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Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily that controls bile acid (BA) homeostasis, has also been proposed as a tumor suppressor for breast and liver cancer. However, its role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis remains controversial. We recently found that FXR attenuates acinar cell autophagy in chronic pancreatitis resulting in reduced autophagy and promotion of pancreatic carcinogenesis. Feeding Kras-p48-Cre (KC) mice with the BA chenodeoxycholic acid (CDCA), an FXR agonist, attenuated pancreatic intraepithelial neoplasia (PanIN) progression, reduced cell proliferation, neoplastic cells and autophagic activity, and increased acinar cells, elevated pro-inflammatory cytokines and chemokines, with a compensatory increase in the anti-inflammatory response. Surprisingly, FXR-deficient KC mice did not show any response to CDCA, suggesting that CDCA attenuates PanIN progression and decelerate tumorigenesis in KC mice through activating pancreatic FXR. FXR is activated in pancreatic cancer cell lines in response to CDCA in vitro. FXR levels were highly increased in adjuvant and neoadjuvant PDAC tissue compared to healthy pancreatic tissue, indicating that FXR is expressed and potentially activated in human PDAC. These results suggest that BA exposure activates inflammation and suppresses autophagy in KC mice, resulting in reduced PanIN lesion progression. These data suggest that activation of pancreatic FXR has a protective role by reducing the growth of pre-cancerous PDAC lesions in response to CDCA and possibly other FXR agonists., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Early-onset pancreatic cancer (EOPC) represents 5-10% of all pancreatic ductal adenocarcinoma (PDAC) cases, and the etiology of this form is poorly understood. It is not clear if established PDAC risk factors have the same relevance for younger patients. This study aims to identify genetic and non-genetic risk factors specific to EOPC., Methods: A genome-wide association study was performed, analysing 912 EOPC cases and 10 222 controls, divided into discovery and replication phases. Furthermore, the associations between a polygenic risk score (PRS), smoking, alcohol consumption, type 2 diabetes and PDAC risk were also assessed., Results: Six novel SNPs were associated with EOPC risk in the discovery phase, but not in the replication phase. The PRS, smoking, and diabetes affected EOPC risk. The OR comparing current smokers to never-smokers was 2.92 (95% CI 1.69-5.04, P = 1.44 × 10 -4 ). For diabetes, the corresponding OR was 14.95 (95% CI 3.41-65.50, P = 3.58 × 10 -4 )., Conclusion: In conclusion, we did not identify novel genetic variants associated specifically with EOPC, and we found that established PDAC risk variants do not have a strong age-dependent effect. Furthermore, we add to the evidence pointing to the role of smoking and diabetes in EOPC., Competing Interests: Conflict of interest M.F.B. has received research funding from Celgene and Lead Pharma and acted as a consultant to Servier. H.W.M.L. reports research funding and/or medication supply from Bristol-Myers Squibb, Bayer Schering Pharma, Celgene, Janssen-Cilag, Lilly, Nordic Pharma, Philips Healthcare, Roche, Merck Sharp and Dohme, Servier, Incyte; and consultant/advisory board member for Lilly, Nordic Pharma, Bristol-Myers Squibb, Dragonfly, Merck Sharp and Dohme, Servier, all outside the submitted work. The other authors do not have any conflict of interest to declare., (Copyright © 2023 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Anastomotic suturing is the Achilles heel of pancreatic surgery. Especially in laparoscopic and robotically assisted surgery, the pancreatic anastomosis should first be trained outside the operating room. Realistic training models are therefore needed., Methods: Models of the pancreas, small bowel, stomach, bile duct, and a realistic training torso were developed for training of anastomoses in pancreatic surgery. Pancreas models with soft and hard textures, small and large ducts were incrementally developed and evaluated. Experienced pancreatic surgeons (n = 44) evaluated haptic realism, rigidity, fragility of tissues, and realism of suturing and knot tying., Results: In the iterative development process the pancreas models showed high haptic realism and highest realism in suturing (4.6 ± 0.7 and 4.9 ± 0.5 on 1-5 Likert scale, soft pancreas). The small bowel model showed highest haptic realism (4.8 ± 0.4) and optimal wall thickness (0.1 ± 0.4 on -2 to +2 Likert scale) and suturing behavior (0.1 ± 0.4). The bile duct models showed optimal wall thickness (0.3 ± 0.8 and 0.4 ± 0.8 on -2 to +2 Likert scale) and optimal tissue fragility (0 ± 0.9 and 0.3 ± 0.7)., Conclusion: The biotissue training models showed high haptic realism and realistic suturing behavior. They are suitable for realistic training of anastomoses in pancreatic surgery which may improve patient outcomes., (Copyright © 2023 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Objective: Endurance athletes are prone to develop flow limitations in iliac arteries (FLIA). Especially in cyclists and ice speed skaters, excessive hemodynamic loading coupled with hip hyperflexion may cause kinking in lengthened iliac arteries necessitating surgical correction. This study investigated the short-term (≤1.5 years) and long-term (≥5 years) satisfaction of operative shortening of the iliac artery in endurance athletes., Methods: All patients who were diagnosed and operated for FLIA owing to lengthened and kinked iliac arteries between 1997 and 2015 in one center were analyzed. Short-term follow-up consisted of an incremental maximal cycling test, ankle-brachial index with flexed hips, echo-Doppler examination with peak systolic velocity measurements and contrast-enhanced magnetic resonance angiography before and 6 to 18 months after surgery. Both short- and long-term satisfaction were assessed using questionnaires., Results: A total of 83 patients (90 operated legs; 96.7% males; median age of 34 years at the time of surgery; interquartile range [IQR], 29-47) were analyzed. In the short-term, 87.5% reported symptom reduction with an 86.4% overall satisfaction rate. Symptom-free cycling improved from 272 ± 84 W to 384 ± 101 W (P < .001), whereas the maximal workload increased from 419 ± 72 W to 428 ± 67 W (P = .01). The ankle-brachial index with flexed hips increased from 0.55 (IQR, 0.45-0.65) to 0.62 (IQR, 0.52-0.74; P = .008), and the peak systolic velocity measured with hips flexed decreased from 2.50 m/s (IQR, 1.77-3.13 m/s) to 1.57 m/s (IQR, 1.20-2.04 m/s; P < .001). After a median of 12 years (IQR, 9.0-15.4 years), symptoms were still decreased in 84.1% of patients with an 81.2% overall satisfaction rate (79.5% response rate). Three patients needed a reintervention (recurrent FLIA, n = 2; failure, n = 1)., Conclusions: Operative shortening of a lengthened and kinked iliac artery causing FLIA is successful both in the short- and long-term., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Hepaticoenterostomy is an important step of reconstruction during hepatopancreatobiliary (HPB) surgery with a subsequent bile leakage rate of up to 5%. The International Study Group of Liver Surgery (ISGLS) proposed a severity grading system for defining bile leakage after HPB surgery, which has not been validated after pancreatic surgery in a large patient cohort. The present study aimed to validate the ISGLS definition for bile leakage in pancreatic surgery and to investigate the postoperative outcomes of bile leakage after pancreatic resections., Materials and Methods: Data from the prospectively maintained database for pancreas surgery were extracted for any type of pancreatectomy with hepaticoenterostomy between 2006 and 2019. The severity of bile leakage was graded according to the ISGLS definition. The influence of our standardized hepaticoenterostomy technique and of the complexity of the surgical procedure on the rate of clinically relevant bile leakages (B and C) were assessed in three different timeframes., Results: Bile leakage was detected in 152 of 5,300 patients (2.9%). Clinically relevant bile leakages included seventy patients with grade B and eighty-two patients with grade C bile leakages (46.1% and 53.9%, respectively). During the study period, the overall rate of bile leakage showed to be stable (from 3.5% to 2.4%). Patients with grade C bile leakage had a higher rate of postoperative wound infection (P < 0.001) and longer ICU stays and hospital stays compared to patients with grade B bile leakage (P = 0.03 and P < 0.001 respectively). These parameters were significantly higher in patients with late grade C bile leakage but were similar between patients with grade B bile leakage and early grade C bile leakage (<5th day POD). In the whole patients' cohort, the 90-day mortality rate was 3.2% (174/5,300), with a rate of 25% in patients with bile leakage (38/152)., Conclusion: The ISGLS classification is a valid method for classifying postoperative bile leak after pancreas surgery. Standardization of our hepaticoenterostomy technique resulted in a stable rate of bile leakage. Although rare, bile leakage following pancreas surgery is a severe complication that has a major impact on patient outcomes and contributes significantly to morbidity and mortality, even in the absence of POPF., (Copyright © 2022 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Background: Chyle leak is a common complication following pancreatic surgery. After failure of conservative treatment, lymphography is one of the last therapeutic options. The objective of this study was to evaluate whether lymphography represents an effective treatment for severe chyle leak (International study Group on Pancreatic Surgery, grade C) after pancreatic surgery., Methods: Patients with grade C chyle leak after pancreatic surgery who received transpedal or transnodal therapeutic lymphography between 2010 and 2020 were identified from a prospectively maintained database. Clinical success of the lymphography was evaluated according to percent decrease of drainage output after lymphography (>50% decrease = partial success; >85% decrease = complete success)., Results: Of the 48 patients undergoing lymphography, 23 had a clinically successful lymphography: 14 (29%) showed partial and 9 (19%) complete success. In 25 cases (52%) lymphography did not lead to a significant reduction of chyle leak. Successful lymphography was associated with earlier drain removal and hospital discharge [complete clinical success: 7.1 days (±4.1); partial clinical success: 12 days (±9.1), clinical failure: 19 days (±19) after lymphography; p = 0.006]. No serious adverse events were observed., Conclusion: Therapeutic lymphography is a feasible, safe, and effective option for treating grade C chyle leak after pancreatic surgery., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Surgical resection is the mainstay of potential cure for patients with pancreatic cancer, however, local recurrence is frequent. Previously, we have described an extended resection technique for pancreatoduodenectomy aiming at a radical resection of the nerve and lymphatic tissue between celiac artery, superior mesenteric artery and mesenteric-portal axis (TRIANGLE operation). Until now, data on postoperative outcome have not been reported, yet., Methods: Patients who underwent either partial (PD) or total pancreatoduodenectomy (TP) applying the TRIANGLE procedure were identified. These cohorts were compared to matched historic cohorts with standard resections., Results: Overall, 330 patients were analysed (PD TRIANGLE and PD STANDARD, each n = 108; TP TRIANGLE and TP STANDARD, each n = 57). More lymph nodes were harvested in TRIANGLE compared to standard resection (PD: 27.5 (21-35) versus 31.5 (24-40); P = 0.0187, TP: 33 (28-49) versus 44 (29-53); P = 0.3174) and the rate of tumour positive resections margins, R1(direct), dropped. Duration of operation was significantly longer and blood loss higher. Postoperative mortality and complications did not differ significantly., Conclusion: Pancreatoduodenectomy according to the TRIANGLE protocol can be performed without increased morbidity and mortality at a high-volume centre. Long-term survival and quality of life need to be investigated in prospective clinical trials with adequate sample size., (Copyright © 2021 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Background: The clinical relevance of hyperamylasemia after distal pancreatectomy (DP) remains unclear and no internationally accepted definition of postoperative acute pancreatitis (POAP) exists. The aim of this study was to characterize POAP after DP and to assess the role of serum amylase (SA) in POAP., Methods: Outcomes of 641 patients who had undergone DP between 2015 and 2019 were analyzed. Postoperative SA was determined in all patients. POAP was defined based on contrast-enhanced computed tomography (CT) or intraoperative findings during relaparotomy., Results: An elevation of SA on postoperative day 1 (hyperamylasemia POD1 ) was found in 398 patients (62.1%). Twelve patients (1.87%) were identified with POAP. Ten patients demonstrated radiologic criteria for POAP and in two patients POAP was diagnosed during relaparotomy. Outcome of POAP patients was worse than that of patients with hyperamylasemia POD1 alone and that with normal SA POD1 without POAP evidence (postoperative pancreatic fistula 50% vs 30.6% vs 18.5%; length of hospital stay 26 days vs 12 vs 11, respectively). The overall 90-day mortality of all 641 patients was 0.6%., Conclusion: POAP is a serious but rare complication after DP. Hyperamylasemia POD1 is of prognostic relevance after DP, but it seems not sufficient as a single parameter to diagnose POAP., (Copyright © 2021 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

