Proteomic biomarkers for psoriasis and psoriasis arthritis.

UNLABELLED: Although several new biomarkers have been recently proposed for psoriasis (Ps) and psoriasis arthritis (PsA), nothing is known about their diagnostic sensitivity and specificity, and their routine use. We therefore searched in-depth for new biomarker candidates using a biobank with EDTA-plasma from 158 individuals, patients and healthy controls. Samples from 6 selected pairs (patients against healthy controls) were searched proteomically using a workflow of extensive and precise design that is highly comprehensive. Subsequent verification was performed using ELISA and the entire biobank. By proteomic methods, 208 altered proteins were identified. Of these, 15 biomarker candidates were selected for verification. Of these 15, 4 individual parameters and 11 combinations significantly discriminated between patient and control groups. These individual parameters were Zn-α2-glycoprotein, complement C3, polymeric immunoglobulin receptor, and plasma kallikrein. Significant discrimination was obtained by combinations of 2 or 3 parameters. One combination seemed suitable for diagnosing PsA. Moreover, several candidates desmoplakin, complement C3, polymeric immunoglobulin receptor, and cytokeratin 17, correlated with PASI in all patients. This first comprehensive proteomic study on non-depleted plasma identified several biomarker candidates that have not been described before as well as some known from previous studies. BIOLOGICAL SIGNIFICANCE: Our non-gel proteomic analysis is based on the highly comprehensive and significantly optimized chromatographic protein pre-fractionation. The method allows a biomarker search in non-depleted plasma. The subsequent verification by ELISA identifies several biomarker-candidates for the unbiased diagnosis of psoriasis and psoriasis arthritis. Four of the identified candidate markers might be used individually. Combinations of several parameters improve the diagnostic sensitivity and specificity. The still not validated candidates form a reserve for further evaluation. Moreover, mass spectrometric data uncover several biomarker-candidates which show diverse protein species of the same protein with opposing changes in the same sample.

Effectiveness of a group diabetes education programme in under-served communities in South Africa: a pragmatic cluster randomized controlled trial.

AIM: To evaluate the effectiveness of group education, led by health promoters using a guiding style, for people with type 2 diabetes in public sector community health centres in Cape Town. METHODS: This was a pragmatic clustered randomized controlled trial with 17 randomly selected intervention and 17 control sites. A total of 860 patients with type 2 diabetes, regardless of therapy used, were recruited from the control sites and 710 were recruited from the intervention sites. The control sites offered usual care, while the intervention sites offered a total of four monthly sessions of group diabetes education led by a health promoter. Participants were measured at baseline and 12 months later. Primary outcomes were diabetes self-care activities, 5% weight loss and a 1% reduction in HbA(1c) levels. Secondary outcomes were self-efficacy, locus of control, mean blood pressure, mean weight loss, mean waist circumference, mean HbA1c and mean total cholesterol levels and quality of life. RESULTS: A total of 422 (59.4%) participants in the intervention group did not attend any education sessions. No significant improvement was found in any of the primary or secondary outcomes, apart from a significant reduction in mean systolic (-4.65 mmHg, 95% CI 9.18 to -0.12; P = 0.04) and diastolic blood pressure (-3.30 mmHg, 95% CI -5.35 to -1.26; P = 0.002). Process evaluation suggested that there were problems with finding suitable space for group education in these under-resourced settings, with patient attendance and with full adoption of a guiding style by the health promoters. CONCLUSION: The reported effectiveness of group diabetes education offered by more highly trained professionals, in well-resourced settings, was not replicated in the present study, although the reduction in participants' mean blood pressure is likely to be of clinical significance.

Colorful quality control of chromatographic sample preparation.

Multidimensional chromatographic separation for proteomic biomarker search generates sets of several hundred homologous fractions, which have to be compared. Due to the high number of sequential steps, deviations between samples may be produced randomly by slight processing differences. These deviations may falsify proteomic results. In order to overcome this problem, we tested the applicability of quality control by colored phycobilins as internal standards. The elution of the used protein standards themselves shows a high reproducibility since their main peak location is practically constant under proper performance of size exclusion and anion exchange chromatography. This applies to runs of one phycobilin alone, combined with another phycobilin, or combined with plasma proteins. Thus, these protein standards do not disturb sample processing. Characteristic peak shifts of phycobilins allow easy observation of deviations caused by typical failures in the elution protocol (aberrant step number, buffer permutation). Mass spectrometric analysis is not influenced by their presence since protein coverage, peptide numbers, and protein numbers are not altered. Thus, colored protein standards may be used for quality control and evaluation of robustness of various chromatographic applications.

