A double-blind, randomized, placebo-controlled study to explore the efficacy of a dietary plant-derived polysaccharide supplement in patients with rheumatoid arthritis.

OBJECTIVES: There is increased interest in the potential benefits of complementary therapies, of which dietary plant-derived polysaccharides (dPPs) are an important component. We examined the impact of oral ingestion of a pre-biotic dPP supplement active compound (AC) on serum glycosylation and clinical variables associated with inflammation and general health in patients with RA. METHODS: A double-blind, placebo-controlled, parallel-group clinical trial was used. Participants were randomly assigned to receive AC (n = 33) or placebo (n = 36) for 6 months. Serum protein N-glycosylation was determined by mass spectrometry. Patient outcomes were assessed by validated clinical trial health questionnaires. The primary clinical efficacy variable was DAS-28. RESULTS: The groups had comparable baseline clinical characteristics. AC was well tolerated with low drop-out rates. Supplementation resulted in a 12% significant drop in the levels of the agalactosylated (G0F) glycans [8.10 (0.89) to 7.16 (0.60); P = 0.03], but had no significant overall effect on patient outcomes. The placebo-treated group showed no change in G0F but exhibited a reduction in the levels of fully digalactosylated (G2) glycans (11%; P = 0.03). Although not clinically significant, DAS scores were, however, marginally lower in the placebo group [difference = 0.63 (0.23) s.e.; 95% CI 0.17, 1.10; P = 0.009], as were two of the secondary variables. CONCLUSIONS: Short-term dietary supplementation with AC resulted in a moderate, but significant, reduction in G0F levels, but did not result in any clinically significant improvement in disease activity when assessing the study group as a whole.

Temperature-dependent modulation of Porphyromonas gingivalis lipid A structure and interaction with the innate host defenses.

Lipid A structure is a critical determinant of the interaction between pathogens and the innate immune system. Previously, we demonstrated the presence of non- and monophosphorylated tetra-acylated lipid A structures in the outer membrane of Porphyromonas gingivalis, an agent of human periodontal disease. These modifications to lipid A structure lead to evasion and suppression of innate defenses mediated by Toll-like receptor 4 (TLR4) and cationic antimicrobial peptides. In this investigation, we examined the influence of growth temperature on P. gingivalis lipid A structure and recognition by TLR4 as an example of an environmental influence which is known to vary between healthy and diseased sites in the periodontium. We demonstrate that P. gingivalis grown at a normal body temperature produces mainly nonphosphorylated and monophosphorylated tetra-acylated lipid A structures, whereas bacteria grown at 39°C and 41°C intended to mimic increasing levels of inflammation, producing increasing proportions of monophosphorylated, penta-acylated lipid A. The temperature-dependent alteration in lipid A renders the bacterium significantly more potent for activating TLR4 and more susceptible to killing by ß-defensins 2 and 3. This is the first report of a lipid A remodeling system linked to temperature shifts associated with a deregulated inflammatory response. Temperature elevation at sites of inflammation in the periodontium may be a significant environmental regulator of the lipid A modification systems of P. gingivalis, which will influence the interaction of this organism with the innate host defense.

Detection of Mycolactone A/B in Mycobacterium ulcerans-Infected Human Tissue.

BACKGROUND: Mycobacterium ulcerans disease (Buruli ulcer) is a neglected tropical disease common amongst children in rural West Africa. Animal experiments have shown that tissue destruction is caused by a toxin called mycolactone. METHODOLOGY/PRINCIPAL FINDINGS: A molecule was identified among acetone-soluble lipid extracts from M. ulcerans (Mu)-infected human lesions with chemical and biological properties of mycolactone A/B. On thin layer chromatography this molecule had a retention factor value of 0.23, MS analyses showed it had an m/z of 765.6 [M+Na(+)] and on MS:MS fragmented to produce the core lactone ring with m/z of 429.4 and the polyketide side chain of mycolactone A/B with m/z of 359.2. Acetone-soluble lipids from lesions demonstrated significant cytotoxic, pro-apoptotic and anti-inflammatory activities on cultured fibroblast and macrophage cell lines. Mycolactone A/B was detected in all of 10 tissue samples from patients with ulcerative and pre-ulcerative Mu disease. CONCLUSIONS/SIGNIFICANCE: Mycolactone can be detected in human tissue infected with Mu. This could have important implications for successful management of Mu infection by antibiotic treatment but further studies are needed to measure its concentration.