MYBPC3 is the most frequently affected gene in hypertrophic cardiomyopathy (HCM), which is an autosomal-dominant cardiac disease caused by mutations in sarcomeric proteins. Bi-allelic truncating MYBPC3 mutations are associated with severe forms of neonatal cardiomyopathy. We reprogrammed skin fibroblasts from a HCM patient carrying a heterozygous MYBPC3 truncating mutation into human induced pluripotent stem cells (iPSC) and used CRISPR/Cas9 to generate bi-allelic MYBPC3 truncating mutation and isogenic control hiPSC lines. All lines expressed pluripotency markers, had normal karyotype and differentiated into endoderm, ectoderm and cardiomyocytes in vitro. This set of three lines provides a useful tool to study HCM pathomechanisms., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: The new 2019 guideline of the European Society for Vascular Surgery (ESVS) recommends consideration for elective iliac artery aneurysm (eIAA) repair when the iliac diameter exceeds 3.5 cm, as opposed to 3.0 cm previously. The current study assessed diameters at time of eIAA repair and ruptured IAA (rIAA) repair and compared clinical outcomes after open surgical repair (OSR) and endovascular aneurysm repair (EVAR)., Methods: This retrospective observational study used the nationwide Dutch Surgical Aneurysm Audit (DSAA) registry that includes all patients who undergo aorto-iliac aneurysm repair in the Netherlands. All patients who underwent primary IAA repair between 1 January 2014 and 1 January 2018 were included. Diameters at time of eIAA and rIAA repair were compared in a descriptive fashion. The anatomical location of the IAA was not registered in the registry. Patient characteristics and outcomes of OSR and EVAR were compared with appropriate statistical tests., Results: The DSAA registry comprised 974 patients who underwent IAA repair. A total of 851 patients were included after exclusion of patients undergoing revision surgery and patients with missing essential variables. eIAA repair was carried out in 713 patients, rIAA repair in 102, and symptomatic IAA repair in 36. OSR was performed in 205, EVAR in 618, and hybrid repairs and conversions in 28. The median maximum IAA diameter at the time of eIAA and rIAA repair was 43 (IQR 38-50) mm and 68 (IQR 58-85) mm, respectively. Mortality was 1.3% (95% CI 0.7-2.4) after eIAA repair and 25.5% (95% CI 18.0-34.7) after rIAA repair. Mortality was not significantly different between the OSR and EVAR subgroups. Elective OSR was associated with significantly more complications than EVAR (intra-operative: 9.8% vs. 3.6%, post-operative: 34.0% vs. 13.8%, respectively)., Conclusion: In the Netherlands, most eIAA repairs are performed at diameters larger than recommended by the ESVS guideline. These findings appear to support the recent increase in the threshold diameter for eIAA repair., (Copyright © 2020 European Society for Vascular Surgery. Published by Elsevier B.V. All rights reserved.)

