Exposure of intestinal explants to NX, but not to DON, enriches the secretome in mitochondrial proteins.

NX is a type A trichothecene produced by Fusarium graminearum with limited information on its toxicity. NX is structurally similar to deoxynivalenol (DON), only differing by the lacking keto group at C8. Because of the structural similarity of the two toxins as well as their potential co-occurrence in food and feed, it is of interest to determine the toxicity of this new compound. In this study, we compared the protein composition of the extracellular media of pig intestinal explants (secretome) exposed to 10 µM of DON or NX for 4 h compared with controls. The combination of two complementary quantitative proteomic approaches (a gel-based and a gel-free approach) identified 18 and 23 differentially abundant proteins (DAPs) for DON and NX, respectively, compared to controls. Functional analysis suggested that, whereas DON toxicity was associated with decreased cell viability and cell destruction, NX toxicity was associated with an enrichment of mitochondrial proteins in the secretome. The presence of these proteins may be associated with the already known ability of NX to induce an intestinal inflammation. Overall, our results indicated that DON- and NX-induced changes in the extracellular proteome of intestinal explants are different. The increased leakage/secretion of mitochondrial proteins by NX may be a feature of NX toxicity.

Toxicity of DDT to the hooded oyster Saccostrea cucullata: Mortality, histopathology and molecular mechanisms as revealed by a proteomic approach.

Dichlorodiphenyltrichloroethane (DDT), a persistent organochlorine pesticide, has been linked to adverse biological effects in organisms. However, there is limited knowledge about its toxic effects on marine organisms and the underlying molecular mechanisms. This study investigated the toxic effects of DDT in the hooded oyster Saccostrea cucullata. The oysters were exposed to DDT at concentrations of 0, 10, 50, 100, 500, 1000 and 2000 µg/L for 96 h and the LC50 (96 h) was 891.25 µg/L. Two sublethal concentrations (10 and 100 µg/L) were used to investigate the histopathological effects and the proteome response. Histopathological results showed that DDT caused the alteration of mantle tissue. This included the induction of mucocytes in the epithelium and the inflammatory effect in the connective tissue indicated by the enlargement of blood sinus and hemocyte aggregation within the sinus. Proteomic results showed that, amongst approximately 500 protein spots that were detected across 2DE gels, 51 protein spots were differentially expressed (P < 0.01; fold change > 1.2). Of these, 29 protein spots were identified by LC-MS/MS. These included 23 up-regulated, 5 down-regulated and 1 fluctuating spots. Thus, we observed that stress response and cytoskeletal proteins are the central targets of DDT action. Furthermore, DDT alters the expression of proteins involved in energy metabolism, calcium homeostasis and other proteins of unknown function. Additionally, proteomic results clearly elucidated the molecular response of the histopathological changes which were driven by the alteration of cytoskeletal proteins. Our results improve the current knowledge of toxicity of the DDT to histology and molecular response of oyster proteome to DDT exposure. In addition, histopathological changes will be beneficial for the development of an appropriate guideline for health assessment of this species in ecotoxicological context.

Quitting behaviours and cessation methods used in eight European Countries in 2018: findings from the EUREST-PLUS ITC Europe Surveys.

