Heparin and EDTA as anticoagulant differentially affect cytokine mRNA level of cultured porcine blood cells.

Cytokine mRNA expression profiles serve to characterize immune cell activation in different test systems. Both, diluted whole blood and isolated PBMC are widely applied for these studies. Comprehensive data regarding the suitability of different anticoagulants for profiling cytokine expression are not available for the pig. Therefore the aim of this study was to compare the effect of two commonly used anticoagulants (heparin and EDTA) on the cytokine expression pattern of porcine blood cells. IL-1alpha, IL-2, IL-4, IL-6, IL-10 and IFN-gamma mRNA levels were detected ex-vivo and upon in-vitro stimulation in diluted porcine whole blood and isolated PBMC by real-time PCR. The cells were stimulated with ConA or LPS, known to act on different target cells and implying different signalling pathways. Additionally the integrity of the isolated RNA was investigated. Ex-vivo cytokine expression pattern of fresh whole blood were not affected by the investigated anticoagulants. In contrast, stimulation of cultured diluted whole blood or PBMC resulted in significant differences depending on the applied anticoagulant. Using EDTA we found a significantly decreased capacity of whole blood to express cytokines. However, isolated PBMC from EDTA anticoagulated blood showed a higher cytokine expression capacity than PBMC from heparinized blood. Comparing diluted whole blood and PBMC we found that cultured porcine whole blood responded better to bacterial products than isolated PBMC, probably because sufficient auxiliary plasma derived factors such as LPS-binding protein, are present. However, isolated PBMC showed a higher T-cell response than diluted whole blood. In conclusion, our findings underline that each application demands a specific assay system.

Clenbuterol affects the expression of messenger RNA for interleukin 10 in peripheral leukocytes from horses challenged intrabronchially with lipopolysaccharides.

On four occasions, four horses with heaves and four horses with small airway inflammatory diseases inhaled 0.9% saline based aerosol mixtures with or without lipopolysaccharides (LPS). Prior to the first saline and LPS inhalation, horses were untreated, while three and a half days prior to the third and forth inhalation horses had received 0.8 microg/kg clenbuterol intravenously twice daily. The messenger RNA (mRNA) expression of tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-4, IL-6, IL-8, IL-10 and interferon- gamma (IFN- gamma) was investigated by RT-PCR, all of which were expressed in the white blood cells of samples collected. Inhalation of LPS only changed the cytokine expression profile of IL-10, IL-4 and TNF-alpha mRNA which were higher after challenge with LPS. However in those horses that were treated with clenbuterol the LPS-induced IL-10 mRNA expression was shown to be suppressed. Further changes in IL-4 and TNF-alpha were not significant. Thus the results of this study indicated that clenbuterol can modulate the expression of IL-10 mRNA in peripheral white blood cells in those horses with small airway diseases that have been exposed to LPS.

Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines.

We have established an easy real-time PCR assay, which allows the precise quantification of changes in the expression level of 6 relevant porcine cytokines, and 3 housekeeping genes. This assay simultaneously detects 9 sequences by measuring 3 x 3 targets in a triplex-format. The mRNA of the lymphokines IL-2, IL-4, IL-10, and IFN-gamma, of the proinflammatory cytokines IL-1alpha and IL-6, and of the housekeeping genes are quantified using TaqMan-probes by means of standard dilution series on the iCycler iQ. The standard consists of equal aliquots of the experimental cDNAs under investigation. Simultaneously the most suitable combination of 3 out of the four housekeeping genes beta-actin, HPRT, GAPDH, and cyclophilin can be selected, and their averaged expression values constitute a normalisation factor. The raw data of all targets of interest is then calculated relative to this normalisation factor, making eventual changes of the relative expression level of the single housekeeping genes controllable and quantifiable. We have applied this assay to quantify changes in the cytokine mRNA levels of porcine stimulated with various concentrations of LPS and ConA, known to induce different cytokine expression patterns. We have shown, that even small differences in the expression level (less than 2-fold) can be precisely quantified, and reveal statistically significant changes, when using the normalisation factor. This assay will be useful for studying changes in the expression of relevant porcine cytokines and will help to further improve the investigation of immune responses in the pig.

Variations in the concentration of zinc in the blood of Icelandic horses.