Three different chemical oxidation processes were investigated in terms of their capability to degrade organic chemical components of real mature landfill-leachate in combination with biological treatment run in a Sequencing Batch Biofilter Granular Reactor (SBBGR). H 2 O 2 , H 2 O 2  + UV and O 3 were integrated with SBBGR and respective effluents were analyzed and compared with the effluent obtained from biological SBBGR treatment alone. In agreement with their respective oxidative power, conventional bulk parameters (residual COD, TOC, N tot , TSS) determined from the resulting effluents evidenced the following efficacy ranking for degradation: SBBGR/O 3  > SBBGR/UV + H 2 O 2  > SBBGR/H 2 O 2  > SBBGR. A more detailed characterization of the organic compounds was subsequently carried out for the four treated streams. For this, effluents were first subjected to a sample preparation step, allowing for a classification in terms of acidic, basic, strongly acidic and strongly basic compounds, and finally to analysis by liquid chromatography/high resolution mass spectrometry (LC/HR-MS). This classification, combined with further data post-processing (non-target screening, Venn Diagram, tri-dimensional plot and Principal Component Analysis), evidenced that the SBBGR/H 2 O 2 process is comparable to the pure biological oxidation. In contrast, SBBGR/O 3 and SBBGR/UV + H 2 O 2 not only resulted in a very different residual composition as compared to SBBGR and SBBGR/H 2 O 2 , but also differ significantly from each other. In fact, and despite of the SBBGR/O 3 being the most efficient process, this treatment remained chemically more similar to SBBGR/H 2 O 2 than to SBBGR/UV + H 2 O 2 . This finding may be attributable to different mechanism of degradation involved with the use of UV radiation. Apart from these treatment differences, a series of recalcitrant compounds was determined in all of the four treatments and partly identified as hetero-poly-aromatic species (humic acids-like species)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Infant cognitive development can be positively influenced by breastfeeding rather than formula feeding. The composition of breast milk, especially lipid quality, and the duration of breastfeeding have been linked to this effect., Objective: We investigated whether the physical properties and composition of lipid droplets in milk may contribute to cognitive development., Methods: From postnatal day (P) 16 to P44, healthy male C57BL/6JOlaHsd mice were fed either a control or a concept rodent diet, in which the dietary lipid droplets were large and coated with milk phospholipids, resembling more closely the physical properties and composition of breast milk lipids. Thereafter, all mice were fed an AIN-93M semisynthetic rodent diet. The mice were subjected to various cognitive tests during adolescence (P35-P44) and adulthood (P70-P101). On P102, mice were killed and brain phospholipids were analyzed., Results: The concept diet improved performance in short-term memory tasks that rely on novelty exploration during adolescence (T-maze; spontaneous alternation 87% in concept-fed mice compared with 74% in mice fed control diet; P < 0.05) and adulthood (novel object recognition; preference index 0.48 in concept-fed mice compared with 0.05 in control-fed mice; P < 0.05). Cognitive performance in long-term memory tasks, however, was unaffected by diet. Brain phospholipid composition at P102 was not different between diet groups., Conclusions: Exposure to a diet with lipids mimicking more closely the structure and composition of lipids in breast milk improved specific cognitive behaviors in mice. These data suggest that lipid structure should be considered as a relevant target to improve dietary lipid quality in infant milk formulas., (© 2016 American Society for Nutrition.)