BACKGROUND: We examined quit attempts, use of cessation assistance, quitting beliefs and intentions among smokers who participated in the 2018 International Tobacco Control (ITC) Europe Surveys in eight European Union Member States (England, Germany, Greece, Hungary, the Netherlands, Poland, Romania and Spain). METHODS: Cross-sectional data from 11 543 smokers were collected from Wave 2 of the ITC Six European Country (6E) Survey (Germany, Greece, Hungary, Poland, Romania and Spain-2018), the ITC Netherlands Survey (the Netherlands-late 2017) and the Four Countries Smoking and Vaping (4CV1) Survey (England-2018). Logistic regression was used to examine associations between smokers' characteristics and recent quit attempts. RESULTS: Quit attempts in the past 12 months were more frequently reported by respondents in the Netherlands (33.0%) and England (29.3%) and least frequently in Hungary (11.5%), Greece (14.7%), Poland (16.7%) and Germany (16.7%). With the exception of England (35.9%), the majority (56-84%) of recent quit attempts was unaided. Making a quit attempt was associated with younger age, higher education and income, having a smoking-related illness and living in England. In all countries, the majority of continuing smokers did not intend to quit in the next 6 months, had moderate to high levels of nicotine dependence and perceived quitting to be difficult. CONCLUSIONS: Apart from England and the Netherlands, smokers made few quit attempts in the past year and had low intentions to quit in the near future. The use of cessation assistance was sub-optimal. There is a need to examine approaches to supporting quitting among the significant proportion of tobacco users in Europe and increase the use of cessation support as part of quit attempts.

ECM Characterization Reveals a Massive Activation of Acute Phase Response during FSGS.

The glomerular basement membrane (GBM) and extra-cellular matrix (ECM) are essential to maintain a functional interaction between the glomerular podocytes and the fenestrated endothelial cells in the formation of the slit diaphragm for the filtration of blood. Dysregulation of ECM homeostasis can cause Focal segmental glomerulosclerosis (FSGS). Despite this central role, alterations in ECM composition during FSGS have not been analyzed in detail yet. Here, we characterized the ECM proteome changes in miR-193a-overexpressing mice, which suffer from FSGS due to suppression of Wilms' tumor 1 (WT1). By mass spectrometry we identified a massive activation of the acute phase response, especially the complement and fibrinogen pathways. Several protease inhibitors (ITIH1, SERPINA1, SERPINA3) were also strongly increased. Complementary analysis of RNA expression data from both miR-193a mice and human FSGS patients identified additional candidate genes also mainly involved in the acute phase response. In total, we identified more than 60 dysregulated, ECM-associated genes with potential relevance for FSGS progression. Our comprehensive analysis of a murine FSGS model and translational comparison with human data offers novel targets for FSGS therapy.

Modulation of posterior intestinal mucosal proteome in rainbow trout (Oncorhynchus mykiss) after Yersinia ruckeri infection.

Yersinia ruckeri is the causative agent of enteric redmouth disease in salmonids. In fish, the intestine represents an important site of nutrient uptake, host-pathogen interactions, and defense. The posterior intestine can be inflamed, reddened, and filled with an opaque, yellowish fluid during Y. ruckeri infection. Herein, we report an investigation on the proteome alteration in the posterior intestinal mucosa of rainbow trout (Oncorhynchus mykiss) after exposure to Y. ruckeri. The intestinal mucosal proteins were identified and quantified by a shotgun proteomic approach by applying data-independent quantification with sequential windowed acquisition of all theoretical mass spectra (SWATH). A total of 437 proteins were found to be differentially up- or downregulated in the posterior intestine. Gene ontology of upregulated proteins pointed to their involvement into exopeptidase, endopeptidase, and hydrolase activities, while the downregulated proteins were involved in lipid metabolism, actin binding, and translation processes. Additionally, upregulated proteins were predicted to be involved in lysosome, oxidative phosphorylation, and metabolic pathways, while downregulated proteins were implicated in focal adhesion, regulation of actin cytoskeleton, protein digestion and absorption pathways. This study showed that Y. ruckeri infection can alter protein abundance involved in serine-type carboxypeptidase, cysteine and aspartic-type endopeptidases, metallopeptidases, antioxidant defense, calcium ion binding, glycolytic and carbohydrate metabolic processes in the proteome of the intestinal mucosa of rainbow trout.

Does free or lower cost smoking cessation medication stimulate quitting? Findings from the International Tobacco Control (ITC) Netherlands and UK Surveys.