The effect of factors including the horses' farm environment, their sex and age and whether they suffered from summer seasonal recurrent dermatitis (sweet itch) on the concentrations of zinc in the plasma, whole blood and blood cells of 104 Icelandic horses was investigated. Its concentration in plasma varied significantly between farms (P<0.01), but its concentration in blood and blood cells was not influenced by any of the variables. The concentration of zinc in the blood cells was 10.5 times greater than in plasma, but its concentration in plasma was not correlated with its concentration in whole blood or blood cells owing to the variability in the proportion of whole blood zinc present in plasma (relative plasma zinc), which ranged between 9 and 24 per cent. This variability was significantly influenced by a three-way interaction between farm, sex and sweet itch (P<0.05). Relative plasma zinc was positively correlated with absolute plasma zinc (r=0.78, P<0.001) and negatively correlated with whole blood and blood cellular zinc (r=-0.58, r=-0.71, P<0.001).

Effect of clenbuterol on the clearance of particles of charcoal (4 to 90 microm) from the uteri of mares.

The failure of clearance mechanisms in the mare's uterus results in persistent inflammation and is considered a major cause of subfertility. Eighteen mares, of which three were susceptible to endometritis and four had been ovariectomised, underwent charcoal clearance testing to evaluate their clearance mechanisms. This consisted of installing 500 mg of charcoal (particle size 4 to 90 microm) added to 50 ml of phosphate-buffered saline (PBS) into the uterus. Forty-eight hours later the uterus was flushed out with 0.0012 per cent methylene blue in 50 ml of PBS for determination of the diluting factor by photospectrometry. Flush volume, pH, specific gravity and pellet size were all analysed. To investigate the effect of a beta2-adrenergic agonist on the ability of genitally healthy oestrous mares to eliminate a suspension of charcoal from the uterus, four genitally healthy mares with appropriate charcoal clearance were also subjected to clearance testing following intravenous administration of 0.8 microg/kg of clenbuterol every 12 hours and 1 microg/kg of clenbuterol every eight hours. All parameters were within their normal range following clenbuterol treatment. However, minimal but significant differences in pre- and post-treatment values regarding fluid volume and extinction rate were recorded.

Acute-phase proteins before cerebral ischemia in stroke-prone rats: identification by proteomics.

BACKGROUND AND PURPOSE: A high degree of proteinuria has been reported in stroke-prone spontaneously hypertensive rats (SHRSP). We studied the effect of salt loading on the detailed protein pattern of serum and urine in 3 rat strains: Wistar-Kyoto, spontaneously hypertensive rats, and SHRSP, an inbred animal model for a complex form of cerebrovascular disorder resembling the human disease. METHODS: Rats were given a permissive diet and received 1% NaCl in drinking water. The protein pattern in body fluids was assessed over time by 2-dimensional electrophoretic analysis. Brain alterations were monitored by MRI and histology. RESULTS: Several proteins were excreted in urine after weeks of treatment and in advance of stroke: transferrin, hemopexin, albumin, alpha(2)-HS-glycoprotein, kallikrein-binding protein, alpha(1)-antitrypsin, Gc-globulin, and transthyretin. Markers of an inflammatory response, including very high levels of thiostatin, were detected in the serum of SHRSP at least 4 weeks before a stroke occurred. CONCLUSIONS: In SHRSP subjected to salt loading, an atypical inflammatory condition and widespread alterations of vascular permeability developed before the appearance of anomalous features in the brain detected by MRI. Urinary concentrations of each of the excreted serum proteins correlated positively with time before stroke occurred.

Delay in processing porcine whole blood affects cytokine expression.

Quantification of cytokine messenger ribonucleic acid (mRNA) in blood samples has become an important tool in the investigation of immune cell activation in a variety of clinical settings. It has been shown that the method of sample collection and processing influences the levels of several cytokine mRNAs. Therefore, it is generally accepted that blood samples for analysis of cytokine expression be processed as soon as possible and under standardised conditions. Since immediate sample processing is not always possible, we investigated the effect of different storage conditions (room temperature (Rt) and 4 degrees C) and storage times (1, 2, 4, 6 and 24 h) on the mRNA level of different cytokines (IL-1alpha, IL-2, IL-6, IL-8, IL-10, IFN-gamma), as well as the IL-2 receptor (IL-2R) in porcine whole blood samples (n=8). Quantification of cytokine expression was performed using simultaneous reverse transcription PCR (RT-PCR) combined with the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference. Our data demonstrate that delays in sample processing longer than 1 h result in significant changes of the mRNA levels of individual cytokines. Expression of the monokines IL-1alpha, IL-6 and IL-10 were increased by storage at both room temperature and 4 degrees C. Expression of IL-8 was increased only in the samples stored at room temperature, and expression of IFN-gamma was raised exclusively in the samples stored at 4 degrees C. We conclude that porcine blood samples should be processed within 2 h to prevent undesired stimulatory effects on the cytokine expression pattern. However, if only selected cytokines are investigated, the undesired effects of prolonged storage can be selectively suppressed by choosing the appropriate temperature of sample storage.