Glucose homeostasis across human airway epithelial cell monolayers: role of diffusion, transport and metabolism.

Glucose in airway surface liquid (ASL) is maintained at low concentrations compared to blood glucose. Using radiolabelled [(3)H]-D: -glucose and [(14)C]-L: -glucose, detection of D: - and L: -glucose by high-performance liquid chromatography and metabolites by nuclear magnetic resonance, we found that glucose applied to the basolateral side of H441 human airway epithelial cell monolayers at a physiological concentration (5 mM) crossed to the apical side by paracellular diffusion. Transepithelial resistance of the monolayer was inversely correlated with paracellular diffusion. Appearance of glucose in the apical compartment was reduced by uptake of glucose into the cell by basolateral and apical phloretin-sensitive GLUT transporters. Glucose taken up into the cell was metabolised to lactate which was then released, at least in part, across the apical membrane. We suggest that glucose transport through GLUT transporters and its subsequent metabolism in lung epithelial cells help to maintain low glucose concentrations in human ASL which is important for protecting the lung against infection.

Serum alpha 2-HS glycoprotein predicts survival in patients with glioblastoma.

BACKGROUND: Glioblastoma, the most common primary brain tumor, has variable prognosis. We aimed to identify serum biomarkers that predict survival of patients with glioblastoma. METHODS: In phase 1 (biomarker discovery), SELDI-TOF mass spectra were studied in 200 serum samples from 58 control subjects and 36 patients with grade II astrocytoma, 15 with anaplastic astrocytoma, and 91 with glioblastoma. To identify potential biomarkers, we searched for peptide peaks that changed progressively in size with increasing malignancy. One peak, identified as the B-chain of alpha 2-Heremans-Schmid glycoprotein (AHSG), was less prominent with increasing tumor grade. We therefore investigated AHSG as a survival predictor in glioblastoma. We measured serum AHSG by turbidimetry and determined indices of malignancy, including tumor proliferation (Ki67 immunolabel) and necrosis (tumor lipids on magnetic resonance spectroscopy). In phase 2 (biomarker validation), the prognostic power of AHSG was validated in an independent group of 72 glioblastoma patients. RESULTS: Median survival was longer (51 vs 29 weeks) in glioblastoma patients with normal vs low serum AHSG concentrations (hazard ratio 2.7, 95% CI 1.5-5.0, P <0.001), independent of age and Karnofsky score. Serum AHSG inversely correlated with Ki-67 immunolabeling and tumor lipids. A prognostic index combining serum AHSG with patient age and Karnofsky score separated glioblastoma patients with short (<3 months) and long (>2 years) median survival. The prognostic value of serum AHSG was validated in a different cohort of glioblastoma patients. CONCLUSIONS: We conclude that serum AHSG concentration, measured before starting treatment, predicts survival in patients with glioblastoma.

Identification of a second lipopolysaccharide in Porphyromonas gingivalis W50.

We previously described a cell surface anionic polysaccharide (APS) in Porphyromonas gingivalis that is required for cell integrity and serum resistance. APS is a phosphorylated branched mannan that shares a common epitope with posttranslational additions to some of the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the surface of P. gingivalis. APS was purified on concanavalin A affinity columns to minimize the loss of the anchoring system that occurred during chemical extraction. (1)H nuclear magnetic resonance spectroscopy of the lectin-purified APS confirmed the previous structure but also revealed additional signals that suggested the presence of a lipid A. This was confirmed by fatty acid analysis of the APS and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the lipid A released by treatment with sodium acetate buffer (pH 4.5). Hence, P. gingivalis synthesizes two distinct lipopolysaccharide (LPS) macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Nonphosphorylated penta-acylated and nonphosphorylated tetra-acylated species were detected in lipid A from P. gingivalis total LPS and in lipid A from A-LPS. These lipid A species were unique to lipid A derived from A-LPS. Biological assays demonstrated a reduced proinflammatory activity of A-LPS compared to that of total LPS. Inactivation of a putative O-antigen ligase (waaL) at PG1051, which is required for the final step of LPS biosynthesis, abolished the linkage of both the O antigen and APS to the lipid A core of O-LPS and A-LPS, respectively, suggesting that WaaL in P. gingivalis has dual specificity for both O-antigen and APS repeating units.

