James LeBlanc

Magnesium supplementation, metabolic and inflammatory markers, and global genomic and proteomic profiling: a randomized, double-blind, controlled, crossover trial in overweight individuals

BACKGROUND Dietary magnesium intake has been favorably associated with reduced risk of metabolic outcomes in observational studies; however, few randomized trials have introduced a systems-biology approach to explore molecular mechanisms of pleiotropic metabolic actions of magnesium supplementation. OBJECTIVE We examined the effects of oral magnesium supplementation on metabolic biomarkers and global genomic and proteomic profiling in overweight individuals. DESIGN We undertook this randomized, crossover, pilot trial in 14 healthy, overweight volunteers [body mass index (in kg/m(2)) ≥25] who were randomly assigned to receive magnesium citrate (500 mg elemental Mg/d) or a placebo for 4 wk with a 1-mo washout period. Fasting blood and urine specimens were collected according to standardized protocols. Biochemical assays were conducted on blood specimens. RNA was extracted and subsequently hybridized with the Human Gene ST 1.0 array (Affymetrix, Santa Clara, CA). Urine proteomic profiling was analyzed with the CM10 ProteinChip array (Bio-Rad Laboratories, Hercules, CA). RESULTS We observed that magnesium treatment significantly decreased fasting C-peptide concentrations (change: -0.4 ng/mL after magnesium treatment compared with +0.05 ng/mL after placebo treatment; P = 0.004) and appeared to decrease fasting insulin concentrations (change: -2.2 μU/mL after magnesium treatment compared with 0.0 μU/mL after placebo treatment; P = 0.25). No consistent patterns were observed across inflammatory biomarkers. Gene expression profiling revealed up-regulation of 24 genes and down-regulation of 36 genes including genes related to metabolic and inflammatory pathways such as C1q and tumor necrosis factor-related protein 9 (C1QTNF9) and pro-platelet basic protein (PPBP). Urine proteomic profiling showed significant differences in the expression amounts of several peptides and proteins after treatment. CONCLUSION Magnesium supplementation for 4 wk in overweight individuals led to distinct changes in gene expression and proteomic profiling consistent with favorable effects on several metabolic pathways. This trial was registered at clinicaltrials.gov as NCT00737815.

A Metaproteomic Approach to Study Human-Microbial Ecosystems at the Mucosal Luminal Interface

Aberrant interactions between the host and the intestinal bacteria are thought to contribute to the pathogenesis of many digestive diseases. However, studying the complex ecosystem at the human mucosal-luminal interface (MLI) is challenging and requires an integrative systems biology approach. Therefore, we developed a novel method integrating lavage sampling of the human mucosal surface, high-throughput proteomics, and a unique suite of bioinformatic and statistical analyses. Shotgun proteomic analysis of secreted proteins recovered from the MLI confirmed the presence of both human and bacterial components. To profile the MLI metaproteome, we collected 205 mucosal lavage samples from 38 healthy subjects, and subjected them to high-throughput proteomics. The spectral data were subjected to a rigorous data processing pipeline to optimize suitability for quantitation and analysis, and then were evaluated using a set of biostatistical tools. Compared to the mucosal transcriptome, the MLI metaproteome was enriched for extracellular proteins involved in response to stimulus and immune system processes. Analysis of the metaproteome revealed significant individual-related as well as anatomic region-related (biogeographic) features. Quantitative shotgun proteomics established the identity and confirmed the biogeographic association of 49 proteins (including 3 functional protein networks) demarcating the proximal and distal colon. This robust and integrated proteomic approach is thus effective for identifying functional features of the human mucosal ecosystem, and a fresh understanding of the basic biology and disease processes at the MLI.

Host–microbe relationships in inflammatory bowel disease detected by bacterial and metaproteomic analysis of the mucosal–luminal interface

Background: Host–microbe interactions at the intestinal mucosal–luminal interface (MLI) are critical factors in the biology of inflammatory bowel disease (IBD). Methods: To address this issue, we performed a series of investigations integrating analysis of the bacteria and metaproteome at the MLI of Crohns disease, ulcerative colitis, and healthy human subjects. After quantifying these variables in mucosal specimens from a first sample set, we searched for bacteria exhibiting strong correlations with host proteins. This assessment identified a small subset of bacterial phylotypes possessing this host interaction property. Using a second and independent sample set, we tested the association of disease state with levels of these 14 “host interaction” bacterial phylotypes. Results: A high frequency of these bacteria (35%) significantly differentiated human subjects by disease type. Analysis of the MLI metaproteomes also yielded disease classification with exceptional confidence levels. Examination of the relationships between the bacteria and proteins, using regularized canonical correlation analysis (RCCA), sorted most subjects by disease type, supporting the concept that host–microbe interactions are involved in the biology underlying IBD. Moreover, this correlation analysis identified bacteria and proteins that were undetected by standard means‐based methods such as analysis of variance, and identified associations of specific bacterial phylotypes with particular protein features of the innate immune response, some of which have been documented in model systems. Conclusions: These findings suggest that computational mining of mucosa‐associated bacteria for host interaction provides an unsupervised strategy to uncover networks of bacterial taxa and host processes relevant to normal and disease states. (Inflamm Bowel Dis 2012;)