Ultrahigh spectral brightness femtosecond XUV and X-ray sources like free electron lasers (FEL) and table-top high harmonics sources (HHG) offer fascinating experimental possibilities for analysis of transient states and ultrafast electron dynamics. For electron spectroscopy experiments using illumination from such sources, the ultrashort high-charge electron bunches experience strong space-charge interactions. The Coulomb interactions between emitted electrons results in large energy shifts and severe broadening of photoemission signals. We propose a method for a substantial reduction of the effect by exploiting the deterministic nature of space-charge interaction. The interaction of a given electron with the average charge density of all surrounding electrons leads to a rotation of the electron distribution in 6D phase space. Momentum microscopy gives direct access to the three momentum coordinates, opening a path for a correction of an essential part of space-charge interaction. In a first experiment with a time-of-flight momentum microscope using synchrotron radiation at BESSY, the rotation in phase space became directly visible. In a separate experiment conducted at FLASH (DESY), the energy shift and broadening of the photoemission signals were quantified. Finally, simulations of a realistic photoemission experiment including space-charge interaction reveals that a gain of an order of magnitude in resolution is possible using the correction technique presented here., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: A deficit in impulse control is a prominent, heritable symptom in several psychiatric disorders, such as addiction, attention-deficit/hyperactivity disorder, and schizophrenia. Here, we aimed to identify genes regulating impulsivity, specifically of impulsive action, in mice., Methods: Using the widely used 5-choice serial reaction time task, we measured impulsive action in 1) a panel of 41 BXD recombinant inbred strains of mice (n = 13.7 ± .8 per strain; n = 654 total) to detect underlying genetic loci; 2) congenic mice (n = 23) to replicate the identified locus; 3) mice overexpressing the Nrg3 candidate gene in the medial prefrontal cortex (n = 21); and 4) a Nrg3 loss-of-function mutant (n = 59) to functionally implicate the Nrg3 candidate gene in impulsivity., Results: Genetic mapping of impulsive action in the BXD panel identified a locus on chromosome 14 (34.5-41.4 Mb), syntenic with the human 10q22-q23 schizophrenia-susceptibility locus. Congenic mice carrying the impulsivity locus (Impu1) confirmed its influence on impulsive action. Increased impulsivity was associated with increased Nrg3 gene expression in the medial prefrontal cortex (mPFC). Viral overexpression of Nrg3 in the mPFC increased impulsivity, whereas a constitutive Nrg3 loss-of-function mutation decreased it., Conclusions: The causal relation between Nrg3 expression in the mPFC and level of impulsive action shown here provides a mechanism by which polymorphism in NRG3 in humans contributes to a specific cognitive deficit seen in several psychiatric diseases, such as addiction, attention-deficit/hyperactivity disorder, and schizophrenia., (Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: The costimulatory molecule B7-H1 (programmed death-1 ligand-1, PD-L1) has been implicated as a potential regulator of antitumor immunity in various human cancers. To date, no data are available on the role of B7-H1 in Barrett carcinoma. Therefore, we investigated the expression pattern and clinical significance of B7-H1 in a large cohort of patients with Barrett carcinoma., Methods: Expression of B7-H1 was evaluated by immunohistochemistry in 101 patients with Barrett carcinoma. Expression data were correlated with clinicopathologic features, including TNM stage, UICC (Union Internationale Contre le Cancer) tumor stage, tumor grade, resection status, and survival, and with the number of tumor-infiltrating CD3(+), CD8(+), and CD45RO(+) T lymphocytes., Results: Aberrant B7-H1 expression was found in Barrett carcinoma cells. High tumor B7-H1 expression was significantly associated with advanced T stage (p = 0.002), advanced UICC tumor stage (p = 0.022), and incomplete resection status (p = 0.009). The median survival of patients with high tumor B7-H1 expression was 38 months compared with 136 months for patients with no or low tumor B7-H1 expression. In the multivariable analysis, high tumor B7-H1 expression was significantly associated with an increased risk of death from Barrett carcinoma (hazard ratio, 3.50; 95% confidence interval, 1.66 to 7.38; p < 0.001)., Conclusions: Our data suggest that B7-H1 may represent a new prognostic marker for patients with Barrett carcinoma. Furthermore, given its immune-inhibitory function, B7-H1 may represent a potential target in the treatment of Barrett carcinoma., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Wildlife organisms are exposed to a combination of chemical, biological and physical stressors. Information about the relative impact of each stressor can support management decisions, e.g., by the allocation of resources to counteract those stressors that cause most harm. The present paper introduces Eco-SpaCE; a novel receptor-oriented cumulative exposure model for wildlife species that includes relevant ecological processes such as spatial habitat variation, food web relations, predation, and life history. A case study is presented in which the predicted mortality due to cadmium contamination is compared with the predicted mortality due to flooding, starvation, and predation for three small mammal species (Wood mouse, Common vole, and European mole) and a predator (Little owl) living in a lowland floodplain along the river Rhine in The Netherlands. Results indicated that cadmium is the principal stressor for European mole and Little owl populations. Wood mouse and Common vole population densities were mainly influenced by flooding and food availability. Their estimated population sizes were consistent with numbers reported in literature. Predictions for cadmium accumulation and flooding stress were in agreement with field data. The large uncertainty around cadmium toxicity for wildlife leads to the conclusion that more species-specific ecotoxicological data is required for more realistic risk assessments. The predictions for starvation were subject to the limited quantitative information on biomass obtainable as food for vertebrates. It is concluded that the modelling approach employed in Eco-SpaCE, combining ecology with ecotoxicology, provides a viable option to explore the relative contribution of contamination to the overall stress in an ecosystem. This can help environmental managers to prioritize management options, and to reduce local risks., (Copyright 2009 Elsevier B.V. All rights reserved.)

The subject of the present study is to find the best way of making a simulation model for the exposure assessment of mobile individuals. For that purpose we consider and apply several very different approaches to model movement patterns in a more or less random way and compare them in terms of the exposure resulting for the individuals. The models combine random movement with agenda-driven movement. We do not aim to involve all details of real conditions into the models, but explain and review the general concepts and provide an inter-comparison of these concepts. Stationary and ergodic behaviour are explained and applied as well as performance criteria for a comparative evaluation of the investigated algorithms. In particular, the present study investigates the exposure to air contaminants of persons moving in heterogeneously polluted urban areas by help of movement simulations. For that purpose we applied four different movement algorithms: Lévy-modulated correlated random walk (LMCRW), Potential field controlled walk (PTW), Reference point mobility model (RPM), and RPM with a pre-defined daily agenda of targets (RPMA). We find that none of the studied algorithm results in purely random trajectories. PTW and RPMA prove to be suitable for human mobility modelling, because they provide conditions for very individual-specific trajectories and exposure. Suggesting these models we demonstrate the plausibility of their results for exposure to air-borne benzene and the combined exposure to benzene and nonane. It appears however that inter-individual variation in the individual-specific short-term exposure diminishes with runtime and when long-term exposure is considered., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: An inability to inhibit behaviors once they become maladaptive is a component of several psychiatric illnesses, and the medial prefrontal cortex (mPFC) was identified as a potential mediator of behavioral inhibition. The current study tested if the mPFC is involved in inhibition of sexual behavior when associated with aversive outcomes., Methods: Using male rats, effects of lesions of the infralimbic and prelimbic areas of the mPFC on expression of sexual behavior and ability to inhibit mating were tested using a paradigm of copulation-contingent aversion., Results: Medial prefrontal cortex lesions did not alter expression of sexual behavior. In contrast, mPFC lesions completely blocked the acquisition of sex-aversion conditioning and lesioned animals continued to mate, in contrast to the robust behavioral inhibition toward copulation in mPFC intact male animals, resulting in only 22% of intact male animals continuing to mate. However, rats with mPFC lesions were capable of forming a conditioned place preference to sexual reward and conditioned place aversion for lithium chloride, suggesting that these lesions did not alter associative learning or sensitivity for lithium chloride., Conclusions: The current study indicates that animals with mPFC lesions are likely capable of forming the associations with aversive outcomes of their behavior but lack the ability to suppress seeking of sexual reward in the face of aversive consequences. These data may contribute to a better understanding of a common pathology underlying impulse control disorders, as compulsive sexual behavior has a high prevalence of comorbidity with psychiatric disorders and Parkinson's disease.