OBJECTIVE: To investigate whether mentioning free or lower cost smoking cessation medication as a trigger for thinking about quitting is related to higher medication use, more quit attempts and quit success, and whether these associations are modified by education and income. METHODS: Data were derived from the 2013 and 2014 surveys of the International Tobacco Control Netherlands (n=1164) and UK (n=768) cohort. Logistic regression analyses were used to assess associations between mentioning in 2013 that free/lower cost smoking cessation medication was a trigger for thinking about quitting smoking and the use of medication, quit attempts and smoking cessation in 2014. RESULTS: 37.0% of smokers in the UK and 24.9% of smokers in the Netherlands mentioned free/lower cost medication as a trigger for thinking about quitting. Smokers who mentioned this trigger were more likely to have used cessation medication during a quit attempt both in the UK (OR=4.19, p<0.001) and in the Netherlands (OR=2.14, p=0.033). The association between mentioning free/lower cost medication as a trigger for thinking about quitting and actual quit attempts was significant in the UK (OR=1.45, p=0.030), but not in the Netherlands (OR=1.10, p=0.587). There was no significant association with quit success. Associations did not differ across income and education groups. CONCLUSION: Free/lower cost smoking cessation medication may increase the use of cessation medication and stimulate quit attempts among smokers with low, moderate and high education and income.

A Distributed Architecture for Human-Drone Teaming: Timing Challenges and Interaction Opportunities.

Drones are expected to operate autonomously, yet they will also interact with humans to solve tasks together. To support civilian human-drone teams, we propose a distributed architecture where sophisticated operations such as image recognition, coordination with humans, and flight-control decisions are made, not on-board the drone, but remotely. The benefits of such an architecture are the increased computational power available for image recognition and the possibility to integrate interfaces for humans. On the downside, communication is necessary, resulting in the delayed reception of commands. In this article, we discuss the design considerations of the distributed approach, a sample implementation on a smartphone, and an application to the concrete use case of bookshelf inventory. Further, we report experimentally-derived first insights into messaging and command response delays with a custom drone connected through Wi-Fi.

Pharmacokinetics of harpagoside in horses after intragastric administration of a Devil's claw (Harpagophytum procumbens) extract.

Devil's claw is used for the treatment of inflammatory symptoms and degenerative disorders in horses since many years, but without the substantive pharmacokinetic data. The pharmacokinetic parameters of harpagoside, the main active constituent of Harpagophytum procumbens DC ex Meisn., were evaluated in equine plasma after administration of Harpagophytum extract FB 8858 in an open, single-dose, two-treatment, two-period, randomized cross-over design. Six horses received a single dose of Harpagophytum extract, corresponding to 5 mg/kg BM harpagoside, and after 7 days washout period, 10 mg/kg BM harpagoside via nasogastric tube. Plasma samples at certain time points (before and 0-24 hr after administration) were collected, cleaned up by solid-phase extraction, and harpagoside concentrations were determined by LC-MS/MS using apigenin-7-glucoside as internal standard. Plasma concentration-time data and relevant parameters were described by noncompartmental model through PKSolver software. Harpagoside could be detected up to 9 hr after administration. Cmax was found at 25.59 and 55.46 ng/ml, t1/2 at 2.53 and 2.32 hr, respectively, and tmax at 1 hr in both trials. AUC0-inf was 70.46 and 117.85 ng hr ml-1 , respectively. A proportional relationship between dose, Cmax and AUC was observed. Distribution (Vz /F) was 259.04 and 283.83 L/kg and clearance (CL/F) 70.96 and 84.86 L hr-1  kg-1 , respectively. Treatment of horses with Harpagophytum extract did not cause any clinically detectable side effects.

Proteome Profiles of Head Kidney and Spleen of Rainbow Trout (Oncorhynchus Mykiss).

The head kidney and spleen are major lymphoid organs of the teleost fish. The authors identify proteome profiles of head kidney and spleen of rainbow trout (Oncorhynchus mykiss) using a shotgun proteomic approach. Gene ontology annotation of proteins is predicted using bioinformatic tools. This study represents detailed proteome profiles of head kidney and spleen of rainbow trout, with a total of 3241 and 2542 proteins identified, respectively. It is found that lymphoid organs are equipped with a variety of functional proteins related to defense, receptor, signal transduction, antioxidant, cytoskeleton, transport, binding, and metabolic processes. The identified proteome profiles will serve as a template for understanding lymphoid organ functions in salmonids and will increase the amount of spectra information of rainbow trout proteins in the public data repository PRIDE. This data can be accessed via ProteomeXchange with identifiers PXD008473 and PXD008478.

