB), fibrin formation is impaired to a greater extent than either thrombin generation or the platelet component of clot strength.9The plasma concentration of fibrinogen10,11and the firmness (elasticity/strength) of the fibrin-based clot9have been reported to decrease by an average of 34–42% in response to CPB. Furthermore, low levels of fibrinogen are associated with an increased risk of postoperative bleeding.10,12,13Overall, there is considerable evidence to support fibrinogen supplementation as a first-line treatment for coagulopathic bleeding among patients undergoing CPB. Several studies have suggested that fibrinogen concentrate therapy may be effective in controlling perioperative bleeding, reducing transfusion requirements as well as blood loss.11,14–19Fibrinogen concentrate is derived from human plasma and does not contain relevant levels of other coagulation factors.20Our group has developed and validated a model for individualized dosing of fibrinogen concentrate,17,18by measuring firmness of the fibrin-based clot, which is mainly dependent on plasma fibrinogen levels.13,21,22Maximum clot firmness (MCF) of the fibrin-based clot can be monitored using a commercially available fibrin-based thromboelastometry test (FIBTEM).11,23A clinical study is needed to investigate the efficacy and safety of fibrinogen concentrate in managing severe perioperative bleeding. We hypothesized that fibrinogen concentrate can reduce the need for blood transfusions without increasing the rate of serious adverse events when given intraoperatively as individualized first-line hemostatic therapy in bleeding patients undergoing aortic replacement surgery.This phase 2, prospective, randomized, double-blind, placebo-controlled, parallel-group, stratified, clinical study was conducted at a single center (Hannover Medical School, Hannover, Germany). It was approved by the Local Ethics Committee in Hannover, Germany, and by the German Regulatory Authorities; it was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. The study was assigned Local Ethics Committee reference code no. 4891M-mono, EudraCT trial no. 2007-004612-31, and ClinicalTrials.govidentifier no. NCT00701142.Patients aged 18 yr or older for whom elective aortic replacement surgery involving CPB was planned and who provided signed informed consent were enrolled between June 2008 and April 2010. All aortic valve operations with root/ascending aorta replacement (thoracic aortic aneurysm [TAA]), with or without aortic arch replacement, and thoracoabdominal replacements were eligible. Patients were excluded from the study if they had undergone previous surgery at the same aortic site, if they had a congenital or acquired coagulation disorder, if they had a myocardial infarction or stroke in the previous 2 months, or if they used aspirin, clopidogrel, or vitamin K antagonists in the previous 2–5 days. All patients received antifibrinolytic prophylaxis with tranexamic acid (30 mg/kg bodyweight as a preoperative loading dose, followed by 1 mg/kg bodyweight/h throughout surgery). CPB was established after aortic cannulation and administration of 400 IU/kg heparin (Heparin-Natrium-25000-ratiopharm®; Merckle GmbH, Blaubeuren, Germany).Before surgery, patients were randomized to receive either fibrinogen concentrate or placebo; study medication was administered if clinically relevant bleeding occurred. This was defined as a 5-min bleeding mass of 60–250 g immediately after removal from CPB, neutralization of heparin with protamine sulfate (Protamin Valeant, Valeant Pharmaceuticals GmbH, Eschborn, Germany; protamine–heparin ratio of 1:1), and completion of surgical hemostasis, which includes suture placement and electrocautery (or diathermy) (fig. 1). Completion of surgical hemostasis consisted of surgical control of focal bleeding. Surgical hemostasis was deemed to be complete if no obvious sources of bleeding were identified. A 5-min bleeding mass was subsequently determined by weighing dry surgical cloths and compresses, applying them to the surgical area for 5 min and weighing them again. No irrigation of the operative field was performed during the measurement of bleeding mass. Bleeding mass in the range 60–250 g was considered to be clinically relevant and coagulopathic in nature; an upper limit was included because of the life-threatening nature of massive bleeding. The thresholds of 60 and 250 g were chosen based on clinical experience at the authors’ center.17,18Randomized patients not eligible for treatment or withdrawn from the study before 24 h after the administration of study medication were replaced until there were sufficient patients to provide primary efficacy endpoint data for each study arm. Stratification was done according to surgery type (thoracoabdominal aortic aneurysm or TAA).Study medication was prepared in 50-ml opaque syringes by the pharmacist, according to a randomization code generated by the contract