Biotinidase determination in serum and dried blood spots--high sensitivity fluorimetric ultramicro-assay.

A miniaturized quantitative biotinidase assay has been developed using biotin 6-amidoquinoline as substrate and the 100-fold enhanced fluorescence of 6-amidoquinoline measured using apolar solvents. Amidoquinoline is measured after deproteinization by ethanol/acetone using individual standardisation and solvent resistant microtiter plates. The assay was optimized for end point determinations of biotinidase activities in serum and for newborn screening using dried blood spots. Serum activities obtained are closely correlated with values obtained using a quantitative validation method (r=0.96). Analytical sensitivity is around 2% of the mean activity (7.01+/-1.92 nmol/min/ml, mean+/-SD) of a healthy control population. With dried blood spots, a close correlation with values obtained using the Wallac-test kit (r=0.92) was found. Biotinidase activities of a healthy population of 651 newborns amount to 0.2429+/-0.07 nmol/min/ml blood. The analytical sensitivity is close to 1% of the mean activity.

Glycosylphosphatidylinositol-specific phospholipase D of human serum--activity modulation by naturally occurring amphiphiles.

The enzymatic properties of glycosylphosphatidylinositol-specific phospholipase D (EC 3.1.4.50) were characterized using a 6,000-fold purified enzyme. This was obtained in 100 microg amounts from human serum with a recovery of 35%. Pure alkaline phosphatase containing one anchor moiety per molecule was used as substrate. The enzyme is stimulated by n-butanol, but in contrast to other phospholipases this activation is not produced by a transphosphatidylation reaction. The previously reported non-linearity of the specific activity with respect to phospholipase concentration in the test was no longer observed upon purification, indicating inhibitor removal. The serum inhibitor(s) co-chromatograph with serum proteins and lipoproteins. The main part of the inhibitory activity was found in the lipid fraction after protein denaturation and can be subfractionated into acid phospholipids, cholesteryl esters and triacylglycerides. Added phosphatidyl-serine, phosphatidylinositol, phosphatidylglycerol, gangliosides, cholesteryl esters, and sphingomyelins turned out to be strong inhibitors, as well as phosphatidic acid. Phosphatidylethanolamine and various monoacylglycerols were found to be activators. The low glycosylphosphatidylinositol-specific phospholipase activity found in native serum did not increase significantly upon 90% removal of phospholipids by n-butanol. High serum concentrations of strongly inhibiting compounds, complex kinetic interactions among aggregates of these substances, and compartmentalization effects are discussed as possible reasons for the observed inactivity.

Gel chromatographic characterization of the hydrophobic interaction of glycosylphosphatidylinositol-alkaline phosphatase with detergents.

The interaction of a glycosylphosphatidylinositol (GPI) protein with different detergents was studied for the first time with a purified protein. Four differently hydrophobic fractions of GPI-alkaline phosphatase (GPI-AP) from calf intestine were used as model proteins. The mode of interaction was determined by investigating (i) the self-aggregation behaviour of the GPI-AP fractions, (ii) the interference of detergents with GPI-AP binding to octyl-Sepharose, and (iii) the elution of GPI-AP bound to octyl-Sepharose. It was shown that polyoxyethylene-type detergents surprisingly interact much stronger than n-octylglucoside with GPI-AP, which is in contrast to the known behaviour of GPI-proteins in natural membranes. Gel filtration chromatography of Triton X-100 at concentrations above the critical micellar concentration yields three different micelle species with apparent molecular weights of about 166, 54, and 16 kDa. GPI-AP fraction II, which is shown to bear only one anchor per dimer, does not bind to any of these micelles. We demonstrate that a complex is formed containing about 150 Triton X-100 molecules and about 4700 molecules of water per molecule of GPI-AP dimer. The experimental findings are in accordance with a simple geometrical model based on the physical data of fatty acids and the arrangement, mean size, and shape of Triton X-100 molecules.