Background: In the setting of severe perigenicular trauma or complicated endoprosthetic knee surgery, primary knee fusion may be the last resort for salvage of the limp. In this case, the patella looses its destination as an anterior knee stabilizer and can become a substantial donor of bone substance, especially if osseous defects are involved., Patients and Methods: 12 formalin fixated cadavers were studied in terms of vascular anatomy, pedicle reliability, arc of rotation and their relation to sex, age, and height. Moreover, the operation was performed on a suitable patient., Results: The quadriceps with the vastus medialis and the patella can be raised from the tibial tuberosity up to the entrance of the osteoarticular branch of the superficial femoral artery into the vastus medialis muscle ca 16 cm (15-19 cm) from the inferior patellar pole. This distance correlated well to the overall height of the cadavers (P=0.009). The vascular prerequisites were always present. In the clinical case, there was a favorable outcome with knee fusion after 4 months, despite of the lateral condylar defect., Discussion: The composite vastus medialis-patellar complex osseomuscular flap can be safely used as a source of vascularized femoral condyle substitute in the setting of primary knee fusion.

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form.

The complement system plays an important role in the humoral immune response. Activation of the classical complement pathway is mediated by its subcomponent, C1q. Among the main C1q-synthesising tissues, macrophages have been attributed as a source of particular importance. We investigated the effects of cytokines (IL-1, IL-6 and Interferon-gamma) on local C1q mRNA expression and C1q secretion in resident and in thioglycollate-stimulated murine peritoneal macrophages in vitro. The macrophages were isolated from murine peritoneal lavage fluid, maintained in culture and incubated with the cytokines. Among the cytokines, only IL-6 had a stimulatory effect on C1q production (25% increase vs. control), while IL-1 and interferon-gamma had an inhibitory effect (50% decrease vs. control), especially in stimulated peritoneal macrophages in culture. Our data suggest that C1q production in macrophages may be differentially regulated by inflammatory cytokines such as IL-1, IL-6 and interferon-gamma, the response being dependent on macrophage activation.

Selective complete C1q deficiencies (SCDC1q) of the complement component C1q are rare genetic disorders with high prevalence of lupus-erythematosus-like symptoms and recurrent infections. Among the 41 published cases from 23 families, 10 derive from 6 Turkish families. One particular mutation leading to a stop codon in the C1q A gene was first identified in members of a Gypsy family from the Slovac Republic. Later the same mutation has been found in all cases in four SCDC1q families from Turkey suggesting that one particular defective allele may be present in the populations of Southeastern Europe and Turkey. This study was undertaken to investigate the frequency of C-->T mutation in exon II of C1qA gene in Turkish population by using allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Among the 1544 patients from 15 pediatric departments and an additional 89 SLE patients of various ages no C1qA gene mutation was found. There were 43 heterozygous and 4 homozygous mutations in 161 family members or relatives investigated from the 4 families known with SCDC1q. Among the 223 inhabitants who were nonrelative to the 3 SCDC1q families living in the same village were screened for mutation and one heterozygous individual was observed. Although this mutant allele appears to be at a low prevalence in the population tested, individuals with recurrent infections or symptoms of lupus erythematosus-like syndrome should be tested for this mutation to rule out this type of C1q deficiency.

The use of biotinylated ligands for the flow cytometric detection of cell surface receptors has become a popular alternative to radioreceptor assays. Although the biotinylation of a protein is a relatively mild chemical reaction several reports have mentioned the fact that the number and location of biotin moieties coupled to amino groups of a protein can alter its physicochemical properties and impair biological activity. In the present study we show for a variety of biotinylated functionally unaltered ligands that biotinylation by N-hydroxysuccinimide (NHS) esters of biotin can induce a binding to cell surfaces, which is not specific for the respective unlabelled ligand. C1q, C1 inhibitor (C1-INH), alpha 1-antitrypsin (AT), ovalbumin (OV), transferrin and soybean trypsin inhibitor (STI) were labelled with S-NHS-LC-biotin and activated C1s (C1s) with NHS-biotin. Biotinylation of C1q, C1s and C1-INH exerted negligible effects on biological function, antigenicity or electrophoretic mobility but when labelled and unlabelled proteins were assayed for binding to monocytic U937 cells, promyelocytic HL-60 cells, monocytes and granulocytes, a remarkable binding was observed for biotinylated C1q, C1-INH and C1s. In contrast, no binding was observed when we used unlabelled C1q, C1s and C1-INH and employed specific antibodies, alpha-mouse-FITC or alpha-rabbit-FITC for detection. Increasing molar ratios of biotin-to-protein (B : P) for biotinylated AT, OV and STI evoked increased fluorescence intensities of the cells. Most importantly the unlabelled ligands did not compete for cell binding with their biotinylated derivatives, with the exception of transferrin. Preincubation of the cells with an excess of free d-biotin did not reduce binding of biotinylated proteins, thus excluding a potential involvement of biotin receptors. Hydrophobic interaction chromatography revealed a remarkable increase in hydrophobicity of the biotinylated proteins compared to their unlabelled counterparts, suggesting that the biotinylation-induced binding is due to increased hydrophobicity. Our findings indicate that biotinylation by the common amino acid esterification method may be critical for proteins if they are to be used as ligands for receptor binding studies.

The major bcr-abl fusion gene is presently seen as the hallmark of chronic myeloid leukemia (CML) and presumably as the cause of its development. Accordingly, long-term disappearance of bcr-abl after intensive therapy is considered to be a probable cure of CML. The nested reverse transcriptase-polymerase chain reaction (RT-PCR) provides a powerful tool for minimal residual CML detection. The RT-PCR was optimized by (1) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells, (2) using a specific abl primer in this reverse reaction, and (3) reamplifying 10% of the RT-PCR product in nested amplification. This optimized RT-PCR permitted us to detect up to 1 copy of RNA bcr-abl synthesised in vitro, mixed with yeast RNA in an equivalent quantity to 10(8) white blood cells (WBCs). Using this highly sensitive RT-PCR during the follow-up of CML patients, a signal was unexpectedly found in healthy controls. Therefore, a systematic study of the possible expression of bcr-abl RNA in the WBCs of healthy adults and children and in umbilical cord blood was undertaken. It showed the presence of bcr-abl transcript in the blood of 22 of 73 healthy adults and in the blood of 1 of 22 children but not in 22 samples of umbilical cord blood.

In situ hybridization, RT-PCR and Northern blot analysis as well immunohistochemistry were used to examine the expression of C1q, a subcomponent of the rat complement system, in brains of rats infected with Borna disease virus (BDV) and rats afflicted with experimental allergic encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein specific T cells. C1q mRNA, which was not detected in normal brain, became clearly detectable using RT-PCR analysis by d14 post infection (p.i.) with BDV. Maximal levels of C1q mRNA were reached 21 days p.i. when inflammatory reactions in the brain were also at a peak. Similarly, C1q mRNA was elevated when the clinical symptoms of EAE became evident 5 days following cell transfer. C1q positive cells, as identified by immunohistology, were preferentially localized in grey and white matter of the hippocampus and basolateral cortex. The C1q positive cells resembled microglial cells in morphology. The correlation of C1q expression with the development of neurological disease as well as the dramatic increase of C1q within brain regions with inflammatory lesions suggest that local biosynthesis of C1q may play a role in the pathogenesis of Borna virus induced and autoimmune encephalomyelitis.

