Effect of the use of OTC as feed additive in the pig serum proteome. 5. Management Committee Meeting and 4 Meeting of Working Groups 1,2 & 3 of COST Action FA 1002

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.

Background In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo. Methods Thrombin (0.0004-0.42IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n=5 animals; 15 experiments). In the dose verification study animals received 0.42IU/kg/min of thrombin vs. saline solution in a cross-over design (n=3 animals; 7 experiments). Results Thrombin at an infusion rate of 0.42IU/kg/min decreased fibrinogen levels by 75% (p<0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p=0.006), prolonged thrombin time by 70% (p=0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p<0.05) and decreased the activity of the coagulation factors VIII and XIII (p<0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p=0.019) and induced a 65% decrease in maximal clot firmness (p<0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism. Conclusion Protracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species.

Tyrosine kinase 2 (Tyk2) is an integral part of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway which relays intracellular signals of various cytokines. Tyk2 crucially contributes to host defense mechanisms against microbial pathogens and to tumor surveillance but also facilitates immune pathologies. Here we investigated the impact of Tyk2 on the macrophage proteome using the synthetic double-stranded RNA analog polyinosinic acid-polycytidylic acid (poly(I:C)) as a mimicry of viral infections. By means of 2D-DIGE in connection with PMF obtained by MALDI-MS and sequence tag determination by MS/MS we unambiguously identified eighteen protein spots corresponding to sixteen distinct proteins that are regulated by poly(I:C) and differentially expressed between wildtype (WT) and Tyk2-deficient macrophages. The majority of these proteins are functionally assigned to cellular immune responses and to metabolism. We show for selected metabolic enzymes, i.e. triosephosphate isomerase (TIM), ATP-citrate synthase (ACLY) and long-chain-fatty-acid-CoA ligase 4 (ACSL4), that Tyk2 affects protein expression transcriptionally and post-transcriptionally. We furthermore confirm the involvement of Tyk2 in the regulation of lipid and carbohydrate metabolism at the level of metabolites. Taken together, our results provide new evidence for important functions of Tyk2 at the molecular interface between innate immunity and cellular metabolism.

2-D DIGE was used to investigate 'fingerprint proteins' in biological medicines. A presumably non-originator human albumin was analysed, and the 2-D DIGE patterns of the non-genuine and the authentic product were compared. The products could be clearly distinguished based on the pattern of minor components, which represent plasma proteins and degradation products remaining in the final products after fractionation and purification. The approach demonstrated that 2-D DIGE is an excellent tool for the analysis of biologicals of different sources and for ensuring the identity and quality of blood products.

IL-1beta is an important proinflammatory cytokine with a major role in several inflammatory diseases. Expression of IL-1beta is tightly regulated at the level of transcription, mRNA stability, and proteolytic processing. In this study, we report that IL-1beta expression in response to LPS is also regulated at the translational level. LPS-induced IL-1beta protein levels in macrophages derived from murine bone marrow are markedly increased in the absence of tyrosine kinase 2 (Tyk2). Increased IL-1beta is found intra- and extracellularly, irrespective of the efficiency of IL-1beta processing. We show that the absence of Tyk2 results both in higher translational rates and in enhanced association of IL-1beta mRNA with polysomes. Induction and stability of IL-1beta mRNA are not affected by the lack of Tyk2. We show further that the Tyk2-dependent translational inhibition is mediated by autocrine/paracrine type I IFN signaling and requires signal transducer and activator of transcription 1. Enhanced IL-1beta production in Tyk2- and IFN receptor 1-deficient macrophages is also observed following Listeria monocytogenes infection. Taken together, the data describe a novel mechanism for the control of IL-1beta synthesis.

Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus . The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus . The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture.

We characterized the two-dimensional electrophoretic patterns of fibrinogen chains α, β, and γ from the plasma of six animal species – Bos taurus, Canis familiaris, Equus caballus, Felis catus, Gallus domesticus and Sus scrofa. Comparing the spots resolved from serum and plasma samples, or exploiting the cross-reactivity of animal fibrinogen with an antiserum raised against the human protein could detect only some of the fibrinogen chains. Conversely, the analysis of the precipitate obtained by heating plasma for some minutes at 56°C was adequate for the recognition of all fibrinogen chains in all samples. Physicochemical properties of the homologous proteins were found to extensively vary across species, with complete separation among the mapping areas for α, β and γ chains and maximal heterogeneity among β chains.

A reference protein map for pig serum was set up using two-dimensional electrophoresis (2-DE). Thirty-nine protein chains or spots deriving from 26 different proteins were identified by immunological and mass spectrometric methods. Thus, the positions of most medium to higher abundance serum proteins could be determined on the 2-DE gels. The plasma protein fibrinogen was also included in our study. The overall pig protein pattern differs in some respect to serum/plasma maps of other mammalian species, e.g. in levels and properties of single proteins such as haptoglobin or IgM or in species-specific proteins like pig major acute phase protein. Serum protein maps are a useful tool to get an overview on expressed proteins, and to monitor changes in concentration as well as isotype distribution of the identified proteins. As a consequence, more detailed knowledge on protein pattern changes may give deeper insights into the metabolic development of some pathologic conditions and may lead to putative biomarkers for further investigation. Selected examples for protein pattern changes in pigs infected by a viral (porcine circovirus type 2) and a bacterial (Actinobacillus pleuropneumoniae) pathogen illustrate the usefulness of the method.