Construct and Predictive Validity of Three Measures of Intention to Quit Smoking: Findings From the International Tobacco Control (ITC) Netherlands Survey.

Introduction: The aim of the study was to compare the construct validity and the predictive validity of three instruments to measure intention to quit smoking: a Stages of Change measure, the Motivation To Stop Scale (MTSS), and a Likert scale. We used the Theory of Planned Behavior as theoretical framework. Methods: We used data from the International Tobacco Control Netherlands Survey. We included smokers who participated in three consecutive survey waves (n = 980). We measured attitude, subjective norm, and perceived behavioral control in 2012, intention to quit with three instruments in 2013, and having made a quit attempt in the last year in 2014. We conducted Structural Equation Modeling with three models for the instruments of intention separately and with one model that included the three instruments simultaneously. Results: All three instruments of intention were significantly and positively related to attitude and perceived behavioral control but none was related to subjective norm. All three instruments were significantly and positively related to making a quit attempt. The relation of the Likert scale with making a quit attempt (ß = 0.38) was somewhat stronger than that of the Stages of Change measure (ß = 0.35) and the MTSS (ß = 0.22). When entering the three instruments together into one model, only the Likert scale was significantly related to making a quit attempt. Conclusions: All three instruments showed reasonable construct validity and comparable predictive validity. Under the studied conditions, the Likert scale performed slightly better than the Stages of Change measure and the MTSS. Implications: An assessment of the Stages of Change, the Motivation To Stop Scale, and a Likert scale showed comparable predictive and construct validity as measures for intention to quit smoking. All three instruments can be used in future research; however, under the studied theoretical framework, that is, the Theory of Planned Behavior, the Likert scale performed slightly better than the other two instruments.

Impact of a negative emotional antitobacco mass media campaign on French smokers: a longitudinal study.

BACKGROUND: Mass media campaigns to encourage smoking cessation have been shown to be effective in a context of comprehensive tobacco control programme. The effectiveness of antismoking ads that evoke negative emotions remains unclear, in particular in countries with high smoking prevalence and among smokers with low perceived susceptibility, low self-efficacy or who are not users of smoking cessation services. OBJECTIVE: To evaluate short-term and long-term effects of a 1-month French national highly emotional media campaign, with a focus on these specific targets. DESIGN: A 6-month longitudinal survey by Internet. A sample of 3000 smokers were interviewed before the media campaign (T0). They were contacted again just after (T1) and 6 months after the campaign (T2). OUTCOMES: Perceived susceptibility to the risks of smoking, self-efficacy to quit smoking, use of smoking cessation services (quitline and website) and 7-day quitting. METHODS: The analysis was carried out on 2241 individuals who answered at T1 and T2. Multiple logistic regressions were computed to test the association between the change in each outcome at T1 and T2 and the level of exposure based on self-reported recall. RESULTS: Self-reported recall was associated with an increase in perceived susceptibility and with use of cessation services. Campaign recall was also associated with higher 7-day quitting immediately after the campaign (OR=1.8 (1.0 to 3.2), P<0.05). CONCLUSIONS: Fear-appeal mass media campaigns can be effective in encouraging cessation among smokers in a country with high smoking prevalence (France), but should be accompanied by convincing self-efficacy messages.

Support for a point-of-sale cigarette display ban among smokers: findings from the international tobacco control (ITC) Netherlands survey.