Mutations at four X-linked visible loci (yellow, white, vermilion and forked) induced by X-irradiation of mature sperm and spermatogonial cells were analysed genetically and cytogenetically. In addition, a fraction of the intragenic vermilion mutations was analysed molecularly. Males of two wild-type strains (Amherst M56i and Berlin-K) were used. A total of 332,651 chromosomes of irradiated mature sperm and 311,567 of irradiated spermatogonial cells were scored. The ratio of F1 female sterile, F2 male lethal and F2 male viable mutations in mature sperm and spermatogonial cells is very similar. The cytogenetic analysis shows equal fractions of multilocus deletions and translocations among the mutations recovered from both stages of spermatogenesis. These data strongly suggest that the spectrum of X-ray mutations is similar in mature sperm and spermatogonial cells, including multilocus deletions and chromosome rearrangements. The molecular analysis of a number of intragenic vermilion mutations showed the presence of three small deletions (1-10 bp), one insertion of two nucleotides and seven single nucleotide changes.

The direct acting mutagen 2-cyanoethylene oxide (CEO), formed in the liver by oxidation of acrylonitrile (ACN), is thought to mediate the extrahepatic carcinogenic effects of ACN in rats. This study determined the tissue distribution of CEO (3 mg/kg p.o.) in F-344 rats and B6C3F1 mice. Radioactivity from [2,3-14C]CEO was widely distributed in the major organs of rodents by 2 h and decreased by 71% to 90% within 24 h, demonstrating that there was no preferential tissue uptake or retention of CEO. CEO was detected in rodent blood and brain 5-10 min after an oral dose of ACN (10 mg/kg), demonstrating that this mutagenic epoxide metabolite circulates to extrahepatic target organs following ACN administration.

In this report we are able to show that intravenous (i.v.) application (day 0) of a nonapeptide (residues 26-34) from the human C1q A-chain (designated peptide A-C1q) prior to intradermal (i.d.) administration of chicken type II collagen (CII) in arthritis-susceptible DBA/1 mice (H2q), leads to abrogation of polymorphonuclear neutrophil (PMN) invasion into the joints. This nonapeptide exhibits epitope characteristics and high homology to residues 137-147 of CB11 (a cyanogen bromide fragment of chicken CII, known to contain both arthritis inducing and suppressing determinants). Arthritis index was lowest in animals pretreated i.v. with CII (as internal control), though animals pretreated i.v. with peptide K (residues 137-147 with an additional glycine residue from CB11) or peptide A-C1q exhibited comparative arthritic indices. Only in the arthritis-positive control group (day 0: PBS i.v.) did i.d. application of CII lead to invasion of PMN into the synovial layer and the joint space. Analysis of antibody (Ab) responses at day 48 after i.v. immunization (day 0) and CII challenge (day 7) revealed IgE-Abs to native CII and also to native C1q. IgG titers to CII were highest in animals pretreated with peptide A-C1q. Abs from this group, exhibiting activity to peptide A-C1q (immunizing antigen), were of mainly IgG1 and IgG3 isotypes. Evaluation of the immune response following i.v. application of peptide A-C1q or CII, prior to i.d. CII administration, in DBA/1 mice, revealed IgM responses to peptide A-C1q and peptide K, but not to CII. Intravenous application of peptide A-C1q led to generation of IgG3-Abs reacting only with peptide A-C1q and peptide K, but not with native CII. Additionally, i.v. application of peptide A-C1q elicited IgG responses to a pentapeptide, resembling amino acid residues 26-30 (K-G-E-Q-G) of the C1q A-chain. This five residue antigenic determinant is present in peptide K, in chicken and human CII as well as in human C1q. No specific IgE response to any of the antigens tested could be detected. Since a peptide from the C1q A-chain is both capable of eliciting immune responses and modulating CII-induced arthritis in mice, we postulate that the collagen-like complement component C1q is involved in the development of CII-induced inflammatory arthritic lesions, and may represent, in vivo, the early antigen responsible for inducing anticollagen antibodies prior to CII in hyaline cartilage becoming available as antigen.

Guinea pig peritoneal macrophages were cultured for 24 h in the presence of two inhibitors of the biosynthesis of collagen-like molecules such as C1q : 10(-3) M 3,4-dehydroproline or 10(-4) M 2,2'-dipyridyl. Their Fc-receptor activity was measured by rosette formation, using sheep erythrocytes (E) coated with rabbit anti-sheep IgG (EAIgG). The Fc-receptor activity was decreased by 40 to 70% of control cultures depending on the amount of IgG on the E. The activity of a second receptor on the macrophages, mediating the binding of C3b coated E, was not altered by this treatment. Rat alveolar macrophages were depleted of their Fc-receptor activity by pronase treatment (1.5 mg/ml) in the presence of 2,2'-dipyridyl. After washing the cells, the EAIgG-binding activity was restored to about half of the initial level within 2 h. With 2,2'-dipyridyl also present during the second incubation, the re-expression of the Fc-receptor activity was suppressed further. Preincubation of guinea pig peritoneal macrophages with anti-C1q-F(ab')2 for 45 min at 37 degrees C caused a dose-dependent reduction of the Fc receptor activity, but not C3b receptor activity. These results support our hypothesis that C1q synthesized and secreted by macrophages serves as an Fc-receptor in the membrane during the secretion.

Human peripheral blood lymphocytes stimulated in vitro were exposed to low level irradiation ('adaptive dose') from radioisotopes ([3H]dThd, [14C]dThd, HTO and 32P). After 50 h in culture, they were irradiated with 50 rad of X-rays ('challenge dose') and fixed 3 h later. In another series, the lymphocytes received an adaptive dose of 5 rad of X-rays at 32 h after stimulation and a challenge dose of 150 rad at 48 h; the cells were fixed at 54 h. In cells that received both the adaptive and challenge doses, the frequencies of chromosomal aberrations (chromatid and isochromatid deletions) were lower than expected on the basis of additivity of the effects of the individual treatments. These results support those published from Wolff's laboratory in showing that human lymphocytes can become 'adapted' by prior exposure to low level irradiation so that they become less sensitive to the chromosome-breaking effects of X-rays delivered subsequently. The magnitude of reduction in frequencies in the 'adapted' cells, however, varied between the blood samples from different donors.

In this study, the effect of sixteen different enzymes on serum C1 and its subcomponents was investigated. The sixteen enzymes could be divided into three groups. First, enzymes which activate native C1: trypsin (optimal concentration 2.4 x 10(-4) mM); alpha-chymotrypsin (2.3 x 10(3) mM); thrombin (1.0 x 10(-5) mM); plasmin (1.9 x 10(-5) mM); elastase (5.8 x 10(-5) mM); pronase (3.0 x 10(-6) mM). All these enzymes are serine esterase and activate native serum C1 bound to EAC4 at the given concentration within 10 min at 30 degrees C. Furthermore, native C1 inhibited by a pentosanpolysulfoester, Sp54, is unable to undergo the internal activation but can be externally activated by the serine esterases. Second, enzymes which do not activate native C1 but result in a dose and time-dependent loss of C1 activity: collagenase; pepsin; carboxypeptidase B. Third, enzymes which have no effect on C1 and C1: Lysozyme; neuraminidase; beta-galactosidase; L-amino acid oxidase; arginase; streptokinase, and acetylcholinesterase.

