Multiparametric detection of autoantibodies in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous disease with respect to disease manifestations, disease progression and treatment response. Therefore, strategies to identify biomarkers that help distinguishing SLE subgroups are a major focus of biomarker research. We reasoned that a multiparametric autoantibody profiling approach combined with data mining tools could be applied to identify SLE patient clusters. We used a bead-based array containing 86 antigens including diverse nuclear and immune defense pathway proteins. Sixty-four autoantibodies were significantly (p < 0.05) increased in SLE (n = 69) compared to healthy controls (HC, n = 59). Using binary cut-off thresholds (95% quantile of HC), hierarchical clustering of SLE patients yields five clusters, which differ qualitatively and in their total number of autoantibodies. In two patient clusters the overall accumulated autoantibody reactivity of all antigens tested was 31% and 48%, respectively. We observed a positive association between the autoantibody signature present in these two patient clusters and the clinical manifestation of glomerulonephritis (GLMN). In addition, groups of autoantibodies directed against distinct intracellular compartments and/or biological motifs characterize the different SLE subgroups. Our findings highlight the relevant potential of multiparametric autoantibody detection and may contribute to a deeper understanding of the clinical and serological diversity of SLE.

HCC-1, a novel chemokine from human plasma.

A novel CC chemokine, HCC-1, was isolated from the hemofiltrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and 29-37% with the other human CC chemokines. Unlike MIP-1 alpha and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34+ myeloid progenitor cells. It was as effective as MIP-1 alpha, but about 100-fold less potent.

Is isolation of comprehensive human plasma peptidomes an achievable quest?

The low molecular weight (LMW; <10kDa)* plasma peptidome has been considered a source of useful diagnostic biomarkers and potentially therapeutic molecules, as it contains many cytokines, peptide hormones, endogenous peptide products and potentially bioactive fragments derived from the parent proteome. The small size of the peptides allows them almost unrestricted vascular and interstitial access, and hence distribution across blood-brain barriers, tumour and other vascular permeability barriers. Therefore, the peptidome may carry specific signatures or fingerprints of an individual's health, wellbeing or disease status. This occurs primarily because of the advantage the peptidome has in being readily accessible in human blood and/or other biofluids. However, the co-expression of highly abundant proteins (>10kDa) and other factors present inherently in human plasma make direct analysis of the blood peptidome one of the most challenging tasks faced in contemporary analytical biochemistry. A comprehensive compendium of extraction and fractionation tools has been collected concerning the isolation and micromanipulation of peptides. However, the search for a reliable, accurate and reproducible single or combinatorial separation process for capturing and analysing the plasma peptidome remains a challenge. This review outlines current techniques used for the separation and detection of plasma peptides and suggests potential avenues for future investigation. This article is part of a Special Issue entitled: HUPO 2014.

Peptidomics technologies for human body fluids.

Peptides play a central role in many physiological processes. In order to analyse comprehensively all peptides and small proteins of a whole organism or a subsystem (peptidome), the use of technologies other than 2D gel electrophoresis is necessary. Approaches that use liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of human body fluids.

hBD-1: a novel beta-defensin from human plasma.

We report the isolation and characterization of a novel peptide with significant sequence homology to beta-defensins from human blood filtrate. The human beta-defensin-1 (hBD-1) is a short basic peptide of 36 amino acid residues. It contains six cysteines forming three intramolecular disulfide bonds. The molecular mass of hBD-1 is 3928.6 Da. Cloning of the specific cDNA confirmed the amino acid sequence of the native peptide. hBD-1 shares the nine conserved amino acids characteristic for beta-defensins from respiratory epithelial cells and neutrophils of cattle and chicken leukocytes. hBD-1 is present in nanomolar concentration in human plasma.

GCAP-II: isolation and characterization of the circulating form of human uroguanylin.

The systematic isolation of circulating regulatory peptides which generate cGMP as second messenger resulted in the identification of a novel member of the guanylin family. In the present study we describe the purification and amino acid sequence of a new guanylate cyclase C activating peptide (GCAP-II). GCAP-II contains 24 amino acids in the following sequence: FKTLRTIANDDCELCVNVACTGCL. Its molecular mass is 2597.7 Da. The 16 C-terminal amino acids are identical to uroguanylin from human urine. native and synthetic GCAP-II activate GC-C, the specific guanylate cyclase receptor, of cultured human colon carcinoma (T84) cells. GCAP-II stimulates chloride secretion in isolated human intestinal mucosa mediated by intracellular cGMP increase. GCAP-II specific antibodies were used to localize the peptide by immunohistochemistry in entero-endocrine cells of the colonic mucosa.

LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity.