research organization (Accovion GmbH, Eschborn, Germany) using SAS (SAS Institute, Cary, NC) (the investigator initially assigned a randomization number to each patient). The randomization ratio of the treatment arms in each stratum (thoracoabdominal aortic aneurysm and TAA) was 1:1, with a block size of 4. Each 50-ml syringe contained either 1 g fibrinogen concentrate (Haemocomplettan P®, RiaSTAPTM; CSL Behring, Marburg, Germany) diluted in 50 ml sterile water, or an equivalent volume of 0.9% saline as placebo. The medication was delivered to the operation room upon request of the study investigator (anesthesiologist) and administered intravenously within 5 min of bleeding measurement. In order for study medication to be administered, patients had to fulfill the following conditions: activated clotting time less than 150 s, body temperature higher than 36°C, pH more than 7.3, and hemoglobin level higher than 8.5 g/dl. To ensure blinding, only the pharmacist had access to the randomization code; the use of opaque syringes ensured that fibrinogen concentrate and placebo were visually identical.Doses were determined from the MCF of the FIBTEM test, using a model developed in previous studies for individualizing fibrinogen concentrate dosing.17,18The FIBTEM test was performed by point-of-care thromboelastometry (ROTEM®device; TEM International, Munich, Germany), using blood samples taken 20 min before the end of CPB. The time taken to obtain the MCF value was 15 min. The fibrinogen concentrate dose was calculated by the unblinded point-of-care laboratory staff as follows: Fibrinogen concentrate dose (g) = (target FIBTEM MCF − actual FIBTEM MCF) (mm) × (bodyweight [kg]/ 70) × 0.5 g/mm.11,17,18The target FIBTEM MCF was 22 mm. None of the blinded personnel (investigator and staff of the operating room and ICU) had access to FIBTEM or fibrinogen concentration values after the administration of study medication.Approximately 5 min after the administration of study medication, bleeding mass was measured again. A transfusion algorithm was initiated in patients with a bleeding mass of 60–250 g as follows: if, upon removal of the aortic clamp, platelet count was lower than 100,000/µl, 2 U of apheresis platelet concentrate was administered; if platelet count was 100,000/µl or higher, 4 U FFP was administered in approximately 10 min. Subsequently, 5-min bleeding mass was reevaluated. If clinically relevant bleeding continued, the patient was given the blood component (platelet concentrate/FFP) not administered after the first bleed, and 5-min bleeding mass was again measured. Transfusion packages of 1 U platelet concentrate and 2 U FFP were administered until the 5-min bleeding mass was less than 60 g. Upon completion of hemostatic therapy, the thorax was closed (last suture = end of surgery), and the patient was transferred to the ICU. In the event of blood drainage higher than 400 ml during 1 h in the ICU, 1 U platelet concentrate and 2 U FFP were administered. Erythrocytes were also administered after CPB to maintain a hemoglobin level higher than 8·5 g/dl. Fibrinogen levels were measured using the Clauss assay, before and after the administration of study medication.The primary endpoint was the total number of units of allogeneic blood components (erythrocytes plus FFP plus platelet concentrate) given to patients between the administration of study medication and 24 h thereafter. Secondary endpoints included the number of units of each individual allogeneic blood component given (erythrocytes, FFP, and platelet concentrate), the proportion of patients who received no allogeneic blood components (total avoidance), the number of days not in the ICU or hospital during the 45 days after surgery (assumed to be zero for patients dying within that period), and mortality at 45 days after surgery.Safety was assessed principally by treatment-emergent adverse events occurring within 10 days of treatment. The follow-up period for serious adverse events was 45 days. Patients were monitored for viral seroconversion by hepatitis A virus, hepatitis B virus, hepatitis B core, hepatitis B surface antigen, hepatitis C virus, human immunodeficiency virus 1 and 2, and parvovirus B19 testing (enzyme immunoassays and polymerase chain reaction were used). In accordance with Good Clinical Practice, the study sponsor evaluated safety data throughout the study.Sixty patients were planned for the primary endpoint based on an assumed difference between the treatment group means of 4·25 ± 5.3 U (where 5.3 is the SD) with a two-sided type I error rate of 5% and more than 80% power for the nonparametric Wilcoxon rank sum test. These assumptions were derived from a previously reported average transfusion of 8·5 ± 5.3 U during TAA surgery.17We assumed that the use of fibrinogen concentrate would reduce transfusion of allogeneic blood products by 50% compared with the standard-of-care group. The efficacy and