BACKGROUND: Displaying tobacco products at point-of-sale (PoS) has become an important marketing strategy for the tobacco industry. This study was designed to (1) examine how support for a PoS cigarette display ban changed among Dutch smokers between 2010 and 2015 and (2) identify the variables that predict support among smokers for a PoS cigarette display ban. METHODS: Longitudinal data from six annual survey waves (2010-2015) from the International Tobacco Control (ITC) Netherlands Survey were analyzed. The sample consisted of between 1279 and 1800 smokers per year. Smokers were asked whether they supported a complete ban on displays of cigarettes inside shops and stores. RESULTS: Support for a PoS cigarette display ban increased from 28.9% in 2010 to 42.5% in 2015 (OR = 1.40, p < 0.001). A multiple logistic regression analysis revealed that support for a PoS display ban of cigarettes was more likely among smokers who had more knowledge about the health risks of smoking (OR = 3.97, p < 0.001), believed smoking-related health risks to be severe (OR = 1.39, p < 0.001), had a more positive attitude towards quitting smoking (OR = 1.44, p = 0.006), reported stronger social norms to quit smoking (OR = 1.29, p = 0.035), had a higher self-efficacy for quitting smoking (OR = 1.31, p = 0.001), and had stronger intentions to quit smoking (OR = 1.23, p = 0.006). CONCLUSIONS: This paper showed that support for a PoS display ban of cigarettes increased among smokers in the Netherlands over the years. To further increase support, educational campaigns about the dangers of smoking, and campaigns that encourage quitting may be needed.

Comprehensive proteomic analysis of Penicillium verrucosum.

Mass spectrometric identification of proteins in species lacking validated sequence information is a major problem in veterinary science. In the present study, we used ochratoxin A producing Penicillium verrucosum to identify and quantitatively analyze proteins of an organism with yet no protein information available. The work presented here aimed to provide a comprehensive protein identification of P. verrucosum using shotgun proteomics. We were able to identify 3631 proteins in an "ab initio" translated database from DNA sequences of P. verrucosum. Additionally, a sequential window acquisition of all theoretical fragment-ion spectra analysis was done to find differentially regulated proteins at two different time points of the growth curve. We compared the proteins at the beginning (day 3) and at the end of the log phase (day 12).

Global proteomic profiling of Yersinia ruckeri strains.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) of salmonids. There is little information regarding the proteomics of Y. ruckeri. Herein, we perform whole protein identification and quantification of biotype 1 and biotype 2 strains of Y. ruckeri grown under standard culture conditions using a shotgun proteomic approach. Proteins were extracted, digested and peptides were separated by a nano liquid chromatography system and analyzed with a high-resolution hybrid triple quadrupole time of flight mass spectrometer coupled via a nano ESI interface. SWATH-MS technology and sophisticated statistical analyses were used to identify proteome differences among virulent and avirulent strains. GO annotation, subcellular localization, virulence proteins and antibiotic resistance ontology were predicted using bioinformatic tools. A total of 1395 proteins were identified in the whole cell of Y. ruckeri. These included proteases, chaperones, cell division proteins, outer membrane proteins, lipoproteins, receptors, ion binding proteins, transporters and catalytic proteins. In virulent strains, a total of 16 proteins were upregulated including anti-sigma regulatory factor, arginine deiminase, phosphate-binding protein PstS and superoxide dismutase Cu-Zu. Additionally, several virulence proteins were predicted such as Clp and Lon pro-teases, TolB, PPIases, PstS, PhoP and LuxR family transcriptional regulators. These putative virulence proteins might be used for development of novel targets for treatment of ERM in fish. Our study represents one of the first global proteomic reference profiles of Y. ruckeri and this data can be accessed via ProteomeXchange with identifier PXD005439. These proteomic profiles elucidate proteomic mechanisms, pathogenicity, host-interactions, antibiotic resistance ontology and localization of Y. ruckeri proteins.

Social Acceptance of Smoking Restrictions During 10 Years of Policy Implementation, Reversal, and Reenactment in the Netherlands: Findings From a National Population Survey.