Glycosylphosphatidylinositol-specific phospholipase D in blood serum: is the liver the only source of the enzyme?

In cases of systemic inflammatory response syndrome, sepsis, and septic shock, the activity of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) in serum amounts to 20 to 25% of the activity found in a healthy control group. The activity of serum GPI-PLD is positively correlated with inflammatory markers and counts of monocytes and stab cells (bands) and negatively correlated with polymorphonuclear neutrophils and lymphocytes in severe diseases. This indicates a yet unknown involvement of the inflammatory system in GPI-PLD liberation and suggests that the liver is not the only source of the plasma enzyme. Plasma was shown to contain an effective inhibitor of GPI-PLD which is soluble in organic solvents. Its concentration in capillary plasma is 20-fold higher than in venous plasma. To find possible other sources of plasma GPI-PLD besides the liver, the GPI-degrading activity was measured in different organs of the rat. Product formation was analysed using [125I]TID-labeled GPI-AP.

Newborn screening for galactosemia: ultramicro assay for galactose-1-phosphate-uridyltransferase activity.

An enzymatically optimized, miniaturized (20 microl) fluorimetric assay of galactose-1-phosphate-uridyltransferase using dried blood spots for newborn screening is presented. The Beutler reaction principle has been adapted to the microtiter plate technology and acetone/methanol was used for complete deproteinization. A special ultramicro multiwell screening plate resistant to organic solvents has been developed and employed. The assay is simple, sensitive and inexpensive, due to small reagent volumes and the low prices of ultramicro screening plates. The reaction is linear with galactose-1-phosphate-uridyltransferase activity up to 120 min of incubation time. It shows low imprecision and good correlation to a quantitative validation test. For standardization the use of plate means or medians of activity or fluorescence values is proposed. Individual blank measurement prevents false negative assessments.

Glycosylphosphatidylinositol-alkaline phosphatase from calf intestine as substrate for glycosylphosphatidylinositol-specific phospholipases--microassay using hydrophobic chromatography in pipet tips.

An electrophoretically homogeneous glycosylphosphatidylinositol- alkaline phosphatase fraction from calf intestine, obtained by hydrophobic chromatography, was used as "enzyme-labeled" substrate for testing phospholipase activity. The reaction products were separated by (i) hydrophobic chromatography in pipet tips and (ii) Triton X-114 phase partitioning. The chromatographic method presented permits high test frequencies, does not need temperature-controlled sample handling, and is only slightly disturbed by detergents, organic solvents, and proteins. The method was used to characterize phosphatidylinositol- specific phospholipase C from Bacillus cereus and phospholipase D from calf serum. Measurement of substrate hydrolysis by phospholipases is apparently linear to enzyme concentration and time. Relative activity of both enzymes is maximum at pH 6.5, corresponding to the optimal pH range found with other glycosylphosphatidylinositol substrates and phosphatidylinositol-specific phospholipases of other sources. Maximum activity of phospholipase C was found at 0.03% Triton X-100, 0.01% Brij 35, and 0.2% n-octylglucoside. The activity is not affected by Ca(2+), NaHCO(3), o-phenanthroline, or EDTA, increasingly inhibited by MgCl(2), MnCl(2), and ZnCl(2), and slightly activated by Na+ and K+. Calf serum phospholipase D shows maximum activity at 0.05% Triton X-100, 0.02% Brij 35, and 0.4% n-octylglucoside. The apparent Km values for phospholipase C (12.25 micron) and phospholipase D (4.94 micron) found with glycosylphosphatidylinositol-alkaline phosphatase are compared with values published for other glycosylphosphatidylinositol substrates.

Characterization of the interaction of alkaline phosphatase with an activity inhibiting monoclonal antibody by progress curve analysis.

Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of calf intestine (AP), the interaction of a macromolecular antigen with the antibody was studied with different reaction conditions and with different conformations of the antigen, i.e. using (i) different pH values, (ii) different temperatures, (iii) different substrate saturation of the enzyme, (iv) different glycosylphosphatidyl-AP (GPI-AP) aggregates, and (v) membrane-bound species. In the case of antibody excess and negligible substrate consumption enzymic product formation proceeds according to [P] = a + b x t - c x exp(-d x t). By direct progress curve fitting and secondary data evaluation using nonlinear regression, omitting numerical derivation and graphic techniques, kinetic constants of the immune reaction have been estimated. The method does not require any artificial labelling nor any separation of bound and free entities. (i) Upon increasing pH from 9.8 to 11.0, the dissociation constant of the enzyme-antibody complex is increased strongly, mainly due to the decreasing association rate constant. (ii) A temperature increase from 25 degrees C to 37 degrees C produces a marked increase of both the association and dissociation rate constant. (iii) To differentiate between the interaction of the antibody with the free (E) and substrate-bound (ES) enzyme, experiments were done at different substrate concentrations. The results were fitted to a model allowing determination of association and dissociation rate constants of the free and substrate-bound enzyme. The inverse variation of association and dissociation rate constants caused by substrate binding produces a marked increase of the dissociation constant of the antibody-enzyme complex. The antibody-bound enzyme shows a nearly three-fold higher Km value and a six-fold lower catalytic constant as compared to the free enzyme. (iv) Investigations of the interaction of the antibody with anchorless AP, different hydrophobic aggregates of purified GPI-AP (fractions II-V). (v) Membrane-bound GPI-AP show that the epitopes of all species are fully accessible to the antibody and not cryptic. Surprisingly the insertion of the GPI-moiety into the membrane and the aggregation of the different GPI-AP fractions II-V seem to improve antibody binding. Such improvement of binding was not found in control experiments with Fab, indicating only for the bivalent antibody a stronger interaction with the multivalent antigen than with the monovalent antigen.

Is the brush border membrane of the intestinal mucosa a generator of "chymosomes"?

1. The microvilli of enterocytes in calf intestine demonstrate high levels of vesiculation activity at the top and at the basal region. 2. The morphology of the vesicles associated with microvilli (100-500 nm diameter, unilamellar, few intramembraneous particles, high AP activity) is very similar to the morphology of vesicles found in the chyme. 3. Vesicles can be purified 6-10 fold from chyme of the calf intestine applying a Mg(++)-precipitation method, used for brush border membrane preparation. 4. Specific activities of alkaline phosphatase and disaccharidases were found to be much higher in chyme vesicles than in the mucosa. 5. Phospholipid content and phospholipid composition is in chyme vesicles different from brush border membrane vesicles. 6. The characterized chyme vesicles are referred to as chymosomes. We consider the mucosa as a large-scale generator of chymosomes, i.e. digestive enzymes bearing vesicles.

Heterogeneity of glycosylphosphatidylinositol-anchored alkaline phosphatase of calf intestine.

A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components. No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regarding Km, Vmax, the type of inhibition and inhibition constants of the amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively. Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatography. Fraction I corresponds to the anchorless enzyme, fractions II-V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns-PLC or PLD action, almost 50% of fraction III is split by PtdIns-PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively. Aggregates of glycosylPtdIns-anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X-100. By gel chromatography in the presence of Brij 35, fractions II-V form stable multiple aggregates of dimers and may bind different amounts of the detergent. These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns-PLC. The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns-anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.

Histamine and peptic ulcer: influence of sample-taking on the precision and accuracy of fluorometric histamine assay in biopsies of human gastric mucosa.

From a methodological point of view the relevance of clinical-biochemical trials depends on the answers to mainly four complexes of questions: (1) the reliability of the assays in the clinical situation to be tested, (2) the precision and accuracy of sample-taking, (3) the qualification of the design and the protocols in the clinical part of the trial and (4) the usefulness of the time concepts in the trial concerning biorhythms, seasonal influences, psychological trauma of diagnostic procedures and treatment. In this study mainly the second complex of questions was studied intensely. The precision of the fluorometric histamine assay in biopsy specimens from human gastric mucosa depended on several conditions: Biochemical technique, sample preparation and removal of biopsies from gastric mucosa via endoscopy. The CV% of the whole procedure was about 8-times higher than that of the biochemical technique. In clinical-biochemical studies on the significance of histamine or any other hormone (such as gastrin) in any disease (uch as duodenal ulcer) it seems therefore useless to describe the precision of an assay only by the variance of the biochemical technique. Calculation of the histamine content as mean of 3 samples reduced the CV% from 27.2 to 14.9% and should therefore be recommended.