Structural basis for complement factor H linked age-related macular degeneration.

Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to approximately 50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulator's surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance-monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG-FH complex.

Resistance to deglycosylation by ammonia of IgA1 O-glycopeptides: implications for the beta-elimination of O-glycans linked to serine and threonine.

Pools of O-glycopeptides (and their deglycosylated analogues) derived from trypsin-digested normal human serum IgA1 have been treated with ammonia under conditions reported to result in complete liberation of O-glycans linked to serine and threonine residues in glycopeptides and glycoproteins. MALDI-TOF MS analysis has revealed that only one of the six glycosylated sites is susceptible to beta-elimination under these conditions. It is likely that resistance to beta-elimination is due to very close proximity of proline to the glycosylated serine or threonine residues. Preliminary results using 0.1M NaOH (instead of ammonia) to perform beta-elimination indicated that there was also selective de-O-glycosylation with this reagent, however, these results were complicated by the concomitant hydrolysis of the peptide bonds. These findings may have implications for similarly O-glycosylated peptides and proteins and possibly for other chemical methods that are used to carry out beta-eliminations of O-glycans.

Integrated membrane protein analysis of mature and embryonic stem cell-derived smooth muscle cells using a novel combination of CyDye/biotin labeling.

Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes followed by biotinylation. After enrichment on avidin columns, proteins were separated on large format gradient gels by SDS-PAGE. A comparison between CyDye-tagged and non-tagged gel bands revealed a substantial increase of protein identifications from membrane, membrane-associated, and extracellular matrix proteins with a corresponding reduction in co-purified intracellular proteins. Notably the majority of identified proteins were involved in cellular adhesion processes. To demonstrate the quantitative potential of this platform, we performed a comparison between mature and embryonic stem cell-derived smooth muscle cells (esSMCs) and identified the membrane proteins E-cadherin, integrin alpha6, and CD98 (4F2) to be significantly up-regulated in esSMCs suggesting that SMCs derived from embryonic stem cells maintain characteristics of their embryonic stem cell origin. This was subsequently confirmed by RT-PCR: despite expressing a panel of smooth muscle markers (calponin, Sm22, and aortic smooth muscle actin), esSMCs remained positive for markers of stem cell pluripotency (Oct4, Nanog, and Rex1). In summary, we describe a novel strategy for the profiling of cell membrane proteins. The procedure combines DIGE technology with biotin/avidin labeling to discriminate membrane and membrane-associated proteins from intracellular contaminants by fluorescence tagging and permits semiquantitative differential expression analysis of membrane proteins.

Expression, purification, cocrystallization and preliminary crystallographic analysis of sucrose octasulfate/human complement regulator factor H SCRs 6-8.

Human plasma protein complement factor H (FH) is an inhibitor of the spontaneously activated alternative complement pathway. An allotypic variant of FH, 402His, has been associated with age-related macular degeneration, the leading cause of blindness in the elderly. Crystals of FH domains 6-8 (FH678) containing 402His have been grown in the presence of a polyanionic sucrose octasulfate ligand (an analogue of the natural glycosaminoglycan ligands of FH) using both native and selenomethionine-derivatized protein. Native data sets diffracting to 2.3 A and SeMet data sets of up to 2.8 A resolution have been collected. An anomalous difference Patterson map reveals self- and cross-peaks from two incorporated Se atoms. The corresponding selenium substructure has been solved.

Identification of diagnostic markers for tuberculosis by proteomic fingerprinting of serum.