safety analyses included all patients who were randomized and received study treatment. This was not strictly an intention-to-treat analysis because of the need to randomize patients before ascertaining whether they met the study inclusion criteria (if randomization had been performed after establishing that patients met the study inclusion criteria, hemostatic therapy would have been delayed unethically).Inferential testing of the primary efficacy endpoint was based on a nonparametric Wilcoxon rank sum test with a two-sided type I error rate of 5%, testing the null hypothesis of no difference between the treatment groups. An unstratified Hodges–Lehman point estimate and the corresponding two-sided 95% CI for the treatment difference were also calculated. The primary endpoint was found to have a positively skewed nonnormal distribution. The proportion of patients receiving no allogeneic blood components within the first 24 h after start of infusion of study medication was analyzed in an exploratory manner applying a chi-square test.Exploratory analysis was performed for selected secondary endpoints (24-h transfusion of erythrocytes, FFP, and platelet concentrate [each component analyzed separately]). Additional analyses were performed for fibrinogen and hemoglobin using the two-sample t test for normally distributed outcomes and the Wilcoxon rank sum test for outcomes with a skewed distribution.All other secondary efficacy endpoints were analyzed by descriptive statistics, with values typically presented as mean ± SD. For the between-group comparison of safety outcomes, risk ratios with 95% CIs were calculated where possible (nonzero incidence).Data for the main efficacy endpoints are presented as median (with interquartile range, between the 25th and the 75th percentiles) values because the data were not normally distributed. SAS version 9.1.3 (SAS Institute) was used for all of the statistical analyses, except the Wilcoxon rank sum test, which was performed using StatXact (version 8.1; Cytel, Cambridge, MA).Of 80 patients screened, 61 were randomized and treated (fig. 2). Of these, 18 patients underwent thoracoabdominal aortic aneurysm surgery, 22 underwent TAA with arch surgery, and 21 underwent TAA without arch surgery (table 1). The two treatment groups had similar perioperative characteristics, and the population was typical for patients undergoing aortic surgery (table 1). Preoperative mean ± SD FIBTEM MCF was 17.9 ± 6.1 mm in the fibrinogen concentrate group and 16.4 ± 3.8 mm in the placebo group.On the basis of MCF values from the FIBTEM test performed during CPB, a median dose of 8 g (interquartile range [IQR], 6–9 g) of fibrinogen concentrate was administered to patients in the fibrinogen concentrate group (median volume, 400 ml; IQR, 300–450 ml). The median volume of study medication administered to the placebo group was also 400 ml (IQR, 300–450 ml). These doses were calculated from the FIBTEM MCF of samples taken approximately 20 min before removal from CPB; the mean MCF was 9.7 ± 3.1 mm in the fibrinogen concentrate group and 9.5 ± 2.7 mm in the placebo group. As shown in table 1, the two study groups were also comparable immediately before the administration of study medication (after removal from CPB, administration of protamine, and completion of surgical hemostasis), in terms of 5-min bleeding mass and activated clotting time.During the 24-h period after start of study medication, patients treated with fibrinogen concentrate received fewer units of allogeneic blood components (median, 2 U; IQR, 0–8 U) than patients treated with placebo (median, 13 U; IQR, 8–21 U); the treatment difference (−9 U; 95% CI, −13 to −6 U) was statistically significant (P &lt;0.001, table 2). A sensitivity analysis was performed, excluding patients who dropped out within 24 h of the administration of study medication (one patient in the placebo group). This showed a similar treatment difference (−8 U; 95% CI, −13 to −6 U). The treatment difference was also generally similar for each of the aortic replacement procedures included in the study. The number of units of erythrocytes, FFP, and platelet concentrate administered during the 24 h after start of study medication was lower in the fibrinogen concentrate group than that in the placebo group (table 2). These treatment differences were statistically significant for erythrocytes (reduction, −2 U; P = 0.007), FFP (reduction, −5 U; P &lt; 0.001) and platelet concentrate (reduction, −2 U; P &lt; 0.001).Total avoidance of transfusion was achieved in 13 of 29 patients (45%) in the fibrinogen concentrate group; in contrast, 32 of 32 patients (100%) in the placebo group received treatment with allogeneic blood components to control their coagulopathic bleeding (P &lt; 0·001). The mean fibrinogen concentration and the mean FIBTEM MCF were similar in the two study groups at all time points except for the first measurement after the