Organ specific formation of nitrosyl complexes under intestinal ischemia/reperfusion in rats involves NOS-independent mechanism(s).

Intestinal ischemia/reperfusion may lead to local and distant organ damage involving nitric oxide (NO). NO rapidly reacts with heme/non-heme-iron-yielding nitrosyl complexes, which can be determined directly by electron paramagnetic resonance spectroscopy. The aim of the present study was to characterize nitrosylation reactions induced by transient intestinal ischemia in blood and tissues. We used electron paramagnetic resonance spectroscopy and reverse transcription polymerase chain reaction analyses to estimate nitrosyl complex levels and inducible NO synthase mRNA expression in rats subjected to superior mesenteric artery occlusion for 60 min followed by the reperfusion. Nitrosyl hemoglobin concentrations in circulating blood were significantly increased during ischemia and reperfusion. Nitrosyl hemoglobin complexes were detected in ischemic intestine, but not in normoxic lung and liver or reperfused intestine. Administration of N-G-monomethyl-L-arginine, a non-specific NO synthase inhibitor, did not affect the formation of circulating nitrosyl complexes. Moreover, inducible NO synthase mRNA was not found in intestinal tissues at 30 min of reperfusion. Our data suggest an organ-specific NO formation indicated by the increased nitrosylation reaction in ischemic intestinal tissue, but not in the distant normoxic organs, in spite of high circulating nitrosyl hemoglobin levels. NO involved in nitrosylation under intestinal ischemia/reperfusion is probably formed by NO synthase-independent mechanism(s).

Optimization of culture conditions for feline x murine heterohybridomas.

Feline splenocytes were fused to the murine myeloma lines NSO or Ag8. Autologous serum and taurine were used as media supplements for the cat x mouse heterohybridomas. The best results were obtained by the use of NSO as fusion line with taurine-supported media.

Zinc and copper plasma levels in Icelandic horses with Culicoides hypersensitivity.

Zinc concentration has been shown to have a potent immunomodulatory capacity, particularly influencing T helper cell organisation and cytokine secretion. Culicoides hypersensitivity (CHS) in horses resembles the early and late phase of type I hypersensitive reactions in man, characterised by a shift from T helper cell subtype 1 to T helper cell subtype 2 cytokine profile. In this pilot study, zinc and copper levels were measured in the plasma of 48 CHS-affected and 56 healthy Icelandic horses age 4-25 years (mean approximately 11 years) kept on 7 farms. Affected horses were divided into 3 groups according to the severity of disease. Time of blood collection and feeding management was constant. No differences in zinc or copper plasma levels and plasma copper/zinc ratio were determined among CHS horses and controls by univariate analysis of variance. Therefore, the most significant influences on zinc and copper plasma levels were affected by the location of housing. However, Spearman correlation showed a negative coefficient between the plasma zinc concentration and the severity of CHS (r = -0.31). Due to a probability value of P = 0.002 the null hypothesis r = 0 is rejected, although only 9% of the total variation of plasma zinc is presently explained by its relationship to CHS. In contrast, the Spearman correlation coefficient between plasma copper levels and severity of CHS was not significant (r = -0.14; P = 0.16). The minor deviations in plasma zinc concentrations in association with the severity of CHS may be real or due to neurohumoral or cytokine-mediated mechanisms, but appear too minimal to be relevant.

Chylopericardium: a rare cause of pericardial effusion.

A 21 year old man presented with asymptomatic, isolated chylopericardium. Despite echocardiography, radionuclide-angiography, computer tomography, and chemical analysis of the chylous effusion, the etiology remained obscure. After patent blue dye infusion into peripheral soft tissues, the appearance of coloring material in the effusion at 4 hours suggested direct communication of the pericardium with an apparently large thoracic duct. Fifteen months later, cardiomegaly persists in site of medium-chain triglyceride dietary restriction.

[Heavy metal and trace element concentrations in patients with idiopathic dilated cardiomyopathy]. / Schwermetall- und Spurenelementkonzentrationen bei Patienten mit idiopathischer dilatativer Kardiomyopathie.