The first component of complement (C1) is a macromolecule consisting of three distinct subcomponents, C1q, C1r, and C1s. In regard to its production site and its role in phagocytic processes it was of interest to find out whether these different subcomponents could be detected in human biopsy material only as a complex in individual cells or whether C1 subcomponents could be found on different cells. To study this question, monospecific fluorescein-labelled anti-human-C1q IgG and monospecific rhodamine-labelled anti-human C1q IgG were used. Biopsy material from human rectum was stained with fluoresceinated antisera, either by use of one antiserum or by double staining. Using this technique, these observations were made: C1q as well as C1s were detectable in individual cells in the subepithelial area of the gut. Furthermore, C1q and C1s could be found together in the same cell or separately in different cells. These findings were supported by experiments with cultured peritoneal macrophages either from human or from guinea pig. The examination of the cultured cells with the two antisera revealed that individual cells were stained either by anti-C1q or by anti-C1s antibodies. The specificity of the detection of the individual subcomponents was also proven by the peroxidase technique and by using fluoresceinated anti-human C1q F(ab')2. The membrane immunofluorescent staining revealed the presence of C1q on the membrane of the macrophage.

New methods have been developed for the isolation, quantitative detection, and functional measurement of human complement C-1-inhibitor (C-1-INH). The two-step purification procedure for C-1-INH from human plasma or serum employs affinity chromatography with a monoclonal anti-C-1-INH antibody coupled to CNBr-activated Sepharose 4B followed by fractionation on a FPLC Mono Q HR 5/5 column. It yields functionally active, homogeneous C-1-INH with about 40% recovery. For quantitative estimation of C-1-INH an ELISA was performed. ELISA plates were coated with a polyclonal anti-C-1-INH antibody, serum or plasma was added and bound C-1-INH was detected with the monoclonal anti-C-1-INH antibody. The method has a sensitivity of 0.4 ng C-1-INH per assay corresponding to 20 ng/ml. For the detection of functionally active C-1-INH an ELISA was developed using C1-s-coated microtiter plates. After incubation with serum or plasma, C1-s-bound C-1-INH was monitored with the monoclonal anti-C-1-INH antibody. With this method it is possible to measure as little as 0.3 ng of functionally active C-1-INH in 20 microliter of a biological sample. All methods described in the present paper are easy to perform, rapid, sensitive, and highly reproducible.

Based on the earlier observation that DNP-HSA interacts directly with C1q, a subcomponent of the first component of complement (Loos and König, 1977), evidence is presented that TNP bound to erythrocytes (E-TNP) can interact with the whole complement sequence leading to the lysis of the TNP-carrying erythrocytes; in this test system the erythrocytes are used as an indicator of the TNP-complement reaction. To exclude any antibody-mediated lysis either the heterologus sera were exhaustively absorbed with the erythrocytes used in the test system or serum and erythrocytes of one individual person were taken. The strongly temperature-dependent interaction of TNP-sulfonic acid with the erythrocytes resulted in the formation of E-TNP as well as of a TNP-protein complex released into the supernatant. The TNP-protein complex strongly inhibited purified C1 similar to DNP-HSA. The antibody independent lysis of E-TNP by complement was dependent upon the TNP concentration per cell, the temperature and time as well as the complement concentration. The reaction of E-TNP with complement showed similar characteristics to those for the reaction of antibody sensitized erythrocytes (EA) with complement. E-TNP is a helpful tool to study antibody-independent activation of the complement system.

A monoclonal antibody directed against the collagenous portion of human C1q was used to detect C1q-bearing immune complexes in patients with rheumatic disorders. Sera of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE), osteoarthritis, as well as normal human sera (NHS) used as controls were tested in an ELISA system. C1q-bearing immune complexes were bound to a solid-phase monoclonal anti-C1q antibody, and detected with F(ab')2 antibodies to human IgG. Heat-aggregated human IgG was adjusted to the same concentration as the WHO standard for immune complexes and used for the standard curve in NHS. The mean value in NHS was 19.5 micrograms/ml equivalents of aggregated IgG. Using 2 SD over the mean as the upper limit for normal values, samples greater than 43 micrograms/ml were considered positive. Patients with osteoarthritis were negative; high levels of C1q-bearing immune complexes were detected in patients with rheumatoid arthritis (up to 800 micrograms/ml equivalents of aggregated IgG). With our assay C1q-bearing immune complexes were detected with high frequency (81%) in the sera of patients with rheumatoid arthritis, while a C1q solid-phase binding assay (C1q SPBA) revealed positive results only in 67% of rheumatoid arthritis sera. Compared to NHS, CH50 titers and C1q values of sera from patients with rheumatoid arthritis were frequently high. In contrast, the sera of SLE patients with low CH50 titers and low C1q levels had IgG immune complexes which could be detected only in the C1q-SPBA. C1q-bearing immune complexes were not detectable in the sera of patients with SLE. Since C1q triggers activation of the classical C pathway, this assay with monoclonal anti-C1q antibody appears to be useful for detecting immune complexes in rheumatoid arthritis patients with normal or elevated CH50 and C1q values, especially in the early stage of the disease.

Three commercially available automated NO2-monitors, based on amperometry, chemiluminescence and coulometry respectively, were compared under simulated field conditions over a period of 45 days of continuous operation. The main purpose of this programme was the study of the reliability of the overall system, sampling line-monitors-data acquisition and handling system (PDP-8/E), the correlation between the individual half-hour averages given by the three monitors in the range 0--700 mug NO2/m3 and the comparison of the global statistics for the same NO2-pollution simultaneously seen by the three different monitors over a period of 45 days. Based on more than 1200 valid half-hour averages, a highly significant correlation between the different monitors was obtained, while the statistics of the simulated NO2-pollution were in good agreement.