administration of study drug (at the “last suture” time point, i.e. , end of surgery), when higher values were in the fibrinogen concentrate group P &lt; table FIBTEM ± ± P &lt; was no between-group difference in hemoglobin at time point (table of days and days were also similar in the two study groups (table of patients in each treatment group reported treatment-emergent adverse events table The of treatment-emergent adverse events reported were typical for patients undergoing surgery, the and None of the treatment-emergent adverse events reported in the study was considered to be to study medication, and to from the study. because of bleeding was in patients in the fibrinogen concentrate group and patients in the placebo group. Although a risk of there was no significant between-group In all a surgical of bleeding was during of serious adverse events was similar in study groups serious adverse events to in the placebo group the fibrinogen concentrate group patients Although the risk was there was no significant between-group None of the serious adverse events was considered to study and to and myocardial infarction were reported in a fibrinogen concentrate patient with a previous of myocardial and who underwent of TAA and was reported the patient was transferred for a with was for no events were reported in the fibrinogen concentrate group. In the placebo two events were reported (one and were no viral in either study study that fibrinogen concentrate, administered intraoperatively as first-line hemostatic the need for transfusion of allogeneic blood products in patients undergoing complex aortic The median transfusion of allogeneic blood components was by among patients receiving fibrinogen concentrate U compared with placebo U avoidance of allogeneic blood components in 13 of 29 patients (45%) treated with fibrinogen concentrate is of clinical that all patients in the placebo group received allogeneic blood outcomes may be to plasma fibrinogen concentration more and to higher levels among patients receiving fibrinogen concentrate compared with who received placebo. The use of fibrinogen concentrate of allogeneic blood products has clinical Fibrinogen concentrate is immediately available for with no need for or blood group and has a low administration can be administered in less time than either FFP or the time to control bleeding. In the of severe bleeding, the administration of g fibrinogen concentrate in min has been reported contrast, FFP administration and the concentration of fibrinogen in FFP 200 the extent to which fibrinogen levels can be a higher concentration of fibrinogen in to a range of concentration is more than with fibrinogen concentrate, that doses be as as with FFP, blood group is before use of fibrinogen concentrate with other coagulation as complex concentrate, in the of a treatment algorithm based on point-of-care coagulation was shown to decrease blood transfusion and events among surgery is of because it the treatment of using coagulation with the for in allogeneic blood transfusions than can be achieved with fibrinogen data that the in fibrinogen to the placebo was only than 24 and may be positively from a safety The safety of fibrinogen concentrate was similar to that of placebo. This study was not to differences between fibrinogen concentrate and placebo in morbidity or the of between-group differences in safety is with the previously reported safety of fibrinogen concentrate, a low risk of an of virus patients in the fibrinogen concentrate group underwent compared with in the placebo group. Surgical bleeding was in all of these In the possible for are and there is no in study that the were to study The data are with the in the use of allogeneic blood products in the fibrinogen concentrate group. all allogeneic blood products administered within 24 h of study medication were included in the primary used during and after and all patients the operating room with a 5-min bleeding mass less than 60 is the randomized, double-blind, study of fibrinogen concentrate It is the first study of fibrinogen concentrate administered intraoperatively as first-line hemostatic therapy, and the first study of fibrinogen concentrate among patients undergoing complex two previous randomized clinical studies of fibrinogen concentrate have been the first patients undergoing = with blood by a significant in MCF after fibrinogen supplementation placebo. Transfusion and blood were similar in the two groups during surgery, erythrocytes were administered to patients in the placebo group two in the fibrinogen group (P &lt; The performed in 20 bypass showed that the administration of fibrinogen concentrate g) blood by (P = transfusions were in patient in the fibrinogen group compared with in the placebo group. The study was and the of operation were distributed between the two treatment that they were as fibrinogen concentration and time the of the two groups before