BACKGROUND: We investigated the potential of proteomic fingerprinting with mass spectrometric serum profiling, coupled with pattern recognition methods, to identify biomarkers that could improve diagnosis of tuberculosis. METHODS: We obtained serum proteomic profiles from patients with active tuberculosis and controls by surface-enhanced laser desorption ionisation time of flight mass spectrometry. A supervised machine-learning approach based on the support vector machine (SVM) was used to obtain a classifier that distinguished between the groups in two independent test sets. We used k-fold cross validation and random sampling of the SVM classifier to assess the classifier further. Relevant mass peaks were selected by correlational analysis and assessed with SVM. We tested the diagnostic potential of candidate biomarkers, identified by peptide mass fingerprinting, by conventional immunoassays and SVM classifiers trained on these data. FINDINGS: Our SVM classifier discriminated the proteomic profile of patients with active tuberculosis from that of controls with overlapping clinical features. Diagnostic accuracy was 94% (sensitivity 93.5%, specificity 94.9%) for patients with tuberculosis and was unaffected by HIV status. A classifier trained on the 20 most informative peaks achieved diagnostic accuracy of 90%. From these peaks, two peptides (serum amyloid A protein and transthyretin) were identified and quantitated by immunoassay. Because these peptides reflect inflammatory states, we also quantitated neopterin and C reactive protein. Application of an SVM classifier using combinations of these values gave diagnostic accuracies of up to 84% for tuberculosis. Validation on a second, prospectively collected testing set gave similar accuracies using the whole proteomic signature and the 20 selected peaks. Using combinations of the four biomarkers, we achieved diagnostic accuracies of up to 78%. INTERPRETATION: The potential biomarkers for tuberculosis that we identified through proteomic fingerprinting and pattern recognition have a plausible biological connection with the disease and could be used to develop new diagnostic tests.

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS.

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.

Proteomic dataset of Sca-1+ progenitor cells.

Embryonic stem cells (ES cells) can differentiate into endothelial cells and smooth muscle cells (SMCs), which participate in vascular angiogenesis. In this study, we differentiated mouse ES cells into Sca-1(+) cells, which have the potential to serve as vascular progenitor cells, and mapped their proteome by 2-DE using a pH 3-10 non-linear gradient and 12% SDS-polyacrylamide gels. A subset of 300 protein spots was analysed and mapped, with 241 protein spots being identified by their PMF using MALDI-TOF MS or by partial amino acid sequencing using MS/MS. Our protein map is the first of Sca-1(+) progenitor cells and will facilitate the identification of proteins differentially expressed during stem cell differentiation. The proteome of adult arterial SMCs is described in an accompanying paper (in this issue, DOI 10.1002/pmic.200402045). All data are made accessible on our website http://www.vascular-proteomics.com.

Proteomic dataset of mouse aortic smooth muscle cells.

In an accompanying study (in this issue, DOI 10.1002/pmic.200402044), we have characterised the proteome of Sca-1(+) progenitor cells, which may function as precursors of vascular smooth muscle cells (SMCs). In the present study, we have analysed and mapped protein expression in aortic SMCs of mice, using 2-DE, MALDI-TOF MS and MS/MS. The 2-D system comprised a non-linear immobilised pH 3-10 gradient in the first dimension (separating proteins with pI values of pH 3-10), and 12%T SDS-PAGE in the second dimension (separating proteins in the range 15,000-150,000 Da). Of the 2400 spots visualised, a subset of 267 protein spots was analysed, with 235 protein spots being identified corresponding to 154 unique proteins. The data presented here are the first map of aortic SMCs and the most extensive analysis of SMC proteins published so far. This valuable tool should provide a basis for comparative studies of protein expression in vascular smooth muscle of transgenic mice and is available on our website hhtp://www.vascular-proteomics.com.

Evaluation of an integrated strategy for proteomic profiling of skeletal muscle.

Proteomic analysis of skeletal muscle presents particular challenges when trying to identify valid biomarkers of phenotypic change in small biopsies from genetically diverse human subjects. Currently, two-dimensional (2-D) gel electrophoresis and mass spectrometry are the chosen analytical strategies but 2-D gels are not appropriate for analyzing proteins less than 11 kDa, they can suffer from problems of reproducibility and in routine use are not a viable high-throughput technique. We have evaluated an integrated proteomic strategy employing Ciphergen ProteinChip arrays, one-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Protein fingerprints characteristic of fast and slow contracting muscles from normal and kyphoscoliosis (ky) mutant mice were obtained from Ciphergen protein arrays. Eight statistically validated protein biomarkers have so far been identified capable of discriminating fast from slow muscle. Five of these showed further differential expression in ky versus normal BDL soleus muscles. Several biomarkers have been formally identified, and were myosin light chain isoforms shown previously to be expressed differentially by fast versus slow skeletal muscles. This integrated experimental approach using a model mouse muscle system shows the potential of Ciphergen protein array technology for proteomic analysis of small proteins in small muscle samples and its applicability for phenotypic characterization of skeletal muscle in general.

Human serum IgA1 is substituted with up to six O-glycans as shown by matrix assisted laser desorption ionisation time-of-flight mass spectrometry.