INTRODUCTION: Little is known about the extent to which smoking restrictions are socially accepted in a country such as the Netherlands where smoking restrictions have been implemented and reversed several times. The current study assessed trends as well as factors associated with two indicators of social acceptance of smoking restrictions in the Netherlands: acceptance of smoking in public places and implementation of home smoking bans. METHODS: We used data from the Dutch Continuous Survey of Smoking Habits (DCSSH) between 2005 and 2014 (n = 182826). The DCSSH is a national population survey with a cross-sectional design in which respondents aged 15 years and older are surveyed weekly. RESULTS: Acceptance of smoking in public places decreased for six out of eight included venues, with the largest decrease for smoking in restaurants. The decrease in acceptance was larger among younger respondents and smokers. Smoking on terraces was an exception: decrease in acceptance there was larger among older respondents and ex-smokers. Implementation of home smoking bans increased over time. Having implemented a home smoking ban was associated with being male, being younger, having a high socioeconomic status, and being ex- or never smoker. CONCLUSIONS: Social acceptance of smoking restrictions has increased in the Netherlands, despite a suboptimal implementation process of smoking restrictions. However, there is still potential for improvement as acceptance of smoking is still quite high for some public venues like bars. It is important to strengthen smoking restrictions in order to further denormalize smoking in the Netherlands. IMPLICATIONS: We examined the extent to which smoking restrictions are socially accepted in the Netherlands where smoking restrictions have been implemented and reversed several times. Acceptance of smoking in public places decreased and implementation of home smoking bans increased between 2005 and 2014. Social acceptance of smoking restrictions increased in the Netherlands despite a suboptimal implementation process of smoking restrictions. However, acceptance of smoking in bars remains relatively high.

Growth promotion in pigs by oxytetracycline coincides with down regulation of serum inflammatory parameters and of hibernation-associated protein HP-27.

The growth promoting effect of supplementing animal feed with antibiotics like tetracycline has traditionally been attributed to their antibiotic character. However, more evidence has been accumulated on their direct anti-inflammatory effect during the last two decades. Here we used a pig model to explore the systemic molecular effect of feed supplementation with sub therapeutic levels of oxytetracycline (OTC) by analysis of serum proteome changes. Results showed that OTC promoted growth, coinciding with a significant down regulation of different serum proteins related to inflammation, oxidation and lipid metabolism, confirming the anti-inflammatory mechanism of OTC. Interestingly, apart from the classic acute phase reactants also down regulation was seen of a hibernation associated plasma protein (HP-27), which is to our knowledge the first description in pigs. Although the exact function in non-hibernators is unclear, down regulation of HP-27 could be consistent with increased appetite, which is possibly linked to the anti-inflammatory action of OTC. Given that pigs are good models for human medicine due to their genetic and physiologic resemblance, the present results might also be used for rational intervention in human diseases in which inflammation plays an important role such as obesity, type 2 diabetes and cardiovascular diseases.

Shotgun proteomic analysis of Yersinia ruckeri strains under normal and iron-limited conditions.

Yersinia ruckeri is the causative agent of enteric redmouth disease of fish that causes significant economic losses, particularly in salmonids. Bacterial pathogens differentially express proteins in the host during the infection process, and under certain environmental conditions. Iron is an essential nutrient for many cellular processes and is involved in host sensing and virulence regulation in many bacteria. Little is known about proteomics expression of Y. ruckeri in response to iron-limited conditions. Here, we present whole cell protein identification and quantification for two motile and two non-motile strains of Y. ruckeri cultured in vitro under iron-sufficient and iron-limited conditions, using a shotgun proteomic approach. Label-free, gel-free quantification was performed using a nanoLC-ESI and high resolution mass spectrometry. SWATH technology was used to distinguish between different strains and their responses to iron limitation. Sixty-one differentially expressed proteins were identified in four Y. ruckeri strains. These proteins were involved in processes including iron ion capture and transport, and enzymatic metabolism. The proteins were confirmed to be differentially expressed at the transcriptional level using quantitative real time PCR. Our study provides the first detailed proteome analysis of Y. ruckeri strains, which contributes to our understanding of virulence mechanisms of Y. ruckeri, and informs development of novel control methods for enteric redmouth disease.