Blood, serum and urine concentrations (24 hour samples) of cadmium, lead, magnesium, calcium, zinc, copper and iron were determined by means of atomic absorption spectrophotometry in 54 patients with dilated cardiomyopathy (CMP). These data were compared with the values in 17 healthy controls. CMP patients showed higher blood cadmium concentrations (+173%, p less than 0.001), higher serum calcium concentrations (+4%, p less than 0.01) and lower serum magnesium levels (-11%, p less than 0.01). In urine samples CMP patients had higher cadmium levels and lower concentrations of calcium (-43%, p less than 0.05) and magnesium (-36%, p less than 0.05). Correlations with clinical data revealed no definite reason for the abnormal laboratory findings in CMP patients. In individual cases, however, the high cadmium concentrations could be of aetiological importance.

[Plasma 25-hydroxycholecalciferol after daily vitamin D administration in comparison with massive single-dose prophylaxis (author's transl)]. / Plasmaspiegel des 25-Hydroxycholecalciferols nach Tages- und "Stossprophylaxe" mit Vitamin D.

The effect of daily administration of 1200 IU vitamin D3 was compared with the response to a single oral dose of 200,000 IU in 21 young infants by determination of the plasma 25-OHCC levels. After about one week the mean value recorded in group 1 was 27 +/- 13 ng/ml; the corresponding value in group 2 was 127 +/- 78.4 ng/ml. At the second control examination about one month subsequently, the mean values were 61 +/- 26.2 ng/ml and 104 +/- 69.0 ng/ml respectively. The wide range of values found in the single-dose group clearly demonstrates the advantage of daily vitamin D administration for routine prophylaxis against rickets in infancy.

[Serum levels of 25-hydroxycholecalciferol (25-OH-D3) in children of different age groups (author's transl)]. / Serumspiegel des 25-Hydroxycholecalciferols (25-OH-D3) bei Kindern verschiedener Altersstufen.

Serum 25-OH-D3 levels were determined in 78 children aged 6 weeks to 15 years, in March 1977 and March 1978 in Vienna. The total number of probands was divided into age groups, each comprising a range of 3 years. The average value with 1 SD. found over the whole age range was 48.3 +/- 25.75 nmol/l (i.e. 19.4 +/- 10.34 ng/ml). Somewhat higher levels were found in the age groups 3 to 6 and 6 to 9 years, with a slight decrease in the later age groups, but no significant differences were detected. The obtained values seem to represent the "normal" 25-OH-D3 serum levels of our population in the above-mentioned age range.

Endocrine disrupters in the aquatic environment: the Austrian approach--ARCEM.

A consortium of Austrian scientists (ARCEM) carried out a multidisciplinary environmental study on Austrian surface and ground waters including chemical monitoring, bioindication, risk assessment and risk management for selected endocrine disrupters: 17beta-estradiol, estriol, estrone, 17alpha-ethinylestradiol, 4-nonylphenol, 4-nonylphenol ethoxylates (4-NP1EO, 4-NP2EO) and their degradation products, ocytlphenol, ocytlphenol ethoxylates (OP1EO, OP2EO) as well as bisphenol A. To obtain data representative for Austria, a material flow analysis served to select relevant compounds and water samples were collected monthly over one year at those sites routinely used in Austrian water quality control. The following results were obtained and conclusions drawn: 1. Chemical monitoring: As compared to other countries, relatively low levels of pollution with endocrine disrupters were detected. 2. Bioindication: In the surface waters under study, male fish showed significant signs of feminization and demasculinization (increased production of the egg-yolk protein and histological changes of the gonads. 3. Risk assessment: For humans, exposure via either drinking water abstraction (ground water) or fish consumption was considered. The exposure levels of the compounds under study were below those considered to result in human health risks. Likewise, for bisphenol A and octylphenols, there was no indication for risk posed upon the aquatic environment (fish). However, nonylphenol or 17alpha-ethinylestradiol exposure along with results of bioindication (2) suggest a borderline estrogenic activity in a considerable number of surface waters. Consequently the emissions of these substances into the surface waters affected have to be reduced. 4. Risk management: Waste water treatment experiments revealed a positive correlation between the removal rate of endocrine disrupters from the waste water and the sludge retention time in the treatment plants. These substances are removed to a higher extend at low loaded plants designed for nutrient removal than at plants that remove carbon and/or employ nitrification only. As to drinking water treatment, chlorine dioxide and ozone were found to eliminate all investigated substances, except nonylphenol ethoxylates. (For the complete study see: www.arcem.at)

[Electrophoretic studies of serum protein fractions in horses with laminitis]. / Elektrophoretische Untersuchungen der Serumproteinfraktionen hufrehekranker Pferde.