the administration of study study medication patients received study medication during a period if treatment consisted only of allogeneic blood the patient would only have been monitored blood products are study may also be considered as a comparison of fibrinogen concentrate with standard hemostatic treatment based on FFP and platelet in study was based on point-of-care coagulation testing to obtain more than standard laboratory are to the use of is as to which are data showed that of , and is to than a single is a test to firmness of a clot in the of a platelet , fibrin-based clot FIBTEM does not provide a measurement of fibrinogen of the fibrin-based clot is measured in units that are from of fibrinogen concentration as to or studies have shown between FIBTEM MCF and of fibrinogen was to FIBTEM MCF to 22 higher than the mean preoperative level of mm and at the end of the range This target was chosen to fibrinogen concentrate to be effective as a hemostatic is evidence that fibrinogen is the primary associated with that fibrinogen supplementation may for platelet typically to a fibrinogen level of approximately is within the range of similar to the value of mg/dl reported for patients older than 60 the plasma concentration for fibrinogen supplementation was at as more than 150 higher target may be because clot firmness to with fibrinogen concentration throughout the study has analysis does not strictly the for to To patients were randomized to treatment before the of clinically significant coagulopathic bleeding was was not because to the study was based on criteria as to clinical and because the criteria were the same for study groups. The use of 5-min bleeding mass as a study inclusion and as a for the transfusion of allogeneic blood products an of bleeding the that it is not the standard for hemostatic and that it may not be in all clinical are and measurement of blood in the Patients with preoperative coagulation or therapy were that the of the to patients may be of the study was comparison of the two treatment groups. This study is also by size and use of a single the of safety , adverse is not the size was determined statistically from an that the use of allogeneic blood products would be by a of clinical and the use of a single center may limit to other it also have in personnel and clinical is a need for studies of fibrinogen concentrate in surgical in patients with preoperative and in of patients to that may efficacy and to the safety studies would be randomized and with intention-to-treat also study in patients undergoing aortic replacement surgery has shown that first-line treatment with fibrinogen concentrate is to reduce the transfusion of allogeneic blood If in fibrinogen concentrate administration would provide a means of reducing or has the to the treatment for perioperative bleeding in patients with life-threatening support was provided by and GmbH, Germany). The for and Medical School, Hannover, Germany) for to blood of coagulation and data

A large number of allergenic proteins have now their complete cDNA sequences determined and in some cases also the 3D structures. It turned out that most allergens could be grouped into a small number of structural protein families, regardless of their biological source. Structural similarity among proteins from diverse sources is the molecular basis of allergic cross-reactivity. The clinical relevance of immunoglobulin E (IgE) cross-reactivity seems to be influenced by a number of factors including the immune response against the allergen, exposure and the allergen. As individuals are exposed to a variable number of allergenic sources bearing homologous molecules, the exact nature of the antigenic structure inducing the primary IgE immune response cannot be easily defined. In general, the 'cross-reactivity' term should be limited to defined clinical manifestations showing reactivity to a source without previous exposure. 'Co-recognition', including by definition 'cross-reactivity', could be used to describe the large majority of the IgE reactivity where co-exposure to a number of sources bearing homologous molecules do not allow unequivocal identification of the sensitizing molecule. The analysis of reactivity clusters in diagnosis allows the interpretation of the patient's reactivity profile as a result of the sensitization process, which often begins with exposure to a single allergenic molecule.

We report on the pathological findings in the brains of 8 Parkinson's disease patients treated with deep brain stimulation (DBS) of the thalamic ventral intermediate nucleus (6 cases) and subthalamic nucleus (2 cases). DBS was performed continuously for up to 70 months. All brains showed well-preserved neural parenchyma and only mild gliosis around the lead track compatible with reactive changes due to surgical placement of the electrode. We conclude that chronic DBS does not cause damage to adjacent brain tissue.