The two forms of human plasma C2 that were described in the preceding report (1) were investigated for their functional and biochemical differences. Incubation with the neuraminidase (NAN'dase) of Clostridium perfringens at 37 degrees C resulted in a four- to fivefold increase in the hemolytic activity of both forms. The increase in activity was different than the increase caused by treatment with iodine. The mechanism of increased activity of NAN'dase-treated C2 was the generation of increased molecules of activated C3 (C3b), resulting in more molecules of C5 binding to (C4b, 2a, 3b)n. Removal of N-acetyl-neuraminate from C2 did not alter its binding to a cationic exchanger. Nonenzymatic glucosylation was used to distinguish the two forms of C2. Incubation of highly pure C2 with 14C-D-glucose resulted in the gradual accumulation of radioactivity in acid-precipitable material. The two forms of C2 were glucosylated in vitro for seven days with 14C-D-glucose in phosphate-buffered saline at 25 degrees C. Form 2 bound twice as much 14C-D-glucose as form 1. Glucosylated form 2, but not form 1, lost some of its affinity to bind to a cationic exchanger. Since the interaction between glucose and protein occurs at free amino groups, we conclude that form 2 of C2 has approximately twice as many free amino groups as form 1. This explains the reason for the existence of two forms of C2 in plasma independent of the allelic variant.

The second component of human complement (C2) in pseudoglobulin prepared from normal plasma eluted as a single peak at high conductivity (30 mS) and pH 4.5 from the cationic exchangers S-Sepharose or Mono S in the Fast Protein Liquid Chromatography (FPLC) System. The C2 was stable at pH 4.5 and 0 degrees C if enzyme inhibitors were used and the pH was raised to 6.0 after elution from the columns. After rechromatography on Mono S in the FPLC System at the median isoelectric point of 5.5 or pH 6.0, the C2 eluted as two distinct hemolytic forms: the first peaked at 16 mS, the second at 30 mS. The two forms of C2 did not correlate with the allotypic variant of C2 in individual, normal human plasmas. After elution at pH 4.5 from S-Sepharose and rechromatography at pH 5.5 or 6.0 on Mono S, the hemolytic activities of the two forms in individual plasmas eluted in 3 patterns: 1) high activity at 16 mS, low activity at 30 mS; 2) low activity at 16 mS, high activity at 30 mS; 3) high activity at 16 mS, high activity at 30 mS. The specific activities of both forms were approximately the same; both eluted the same after gel filtration at pH 5.5, and both had the same pattern on SDS-PAGE and immunoblots. The pattern of elution was characteristic for each individual plasma, and the first hemolytic form appeared to elute independent of the second form. At pH 4.5, C2 was completely separated from Factor B, a functionally and structurally similar protein of the alternative complement pathway, whereas at pH 5.5 or 6.0, the two proteins eluted together. From these results, the two forms of hemolytic C2 can be purified for structural and functional analyses.

A simple and rapid procedure for the purification of C1q from human, guinea pig and mouse serum is described. This procedure allows the purification of C1q within one and a half days using euglobulin precipitation, chromatography on Superose 6B, followed by chromatography on Mono S by Fast Protein Liquid Chromatography (FPLC). The highly purified, hemolytically active C1q is free of any immunoglobulins. Since the purification of C1q of three different species was performed by the same purification procedure, a comparison of the subunit compositions was made under reducing and non-reducing conditions on SDS-PAGE. The yield was found to be more than 50%.

A sandwich ELISA system has been developed for the detection of C1q in human serum. It is specific, uses monoclonal antibodies, is sensitive into the nanogram range and is rapidly performed. Therefore, it may be a helpful tool for clinical routine diagnosis, e.g., detecting abnormal C1q levels in patients with rheumatic disorders. Various combinations of poly- and monoclonal antibodies were tested in a sandwich assay. One of these combinations, in particular, resulted in a highly reproducible standard curve: C1q bound to solid-phase polyclonal anti-C1q was detected by the monoclonal antibody 242 G3. In this assay, the C1q concentration in sera of normal individuals was found to be 160 micrograms/ml (mean value of 70 normal human sera). This ELISA detected nanogram levels of C1q and gave results comparable to those obtained by haemolytic C1q titration. One nanogram of C1q corresponded to ca. 2.6 X 10(10) effective C1q molecules. With this technique, selective C1q deficient sera as well as sera from patients with rheumatoid diseases were analysed.

Classical pathway (CP)-triggered reactions of complement-modulated immune complex (IC) aggregation (tetanus toxoid/human anti-tetanus toxoid-IgG; ICs of equivalence) were investigated turbidimetrically during the early stages of reaction. Monospecific Fab'- or Fab-fragments (rabbit) directed against certain complement components were used to block the complement function in normal human serum (NHS). Additionally, parts of the reactions were studied using purified complement components. C1q in serum generated by the addition of EDTA as well as purified C1q were found to increase the IC aggregation. In contrast to C1q, macromolecular C1 is able to inhibit IC aggregation, whereas additional participation of C-1 INH reversed this process. The cooperation of the remaining CP proteins (C4, C2, C4bp, and I) reconstituted the inhibition capacity of the complement. Whereas C3 supported significantly inhibition, a significant influence of other effector pathway (EP) components (C5-C9) was not detectable turbidimetrically.

The first component of complement, C1, is a calcium-dependent complex of the three distinct subcomponents, C1q, C1r, and C1s. Earlier observations revealed that treatment of C1 with EDTA led to a loss of hemolytic C1 activity even after recalcification. Therefore, it was of interest to study whether EDTA has an additional effect on C1 and its subcomponents, beside its chelating capacity. The chelating effect of EDTA was compared to that of citrate. It was found that treatment of C1 or C1 with EDTA followed by addition of Ca++ led to a loss of hemolytic activity up to 90%, depending on EDTA concentration. Even pretreatment of EDTA with varying amounts of Ca++ did not prevent the inactivation of C1 or C1. In contrast, after dissociation of C1 or C1 by citrate, 100% of the original C1q activity is recoverable on addition of C1q deficient serum as source of C1r and C1s. EDTA-treated serum, however, showed a concentration-dependent loss of hemolytic C1q activity, indicating an inhibitory effect of EDTA on C1q. EDTA-treated C1q, fluid phase or bound to EA, was no longer able to form an hemolytically active C1 complex by interaction with C1r and C1s.

A rapid and simple ELISA has been developed for identifying the specificities of two monoclonal antibodies recognizing either similar or distinct epitope(s) of an antigen. The method utilizes microtiter plates coated with one of the monoclonal antibodies either by direct adsorption of the purified antibody to the plastic or by immobilization of the antibody from ascites or hybridoma supernatants via immobilized polyclonal anti-mouse immunoglobulin antibodies. After preincubation of the antigen with the second monoclonal antibody, the mixture is added to the surface-immobilized first antibody. The amount of antigen bound to the first antibody is subsequently measured by rabbit polyclonal antibodies to the antigen and peroxidase-conjugated anti-rabbit immunoglobulin antibodies. Binding of antigen to the first antibody is only observed when the second monoclonal antibody binds to a distinct epitope. The major advantages of this procedure are its simplicity, rapidity and independence of radioisotopes. Using this method a library of monoclonal antibodies against human C1(-)-INH has been tested and several duplicate monoclonal antibodies have been identified. Furthermore, the above analytical procedure was capable of detecting conformational changes of the C1(-)-INH molecule induced either by binding of a monoclonal antibody to C1(-)-INH or by enzymatic cleavage of C1(-)-INH.