We report the isolation and characterization of a novel human peptide with antimicrobial activity, termed LEAP-1 (liver-expressed antimicrobial peptide). Using a mass spectrometric assay detecting cysteine-rich peptides, a 25-residue peptide containing four disulfide bonds was identified in human blood ultrafiltrate. LEAP-1 expression was predominantly detected in the liver, and, to a much lower extent, in the heart. In radial diffusion assays, Gram-positive Bacillus megaterium, Bacillus subtilis, Micrococcus luteus, Staphylococcus carnosus, and Gram-negative Neisseria cinerea as well as the yeast Saccharomyces cerevisiae dose-dependently exhibited sensitivity upon treatment with synthetic LEAP-1. The discovery of LEAP-1 extends the known families of mammalian peptides with antimicrobial activity by its novel disulfide motif and distinct expression pattern.

The circulating bioactive form of human guanylin is a high molecular weight peptide (10.3 kDa).

Guanylin is a peptide isolated from rat intestine that stimulates intestinal guanylate cyclase. We describe here the purification of circulating guanylin from human hemofiltrate. By N-terminal protein sequence analysis 47 amino acids were determined. This sequence corresponds to the positions 22 to 68 of the prohormone deduced from the cDNA sequence of human proguanylin. Mass spectral analysis of the circulating peptide showed the molecular weight to be 10,336 Da, which corresponds to the mass calculated from position 22 to the C-terminus of the peptide predicted from the cDNA sequence. Circulating guanylin markedly increased the cyclic GMP content of T84 cells. Our data show that the hormonal form of guanylin is circulating as a 10.3-kDa peptide in human blood.

Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate.

We present a multidimensional approach to map the composition of complex peptide mixtures obtained as crude extract from biological liquids by (1) cation exchange chromatography and (2) subsequent microbore reversed-phase liquid chromatography and electrospray mass spectrometry coupling (LC-MS). Human hemofiltrate is an equivalent to blood and is used to obtain peptide material in large quantities from patients with chronic renal failure. The upper exclusion limit of the filtration membranes used results in a protein-free filtrate containing peptides in a range up to 20 ku. Using this unique peptide source, several thousand peptides were detected and an LC-MS data base of circulating human peptides was created. The search for known peptides by their molecular mass is a reliable method to guide peptide purification.

Peptidomics: the comprehensive analysis of peptides in complex biological mixtures.

Progress in the sequencing of genomes has resulted in an increasing demand for a functional analysis of gene products in order to understand the underlying physiology. Proteomics has established itself as a highly valuable technology for producing functionally related data in an unparalleled fashion, but is methodologically restricted to the analysis of proteins with higher molecular masses (>10 kDa). The development of a technology which covers peptides with low molecular weight and small proteins (0.5 to 15 kDa) was necessary, since peptides, amongst them families of hormones, cytokines and growth factors, play a central role in many biological processes. To summarise the technologies used for this approach the term "peptidomics" is introduced. In this article, we present the rationale and first results of a novel, universal peptide display approach for the analysis and visualisation of peptides and small proteins from biological samples. Special attention is given to samples derived from extracellular fluids such as blood plasma and cerebrospinal fluid. Additionally, a high throughput identification procedure for the analysis of peptides in their native and processed molecular form is outlined.

Porcine detrusor cyclic nucleotide phosphodiesterase isoenzymes: characterization and functional effects of various phosphodiesterase inhibitors in vitro.

OBJECTIVES: This study was undertaken to characterize adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) phosphodiesterases (PDEs) in porcine detrusor smooth muscle and to define their possible role in tension regulation. METHODS: PDEs were isolated from porcine detrusor homogenate by Q-Sepharose anion exchange and calmodulin affinity chromatography. The effects of selective inhibitors of cAMP and cGMP PDEs were investigated on isolated PDEs and on carbachol (1 microM) precontracted detrusor strips. RESULTS: Six PDE isoenzymes were isolated by Q-Sepharose anion exchange and calmodulin affinity chromatography: one calmodulin-stimulated PDE (PDE I) which hydrolyzed mainly cGMP, one cGMP-stimulated cAMP PDE (PDE II), two cAMP-specific PDE (PDE IV alpha and IV beta), and two cGMP-specific PDE (PDE V alpha and V beta). PDE I was potently inhibited in a dose-dependent fashion by papaverine, vinpocetine, and zaprinast; the PDE IVs were potently inhibited by papaverine and rolipram; and the PDE Vs were weakly inhibited by papaverine. In organ bath studies, inhibitors of PDE III (milrinone), IV (rolipram), and V (zaprinast) caused only minor relaxations at high concentrations (200 microM), whereas papaverine and vinpocetine caused relaxations of more than 50%. CONCLUSIONS: Our findings support the involvement of cyclic nucleotide metabolism in the regulation of the detrusor smooth muscle tone in the pig and its regulation by PDEs. The weak action of PDE IV and V inhibitors in vitro may be explained by a possible intracellular compartmentalization of such PDEs and the low cyclic nucleotide turnover rate at the conditions used.

