Distinct Metabolic Profile of Primary Focal Segmental Glomerulosclerosis Revealed by NMR-Based Metabolomics

Background

Primary focal segmental glomerulosclerosis (FSGS) is pathological entity which is characterized by idiopathic steroid-resistant nephrotic syndrome (SRNS) and progression to end-stage renal disease (ESRD) in the majority of affected individuals. Currently, there is no practical noninvasive technique to predict different pathological types of glomerulopathies. In this study, the role of urinary metabolomics in the diagnosis and pathogenesis of FSGS was investigated.

Methods

NMR-based metabolomics was applied for the urinary metabolic profile in the patients with FSGS (n = 25), membranous nephropathy (MN, n = 24), minimal change disease (MCD, n = 14) and IgA nephropathy (IgAN, n = 26), and healthy controls (CON, n = 35). The acquired data were analyzed using principal component analysis (PCA) followed by orthogonal projections to latent structure discriminant analysis (OPLS-DA). Model validity was verified using permutation tests.

Results

FSGS patients were clearly distinguished from healthy controls and other three types of glomerulopathies with good sensitivity and specificity based on their global urinary metabolic profiles. In FSGS patients, urinary levels of glucose, dimethylamine and trimethylamine increased compared with healthy controls, while pyruvate, valine, hippurate, isoleucine, phenylacetylglycine, citrate, tyrosine, 3-methylhistidine and β-hydroxyisovalerate decreased. Additionally, FSGS patients had lower urine N-methylnicotinamide levels compared with other glomerulopathies.

Conclusions

NMR-based metabonomic approach is amenable for the noninvasive diagnosis and differential diagnosis of FSGS as well as other glomerulopathies, and it could indicate the possible mechanisms of primary FSGS.     

The Vitamin D Status in Inflammatory Bowel Disease

Context

There is no consensus on the vitamin D status of children and adolescents with inflammatory bowel disease (IBD).

Aim

To determine the vitamin D status of patients with IBD by comparing their serum 25(OH)D concentration to that of healthy controls.

Hypothesis

Serum 25(OH)D concentration will be lower in patients with IBD compared to controls.

Subjects and Methods

A case-controlled retrospective study of subjects with IBD (n = 58) of 2–20 years (male n = 31, age 16.38±2.21 years; female n = 27, age16.56±2.08 years) and healthy controls (n = 116; male n = 49, age 13.90±4.59 years; female n = 67, age 15.04±4.12years). Study subject inclusion criteria: diagnosis of Crohn’s disease (CD) or ulcerative colitis (UC). Vitamin D deficiency was defined as 25(OH)D of (<20 ng/mL) (<50 nmol/L), overweight as BMI of ≥85^th but <95^th percentile, and obesity as BMI ≥95^th percentile. Data were expressed as mean ± SD.

Results

Patients with CD, UC, and their controls had mean serum 25(OH)D concentrations of 61.69±24.43 nmol/L, 53.26±25.51, and 65.32±27.97 respectively (ANOVA, p = 0.196). The overweight/obese controls had significantly lower 25(OH)D concentration compared to the normal-weight controls (p = 0.031); whereas 25(OH)D concentration was similar between the normal-weight and overweight/obese IBD patients (p = 0.883). There was no difference in 25(OH)D between patients with UC and CD, or between subjects with active IBD and controls. However, IBD subjects with elevated ESR had significantly lower 25(OH)D than IBD subjects with normal ESR (p = 0.025), as well as controls (65.3±28.0 nmol/L vs. 49.5±25.23, p = 0.045).

Conclusion

There is no difference in mean serum 25(OH)D concentration between children and adolescents with IBD and controls. However, IBD subjects with elevated ESR have significantly lower 25(OH)D than controls. Therefore, IBD subjects with elevated ESR should be monitored for vitamin D deficiency.     

Overexpression of CD163, CD204 and CD206 on Alveolar Macrophages in the Lungs of Patients with Severe Chronic Obstructive Pulmonary Disease

We have previously reported that the lungs of patients with very severe chronic obstructive pulmonary disease (COPD) contain significantly higher numbers of alveolar macrophages than those of non-smokers or smokers. M1 and M2 macrophages represent pro- and anti-inflammatory populations, respectively. However, the roles of M1 and M2 alveolar macrophages in COPD remain unclear. Immunohistochemical techniques were used to examine CD163, CD204 and CD206, as M2 markers, expressed on alveolar macrophages in the lungs of patients with mild to very severe COPD (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I (mild) n = 11, II (moderate) n = 9, III (severe) n = 2, and IV (very severe) n = 16). Fifteen smokers and 10 non-smokers were also examined for comparison. There were significantly higher numbers of alveolar macrophages in COPD patients than in smokers and non-smokers. The numbers and percentages of CD163⁺, CD204⁺ or CD206⁺ alveolar macrophages in patients with COPD at GOLD stages III and IV were significantly higher than in those at GOLD stages I and II, and those in smokers and non-smokers. In patients with COPD, there was a significant negative correlation between the number of CD163⁺, CD204⁺ or CD206⁺ alveolar macrophages and the predicted forced expiratory volume in one second. Overexpression of CD163, CD204 and CD206 on lung alveolar macrophages may be involved in the pathogenesis of COPD.     