Tyrosine kinase 2 (Tyk2) belongs to the Janus kinase (Jak) family and is involved in signalling via a number of cytokines. Tyk2-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxin shock. Macrophages are key players in the pathogenesis of endotoxin shock and, accordingly, defects in the LPS responses of Tyk2(-/-) macrophages have been reported. In the present study, the molecular role of Tyk2 is investigated in more detail using a proteomics approach. 2-D DIGE was applied to compare protein patterns from wild-type and Tyk2(-/-) macrophages and revealed significant differences in protein expression patterns between the genotypes before and after LPS treatment. Twenty-one proteins deriving from 25 differentially expressed spots were identified by MALDI/ESI MS. Among them, we show for N-myc interactor that its mRNA transcription/stability is positively influenced by Tyk2. In contrast, LPS-induced expression of plasminogen activator 2 protein but not mRNA is strongly enhanced in the absence of Tyk2. Our data furthermore suggest an influence of Tyk2 on the subcellular distribution of elongation factor 2 and on LPS-mediated changes in the peroxiredoxin 1 spot pattern. Thus, our results imply regulatory roles of Tyk2 at multiple levels and establish novel connections between Tyk2 and several cellular proteins.

Mitochondria are involved in the development of organ failure in critical care diseases. However, the mechanisms underlying mitochondrial dysfunction are not clear yet. Inducible hemoxygenase (HO-1), a member of the heat shock protein family, is upregulated in critical care diseases and considered to confer cytoprotection against oxidative stress. However, one of the products of HO-1 is Fe2+ which multiplies the damaging potential of reactive oxygen species catalyzing Fenton reaction. The aim of this study was to clarify the relevance of free iron metabolism to the oxidative damage of the liver in endotoxic shock and its impact on mitochondrial function. Endotoxic shock in rats was induced by injection of lipopolysaccharide (LPS) at a dose of 8 mg/kg (i.v.). We observed that the pro-inflammatory cytokine TNF-α and the liver necrosis marker aspartate aminotransferase were increased in blood, confirming inflammatory response to LPS and damage to liver tissue, respectively. The levels of free iron in the liver were significantly increased at 4 and 8 h after onset of endotoxic shock, which did not coincide with the decrease of transferrin iron levels in the blood, but rather with expression of the inducible form of heme oxygenase (HO-1). The proteins important for sequestering free iron (ferritin) and the export of iron out of the cells (ferroportin) were downregulated facilitating the accumulation of free iron in cells. The temporarily increased concentration of free iron in the liver correlated with the temporary impairment of both mitochondrial function and tissue ATP levels. Addition of exogenous iron ions to mitochondria isolated from control animals resulted in an impairment of mitochondrial respiration similar to that observed in endotoxic shock in vivo. Our data suggest that free iron released by HO-1 causes mitochondrial dysfunction in pathological situations accompanied by endotoxic shock.

Spontaneously hypertensive stroke-prone rats (SHRSP) feature an established model for human cerebrovascular disease. SHRSP, kept on a high-salt permissive diet (JPD), develop hypertension, renal and brain damage. In this report we compared the behavior of female and male SHRSP regarding the main aspects of their pathological condition. Brain abnormalities, detected by magnetic resonance imaging, developed spontaneously in males after 42+/-3 days, in females after 114+/-14 days from the start of JPD. Survival was >3-fold longer for females than for males. The development of brain damage was preceded, in both genders, by an inflammatory condition characterized by the accumulation in serum and urine of acute-phase proteins. The increase in thiostatin level was significantly lower and delayed in female in comparison to male SHRSP. During JPD female and male SHRSP developed massive proteinuria, its worsening being significantly slower in females. The alterations of vasculature-bound barriers in kidney and brain were connected with endothelial dysfunction and relative deficiency in nitric oxide (NO). In thoracic aortic rings, basal release of NO was significantly higher in female than in male SHRSP, both if receiving and if not receiving JPD. The gender differences in SHRSP thus appear to be connected to a more efficient control in females of inflammation and of endothelial dysfunction.

Low levels of fetal calf serum (FCS), used as protein supplement in cell culture medium, were traced in preparations of primary murine macrophages (bone-marrow-derived macrophages (BMM) and peritoneal macrophages (PM)). Main components of this common additive were mapped in 2-DE by means of differential image gel electrophoresis and immunoblotting. Additional washing steps in cell preparation helped to decrease the levels of the four highest abundance foetal serum proteins (serum albumin (SA), alpha1-fetoprotein (AFP), alpha1-antitrypsin (alpha1AT) and transferrin (Tf)) to less than 1% of total protein. Macrophage spot pattern was recorded in parallel and showed little variation. Results presented are supposed to be of general interest for cell preparations with similar background.

In this study, we determined functional integrity and reactive oxygen species generation in mitochondria and endoplasmic reticulum in liver of rats subjected to endotoxic shock to clarify whether intracellular reactive oxygen species (ROS) destabilize cellular integrity causing necrosis in rats challenged with lipopolysaccharide (LPS). LPS caused drastically increased plasma levels of alanine aminotransferase, suggesting damage to plasma membranes of liver cells. Liver necrosis was confirmed by histological examination. LPS induced a significant increase in ROS production in rat liver mitochondria (RLM), but did not impair mitochondrial function. In contrast to mitochondria, enzymatic activity and ROS production of cytochrome P450 were lower in microsomal fraction obtained from LPS-treated animals, suggesting the dysfunction of endoplasmic reticulum. Protein patterns obtained from RLM by two-dimensional electrophoresis showed significant upregulation of mitochondrial superoxide dismutase by LPS. We hypothesize that upregulation of this enzyme protects mitochondria against mitochondrial ROS, but does not protect other cellular compartments such as endoplasmic reticulum and plasma membrane causing necrosis.