High-performance liquid chromatographic determination of sulfated peptides in human hemofiltrate using a radioactivity monitor.

Specific labeling of tyrosine sulfate-containing peptides was achieved using a differential iodination approach. In a complex peptide mixture from human hemofiltrate, cold iodination to saturate free iodine binding sites was followed by mild acidic desulfation of tyrosine sulfate and subsequent radioiodination using iodine-125. Reaction steps were controlled by amino acid analysis using o-phthaldialdehyde precolumn derivatization and by spiking with a sulfated cholecystokinin fragment (CCK4-S). Separation of the peptide mixture with RP-HPLC on a C18 column coupled to a radioactivity monitor led to the sensitive (< or = 5 pM) and specific determination of tyrosine sulfate-containing peptides.

Peptide bank generated by large-scale preparation of circulating human peptides.

Human hemofiltrate (HF) is a source for the purification of circulating regulatory peptides. HF is obtained in large quantities during treatment of patients suffering from chronic renal failure. We have developed a large-scale method for separating peptides from amounts up to 10,000 1 HF into 300 fractions in a standardized two-step procedure, employing cation-exchange separation, followed by reversed-phase chromatography. These fractions represent a peptide bank containing bioactive, desalted and lyophilized peptides of blood. Screening for and isolation of regulatory human peptides is simplified by using this peptide bank.

Matrix-assisted laser desorption/ionisation mass spectrometry guided purification of human guanylin from blood ultrafiltrate.

The purification of the human peptide hormone guanylin 22-115 from blood ultrafiltrate (hemofiltrate, HF) was achieved using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) as the assay system. Screening a peptide bank generated from 5000 1 HF guanylin 22-115 was detected by its molecular mass when adequate conditions for MALDI-MS analysis were chosen. The sensitivity was even better than of the established biological assay system. In addition, the susceptibility towards solvents and salts is strongly reduced. 1.2 mg of the peptide hormone was purified from 10% of the starting material.

Specific determination of tyrosine-phosphorylated proteins and peptides by differential iodination.

A new method for the selective and quantitative determination of phosphotyrosine residues is presented using a differential iodination technique. Characterization of tyrosine-phosphorylated proteins was performed in a biological system using human U937 myeloid leukemia cells. The method is based on the saturation of free iodine binding sites using non-radioactive iodine. Samples are then treated with alkaline phosphatase. New iodine binding sites in dephosphorylated tyrosines are subsequently radio-iodinated, resulting in specific labeling of tyrosine phosphates. Separation is performed by RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled proteins are then identified using a radioactivity detector or autoradiography.

Systematic isolation of circulating human peptides: the concept of peptide trapping.

The structural determination of circulating human peptides is essential to determine their correct posttranslationally processed form. Human hemofiltrate from patients with end stage renal disease is accessible in large quantities and is used as a source for the preparation of circulating peptides. After complete peptide extraction from hemofiltrate, a systematic separation with different chromatographic techniques is achieved. Single peptides are selected according to their mass and chromatographic elution position. Following chromatographic purification, amino acid sequence analysis is performed in combination with data base search. The identification of circulating peptides leads to numerous fragments resulting from cleavage of larger plasma proteins as well as to the discovery of new peptide hormones. The results obtained so far give insight into the degradation of plasma proteins such as fibrinogen, which results in the generation of fragments with biological activity themselves and in the identification of a novel cytokine HCC-1, the first member of beta-defensins in humans, hBD-1, and different peptides not present in any data base.

Human beta-defensin-1: A urinary peptide present in variant molecular forms and its putative functional implication.

Human beta-defensin-1 (hBD-1) was first isolated from blood filtrate by our group. Further studies elucidate the significance of this peptide in the human urogenital tract. The hBD-1 gene is expressed in urogenital epithelial organs such as urinary bladder, ureter, vagina and particularly in distal tubular cells of the kidney. Functional characterization of hBD-1 was carried out with native hBD-1 purified from human body fluids. Several different N-terminally truncated variants derived from the 68-amino acid-containing precursor of hBD-1 occur in blood filtrate and in urine. The generation of these variants can be explained by digestion through a chymotrypsin-like protease. Unlike the alpha-defensins which are structurally related peptide antibiotics, our results indicate that native hBD-1 exhibits minor antimicrobial activity which is not related to the extension of the N-terminus. Only few microorganisms, for example bacilli, are significantly inhibited by hBD-1. Moreover, antibiotic activity is suppressed in solutions containing physiological sodium chloride concentrations. This is in contrast to previous reports assuming a pivotal role of hBD-1 in antimicrobial host defense. In contrast to its weak antimicrobial activity, it is shown that hBD-1 has a strong cytotoxic potential towards mammalian cells like NIH-3T3 fibroblasts. We assume that this property might be important during eradicative processes at epithelia in particular when the synthesis rate of this peptide is upregulated.