The micro-heterogeneity of human serum IgA1 results from variable O-glycan substitutions in the 'hinge region' of the molecule and this O-glycosylation may be altered in a number of medical conditions. This micro-heterogeneity has been monitored by analysis of IgA1-derived tryptic O-glycopeptides using matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) analysis. With ammonium citrate-trihydroxyacetophenone matrix, individual compositional glycoforms have been baseline resolved in more than 70 samples and these spectra revealed for the first time that, in addition to expected substitution with 3,4 and 5 GalNAcs, a sixth GalNAc substitution was also present in the hinge region of the molecule. The spectra obtained from subsequent exoglycosidase-treated samples confirmed hexa-O-substitution. Following endoprotease digestions of the exoglycosidase treated samples, possible locations for the sixth GalNAc were indicated from further MALDI-ToF-MS analysis. Hexa-substitution accounts for around 5-10% the glycoforms. This is, we believe, the first report of hexa-O-substitution with GalNAc of human serum IgA1.

Glycoform composition profiling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry.

Pools of O-glycopeptides prepared from trypsin-digested reduced and alkylated human serum IgA1 have been analyzed using matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-ToF-MS) in the positive-ion mode, using 2,4,6-trihydroxy acetophenone-ammonium citrate matrix. Dozens of such pools prepared from normal serum IgA1 and from serum of patients with a number of different medical conditions have been routinely analyzed in this manner. The glycopeptides present in these pools possess identical amino acid sequences but are substituted with a variety of neutral and sialylated glycans and the spectra obtained were such that individual compositional glycoforms were baseline resolved. In addition, the spectra were reproducible, exhibiting a relative peak intensity and area variation of around 11-16%, enabling the technique to be used for the relative quantitation of the different compositional glycoforms present. This could be achieved manually or by applying a Java program especially developed for this purpose. The MS analysis described here is a major improvement over present MALDI methods used for profiling the O-glycosylation of IgA1. The MS methodology together with the Java data analysis are expected to be generally applicable for profiling O-linked glycopeptides derived from other glycoproteins and probably for N-linked glycopeptide pools.

A novel and accurate diagnostic test for human African trypanosomiasis.

INTRODUCTION: Human African trypanosomiasis (sleeping sickness) affects up to half a million people every year in sub-Saharan Africa. Because current diagnostic tests for the disease have low accuracy, we sought to develop a novel test that can diagnose human African trypanosomiasis with high sensitivity and specificity. METHODS: We applied serum samples from 85 patients with African trypanosomiasis and 146 control patients who had other parasitic and non-parasitic infections to a weak cation exchange chip, and analysed with surface-enhanced laser desorption-ionisation time-of-flight mass spectrometry. Mass spectra were then assessed with three powerful data-mining tools: a tree classifier, a neural network, and a genetic algorithm. FINDINGS: Spectra (2-100 kDa) were grouped into training (n=122) and testing (n=109) sets. The training set enabled data-mining software to identify distinct serum proteomic signatures characteristic of human African trypanosomiasis among 206 protein clusters. Sensitivity and specificity, determined with the testing set, were 100% and 98.6%, respectively, when the majority opinion of the three algorithms was considered. This novel approach is much more accurate than any other diagnostic test. INTERPRETATION: Our report of the accurate diagnosis of an infection by use of proteomic signature analysis could form the basis for diagnostic tests for the disease, monitoring of response to treatment, and for improving the accuracy of patient recruitment in large-scale epidemiological studies.

Immunoglobulin A multiple myeloma presenting with Henoch-Schönlein purpura associated with reduced sialylation of IgA1.

Henoch-Schönlein purpura is characterized by immunoglobulin A1 (IgA1) depositions in blood vessels of the skin or in glomeruli, resulting from altered hinge region O-glycosylation. Henoch-Schönlein purpura is seldom reported as a complication of IgA1 myeloma, even when the circulating IgA concentration is very high. We report two patients with IgA1 myeloma presenting with Henoch-Schönlein purpura. The O-glycosylation of these patients' IgA1 was studied. Both patients showed increased binding to peanut agglutinin lectin, suggesting a low degree of sialylation of the hinge region of IgA1 that was confirmed by mass spectrometry. IgA multiple myeloma, secreting IgA1 molecules with decreased sialylation, presenting with a Henoch-Schönlein purpura-like syndrome was diagnosed.