N-Acetylglucosaminylation of Serine-Aspartate Repeat Proteins Promotes Staphylococcus aureus Bloodstream Infection

Background: Staphylococcus aureus agglutinates in plasma in a manner that requires host fibrinogen and clumping factor A, a bacterial surface protein with serine-aspartate (SD) repeats. Results: SdgB modifies serine residues in SD repeats with GlcNAc, and this glycosylation contributes to the pathogenesis of sepsis. Conclusion: Glycosylation of SD repeats aids bacterial escape from host defenses. Significance: Interference with glycosylation may alter staphylococcal infections. Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.

Proteomic Analysis Reveals Innate Immune Activity in Intestinal Transplant Dysfunction

Background. Many patients with intestinal failure require intestinal transplantation (ITx) to survive. Acute cellular rejection poses a challenge in ITx because its biologic components are incompletely understood. New methodologies for its integrative and longitudinal analysis are needed. Methods. In this study, we characterized episodes of acute cellular rejection in ITx recipients using a noninvasive proteomic analysis. Ostomy effluent was obtained from all patients undergoing ITx at University of California, Los Angeles from July 2008 to September 2009 during surveillance endoscopies in the first 8 weeks post-ITx. Effluent was analyzed using 17-plex Luminex technology and matrix-assisted laser desorption/ionization proteomics. Results. Of 56 ostomy effluent samples from 17 ITx recipients, 14% developed biopsy-proven rejection at a median of 25 days post-ITx. Six had mild rejection, two were indeterminate for rejection, and no graft loss was seen in the first 3 months posttransplantation. Effluent levels of five innate immune cytokines were elevated in the posttransplantation phase: granulocyte colony-stimulating factor, interleukin-8, tissue necrosis factor-&agr;, interleukin-1&bgr;, and interferon-&ggr;. Proteomic analysis revealed 17 protein features differentially seen in rejection, two identified as human neutrophil peptide 1 and 2. This was confirmed by the presence of human neutrophil peptide-positive lamina propria neutrophils in biopsy tissue samples. Conclusions. Proteomic and cytokine analysis of ostomy effluents suggests an early and unappreciated role of innate immune activation during rejection.

PLASMA PROTEOMIC BIOMARKER CANDIDATES THAT PREDICT ACUTE REJECTION IN PEDIATRIC RENAL TRANSPLANTATION: 1192

Introduction: Successful management of acute rejection after renal transplantation requires early detection and therapeutic intervention. Current methods for detecting rejection, which include clinical presentation, serum creatinine and percutaneous renal biopsy, can be nonspecific, subjective, and invasive. Therefore, we aimed to identify biomarker candidates using plasma high-throughput proteomics that could predict acute cellular rejection (ACR) in pediatric renal transplant patients. Methods: We analyzed the plasma proteome of 19 pediatric renal transplant patients (10 with ACR/9 without rejection) at 5 serial time points. Plasma samples were depleted of the 14 most abundant proteins and then bound to C18 reversed phase magnetic beads. Proteins were then extracted from the beads and their intact masses were accurately measured by high resolution MALDI-TOFMS. Patienets were induced with dacluzimab and maintained on either steroid-free or steroid-based immunosupression with tacrolimus and mycophenolate mofetil. All patients had protocol biopsies at 6 month, 1 year, and 2 year in addition to clinical suspicion of rejection. Biopsies were classified by the Banff 2007 criteria. Results: We identified several significant protein masses that differentiate rejectors (rj) from non-rejectors (nr)(Table). A 3,143 Da peptide showed the potential of predicting rejection prior to the rejection episode. This protein signature is significantly different as early as 2 month prior to biopsy, which is maintained at time of biopsy and then normalizes after treatment in the rejectors (Figure). The follow-up study with a second set of patients as well as isolation and ID of the biomarker candidates has begun. Conclusion: We found several protein signatures that could differentiate between ACR versus no rejection in pediatric renal transplant patients. Moreover, a 3,143 Da peptide was identified that could predict ACR as early as 2 months prior to biopsy and potentially be used as therapeutic monitoring tool.