Attrition analysed in five waves of a longitudinal yearly survey of smokers: findings from the ITC Netherlands survey.

BACKGROUND: Attrition bias can affect the external validity of findings. This article analyses attrition bias and assesses the effectiveness of replenishment samples on demographic and smoking-related characteristics for the International Tobacco Control Netherlands Survey, a longitudinal survey among smokers. METHODS: Attrition analyses were conducted for the first five survey waves (2008-12). We assessed, including and excluding replenishment samples, whether the demographic composition of the samples changed between the first and fifth waves. Replenishment samples were tailored to ensure the sample remained representative of the smoking population. We also constructed a multivariable survival model of attrition that included all five waves with replenishment samples. RESULTS: Of the original 1820 respondents recruited in 2008, 46% participated again in 2012. Demographic differences between waves due to attrition were generally small and replenishment samples tended to minimize them further. The multivariable survival analysis revealed that only two of the 10 variables analysed were significant predictors of attrition: a weak effect for gender (men dropped out more often) and weak to moderate effects for age (respondents aged 15-24 years dropped out more than aged 25-39 years, who dropped out more than those aged 40+ years). CONCLUSIONS: Weak to moderate attrition effects were found for men and younger age groups. This information could be used to minimize respondent attrition. Our findings suggest that sampling weights and tailored replenishment samples can effectively compensate for attrition effects. This is already being done for the International Tobacco Control Netherlands Survey, including the categories that significantly predicted attrition in this study.

Concentration and pattern changes of porcine serum apolipoprotein A-I in four different infectious diseases.

Apolipoprotein A-I (Apo A-I) is a major protein in lipid/lipoprotein metabolism and decreased serum levels have been observed in many species in response to inflammatory and infectious challenges. Little is known about the porcine homologue, therefore in this work we have characterized it through biochemical and proteomic techniques. In 2DE, porcine serum Apo A-I is found as three spots, the two more acidic ones corresponding to the mature protein, the more basic spot to the protein precursor. Despite high sequence coverage in LC-MS/MS, we did not find a sequence or PTM difference between the two mature protein species. Besides this biochemical characterization, we measured overall levels and relative species abundance of serum Apo A-I in four different viral and bacterial porcine infectious diseases. Lower overall amounts of Apo A-I were observed in Salmonella typhimurium and Escherichia coli infections. In the 2DE protein pattern, an increase of the protein precursor together with a lower level of mature protein species were detected in the porcine circovirus type 2-systemic disease and S. typhimurium infection. These results reveal that both the porcine serum Apo A-I concentration and the species pattern are influenced by the nature of the infectious disease.

Contamination of therapeutic human immunoglobulin preparations with apolipoprotein H (ß2-glycoprotein I).

Polyclonal immunoglobulin (Ig) concentrates are important biological medicinal products and the assurance of their quality and safety is crucial. In our present approach we used proteomic methods to check the purity of commercial Ig products of different origin. The experimental setup included nonreducing 2DE or DIGE combined with MALDI-TOF and the thrombin generation assay, a routine safety test for pharmaceutical Ig preparations, and was complemented by a specific immunoassay. 2DE patterns displayed contaminations with trace amounts of human apolipoprotein H (Apo-H), transferrin, albumin, and its fragments. In contrast to the latter, Apo-H is a protein that is active in the coagulation cascade, and thus a potential involvement in thromboembolic events in vivo cannot be excluded. It was found by 2DE and MALDI-TOF to be a contaminant of several Ig preparations. Spiking experiments of Ig preparations with pure Apo-H demonstrated an Apo-H concentration dependent increase in thrombin generation assay values. Traces of Apo-H are possibly also contributing to unwanted side effects, as already known for factor XIa. The significance of Apo-H contaminations for these side effects might be verified by detailed analyses of pharmacovigilance data.