The spectrum of serum proteins was evaluated in 46 horses affected with spontaneous laminitis and correlations between the severity of the disease and changes of the protein pattern were analyzed. The investigation was made in two groups; group A consisted of 21 horses of various breeds (warmblood, thoroughbred, standardbred) and group B of 25 ponys. Each group was subdivided according to the severity of the disease, using the OBEL-grade (OG) classification system. Serum proteins were separated by different one- and two-dimensional electrophoretic methods. Sera analysed by cellulose acetate electrophoresis showed a significant difference in the alpha 1-globulin fraction between OG II and OG IV affected horses. An increasing severity of the disease was correlated with a decrease of the alpha 1-globulins. The other protein fractions didn't show a uniform tendency. In group B there was a significant difference in the alpha 1-globulin fractions of OG II and OG III and in the beta 2-globulin fractions of OG I and OG II affected ponys. The acute phase proteins C3c, C4, Hp and fibronectin could be determined in a preliminary study in horse serum using the cross-reactivity of antibodies against the homologous human proteins.

Detection of potential markers for systemic disease in saliva of pigs by proteomics: a pilot study.

Animals with different health status have been studied in order to extend the knowledge about protein composition of porcine saliva samples and to discover potential salivary markers for systemic disease in porcine production. Clinical examination of animals was performed at farm level where 10 healthy pigs and 10 animals with evident clinical signs of disease were randomly selected. Saliva and blood samples were obtained and afterwards animals were humanely sacrificed to perform a complete necropsy. Levels of two acute phase proteins, haptoglobin and C-reactive protein, were used to identify possible active infections of the animals. Moreover, serological analysis, to the main porcine infectious diseases in the area, was performed. Salivary proteins were separated by two-dimensional gel electrophoresis followed by mass spectrometry for the identification of specific proteins. A total of 58 spots out of 75 were successfully identified by MS, which correspond to 20 unique proteins. Two different approaches were used to perform a statistical comparison of saliva protein patterns from healthy and diseased animals using the relative spot volume (% spot volume/total volume of all spot in the gel, approach "A") or taking also into account the total protein content of each saliva sample (µg of spot/mL of saliva, approach "B"). Both analyses showed three proteins in common that are differentially regulated between states. However, approach B was selected for biomarker searching since it gave an estimation of protein concentration and showed differential expression of proteins between both health states in a total of 10 proteins, which were up-regulated in disease. Mass spectrometric analysis identified those proteins as salivary lipocalin, lipocalin 1, double headed protease inhibitor protein, adenosine deaminase, haptoglobin, albumin fragments, S100-A8, S100-A9, S100-A12 and pancreatic alpha amylase. These proteins could be considered as potential salivary markers of disease.

Proteomic analysis of porcine saliva.

Saliva contains a number of proteins that may be useful as biomarkers of health and disease and can be easily obtained from large numbers of animals in a non-invasive, stress-free way. The objective of this study was to explore the protein composition of porcine saliva from 10 specific pathogen free pigs using first one-dimensional SDS-PAGE and then two-dimensional electrophoresis and mass spectrometry. A reference proteome pattern for porcine saliva was established with the identification of 13 different, mainly saliva-specific, proteins. These reference data will facilitate the investigation of salivary proteins potentially altered in disease and could serve as novel diagnostic biomarkers.

Redox options in two-dimensional electrophoresis.

Two-dimensional electrophoresis is usually run on fully reduced samples. Under these conditions even covalently bound oligomers are dissociated and individual polypeptide chains may be fully unfolded by both, urea and SDS, which maximizes the number of resolved components and allows their pI and M(r) to be most accurately evaluated. However, various electrophoretic protocols for protein structure investigation require a combination of steps under varying redox conditions. We review here some of the applications of these procedures. We also present some original data about a few related samples -- serum from four species: Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus -- which we run under fully unreduced and fully reduced conditions as well as with reduction between first and second dimension. We demonstrate that in many cases the unreduced proteins migrate with a better resolution than reduced proteins, mostly in the crowded 'alpha-globulin' area of pI 4.5-6 and M(r) 50-70 kDa.