[Acid reduction and ulcer healing through vagotomy: is an inhibition of histamine release an essential reason for this?]. / Säurereduktion and Ulcusheilung durch Vagotomie: Ist eine Hemmung der Histaminfreisetzung hierfür eine wesentliche Ursache?

In a prospective, consecutive study the histamine content of corpus mucosa and the basal and maximally stimulated acid output were measured in male patients who had selective vagotomy to correct a chronic duodenal ulcer. The increase in histamine content after vagotomy was directly proportional to a decrease in maximum acid output. The relationships between histamine content, recurrent ulcer, and reduction in acid output point to a causal relationship between inhibition of histamine release and successful vagotomy.

[General adaptation syndrome (Selye) in cattle. 6. Influence of stress conditions on antibody levels after active and passive immunization as well as on the topographic distribution of various groups of pathogens in the gastrointestinal canal]. / Allgemeines Adaptationssyndrom (Selye) beim Kalb 6. Mitteilung: Einfluss von Stresszuständen auf den Antikörperspiegel nach aktiver und passiver Immunisierung sowie auf die topographische Verteilung einiger Keimgruppen im Magen-Darm-Kanal

The action of experimental and natural stressors on the humoral immune response of calf was studied. Particular attention was given to the H-antigen of S.dublin and equine erythrocytes,the degradation rate of passively acquired humoral antibody, as well as the quantity and topographic distribution of certain groups of germs in the gastrointestinal tract. The following results were obtained:1. Antibody formation was impededby repeated or lasting stressor effect (ACTH injections). 2. The immunological reactions of the calves involved to antigen injection immediately after transport into the rearing unit were stronger than those to antigen application three days from transfer. Immunisation of animals transferred to rearing or fattening units, therefore, should be applied immediately after arrival in the new accomodation, but no interval of three or four days should be allowed. 3. Antibody formation was no longer impaired in calves immunised two weeks from transfer, as compared to those immunised immediately after arrival in the rearing unit. This seemed to suggest that by that time adaptation of the animals to their new environment had been almost complete. 4. Lasting stress (slow drip infusion of ACTH, cortisol, colibacteria or coli-endotoxin) led to no detectable by paper electrophoresis. 5. Calves that had been given three weekly dosesof 1 IU ACTH per kilogram of live weight through four weeks,did not differ,withthe authors'method,from the controls regarding the decomposition rate of passively acquired humoral antibody. 6. ACTH slow drip infusions of calves over several days caused higher concentrations of colibacteria throughout the intestinal tract, including those proximal sections of the small intestine in which little or no colibacteria should occur under physiological conditions in calves of that age.

Poly(adenosine diphosphate-ribose) polymerase in quail oviduct. Changes during estrogen and progesterone induction.

THE ACTIVITIES OF THE FOLLOWING ENZYMES HAVE BEEN DETERMINED IN NUCLEI OF QUAIL OVIDUCTS IN RESPONSE TO EXOGENOUS STIMULATION OF THE BIRDS WITH DIETHYLSTILBESTROL, USED AS AN ESTROGEN ANALOGUE AND PROGESTERONE: DNA dependent DNA polymerase, DNA dependent RNA polymerase I and II and poly(adenosine diphosphate-ribose) [=poly(ADP-Rib)] polymerase.During primary stimulation with the estrogen analogue the activities of the four DNA dependent polymerases increase to about the same degree. Upon withdrawal of the hormones the levels of the enzymes drop to values known from nuclei from unstimulated quail oviducts. The secondary stimulation with the estrogen analogue causes a significant increase only of the RNA polymerase II. The in vivo induction of avidin by progesterone in oviduct mucosa cells from quails, during the period of primary estrogen stimulation, is accompanied by an increase of RNA polymerase II activity and a marked decrease of poly(ADP-Rib) polymerase activity. The activities of RNA polymerase I and of poly(ADP-Rib) polymerase are not affected significantly by an exogenous administration of progesterone.