Significance of prophylactic urodilatin (INN: ularitide) infusion for the prevention of acute renal failure in patients after heart transplantation.

Acute renal failure is a serious problem following heart transplantation. In first uncontrolled clinical trials, Urodilatin revealed beneficial effects in the prophylaxis and therapy of acute renal failure following heart and liver transplantation. Here, we present the first randomized, placebo-controlled, double-blind study on 24 patients following heart transplantation to investigate whether prophylactic i.v. Urodilatin infusion can prevent acute renal failure requiring renal replacement therapy. Postoperative drug management was characterized by intravenous application of high furosemide, cyclosporine, and vancomycin doses. Urodilatin infusion was started postoperatively with a dose of 40 ng / kg bw / min for 6 days. 6 of the 12 patients in the Urodilatin group and 6 of the 12 patients in the placebo group had a stable diuresis (3 - 4 l / day) during the study period of 6 days. In contrast, the remaining 6 patients of each group developed oliguria / anuria and required subsequent hemofiltration / hemodialysis. Cumulative duration of hemofiltration (88 +/- 7.39 hours in the placebo treated patients versus 44 +/- 5.35 hours in the Urodilatin treated patients, p < 0. 05) as well as frequency of hemodialysis (3.0 +/- 0.49 times in the placebo group vs 1.2 +/- 0.29 times in the Urodilatin group, p < 0. 05) were significantly reduced using Urodilatin. Mean arterial blood pressure was stable during the Urodilatin infusion period and was not different to that observed in placebo patients. We conclude that Urodilatin does not reduce the incidence of acute renal failure and the subsequent requirement for hemofiltration / hemodialysis in our patient population, but seems to reduce the duration of hemofiltration and frequency of hemodialysis compared to the placebo group.

Challenges in planning and conducting diagnostic studies with molecular biomarkers.

Biomarkers are of increasing importance for personalized medicine in many areas of application, such as diagnosis, prognosis, or the selection of targeted therapies. In many molecular biomarker studies, intensity values are obtained from large scale ­omics experiments. These intensity values, such as protein concentrations, are often compared between at least two groups of subjects to determine the diagnostic ability of the molecular biomarker. Various prospective or retrospective study designs are available for molecular biomarker studies, and the biomarker used may be univariate or may even consist in a multimarker rule. In this work, several challenges are discussed for the planning and conduct of biomarker studies. The phases of diagnostic biomarker studies are closely related to levels of evidence in diagnosis, and they are therefore discussed upfront. Different study designs for molecular biomarker studies are discussed, and they primarily differ in the way subjects are selected. Using two systematic reviews from the literature, common sources of bias of molecular diagnostic studies are illustrated. The extreme selection of patients and controls and verification bias are specifically discussed. The pre-analytical and technical variability of biomarker measurements is usually expressed in terms of the coefficient of variation, and is of great importance for subsequent validation studies for molecular biomarkers. It is finally shown that the required sample size for biomarker validation quadratically increases with the coefficient of variation, and the effect is illustrated using real data from different laboratory technologies.

Isolation and structural analysis of "urodilatin", a new peptide of the cardiodilatin-(ANP)-family, extracted from human urine.

Two major forms of cardiac peptides have been established in the last few years: (a) a prohormone of 126 amino acids (CDD/ANP-1-126) in the endocrine heart and (b) the circulating CDD/ANP-99-126 (= alpha ANP) in blood plasma. The method we applied earlier to isolate the circulating form of cardiodilatin from human blood was used to detect and analyze the biologically active, predominant form of the same polypeptide family excreted by the kidneys. Each step of the isolation procedure was followed up by a bioassay using an in vitro vascular smooth muscle relaxation test and a highly specific RIA against cardiodilatin (CDD-99-126) for the initial purification steps. The polypeptides excreted in 1000 l of normal human urine were adsorbed to 2.5 kg of alginic acid, and after elution and lyophilization processed on a G-25 Sephadex column. The obtained crude polypeptide fractions were applied to ion-exchange chromatography. Thereafter four steps of HPLC were carried out to purify the polypeptide which was the suggested form of cardiodilatin (CDD) in human urine. The amino acid analysis and gas phase sequence analysis showed that the main form of urinary cardiodilatin is a 32 amino acid residue containing molecule, cardiodilatin-95-126. The molecule is N-terminally extended compared to the circulating CDD-99-126. This suggests that the analyzed urinary peptide is not the residual plasma form, filtrated and renally cleared from blood, but probably a polypeptide produced and processed in the kidney tubules and cleaved by a different postranslational process. Therefore, this vasorelaxant polypeptide is called urodilatin.