1H NMR metabolic profiling of human serum associated with benign and malignant gallstone diseases

Proton NMR based metabolic profile of serum associated with different gallbladder pathologies is presented. Quantitative and qualitative variations in the metabolic profile of serum in control samples and three different pathologies of gallbladder, chronic cholecystitis, xanthogranulomatous cholecystitis and carcinoma of gallbladder has been evaluated by use of ¹H NMR based metabonomics and multivariate chemometric methods. Multivariate partial least square discriminant analysis of ¹H NMR spectra showed a clear discrimination between control and diseased groups on the basis of quantitative and qualitative metabolic variations. Increased levels of lactate and pyruvate whereas decreased levels of glucose, some amino acids and low density lipoprotein/very low density lipoprotein (LDL/VLDL) were observed. These metabolites, responsible for class discrimination, from different metabolic pathways could be considered as the signatures of the carcinoma of gallbladder.     

A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants

Background

Diagnosis of urogenital schistosomiasis by microscopy and serological tests may be elusive in travelers due to low egg load and the absence of seroconversion upon arrival. There is need for a more sensitive diagnostic test. Therefore, we developed a real-time PCR targeting the Schistosoma haematobium-specific Dra1 sequence.

Methodology/Principal Findings

The PCR was evaluated on urine (n = 111), stool (n = 84) and serum samples (n = 135), and one biopsy from travelers and migrants with confirmed or suspected schistosomiasis. PCR revealed a positive result in 7/7 urine samples, 11/11 stool samples and 1/1 biopsy containing S. haematobium eggs as demonstrated by microscopy and in 22/23 serum samples from patients with a parasitological confirmed S. haematobium infection. S. haematobium DNA was additionally detected by PCR in 7 urine, 3 stool and 5 serum samples of patients suspected of having schistosomiasis without egg excretion in urine and feces. None of these suspected patients demonstrated other parasitic infections except one with Blastocystis hominis and Entamoeba cyst in a fecal sample. The PCR was negative in all stool samples containing S. mansoni eggs (n = 21) and in all serum samples of patients with a microscopically confirmed S. mansoni (n = 22), Ascaris lumbricoides (n = 1), Ancylostomidae (n = 1), Strongyloides stercoralis (n = 1) or Trichuris trichuria infection (n = 1). The PCR demonstrated a high specificity, reproducibility and analytical sensitivity (0.5 eggs per gram of feces).

Conclusion/Significance

The real-time PCR targeting the Dra1 sequence for S. haematobium-specific detection in urine, feces, and particularly serum, is a promising tool to confirm the diagnosis, also during the acute phase of urogenital schistosomiasis.     

Wilm's Tumor-1 Protein Levels in Urinary Exosomes from Diabetic Patients with or without Proteinuria

Background

Podocyte injury is an early feature of diabetic nephropathy (DN). Recently, urinary exosomal Wilm''s tumor-1 protein (WT1), shed by renal epithelial cells, has been proposed as a novel biomarker for podocyte injury. However, its usefulness as biomarker for early diabetic nephropathy has not been verified yet. We investigated urinary exosomal WT1 in type-1 diabetic patients to confirm its role as a non-invasive biomarker for predicting early renal function decline.

Methods

The expression of WT1 protein in urinary exosomes from spot urine samples of type-1 diabetes mellitus patients (n = 48) and healthy controls (n = 25) were analyzed. Patients were divided based on their urinary albumin excretion, ACR (mg/g creatinine) into non- proteinuria group (ACR<30 mg/g, n = 30) and proteinuria group (ACR>30 mg/g, n = 18). Regression analysis was used to assess the association between urinary exosomal levels of WT1 with parameters for renal function. Receiver Operating Characteristic (ROC) curve analysis was used to determine the diagnostic performance of exosomal WT-1.

Results

WT1 protein was detected in 33 out of 48 diabetic patients and in only 1 healthy control. The levels of urinary exosomal WT1 protein is significantly higher (p = 0.001) in patients with proteinuria than in those without proteinuria. In addition, all the patients with proteinuria but only half of the patients without proteinuria were positive for exosomal WT1. We found that the level of exosomal WT1 were associated with a significant increase in urine protein-to-creatinine ratio, albumin-to-creatinine ratio, and serum creatinine as well as a decline in eGFR. Furthermore, patients exhibiting WT1-positive urinary exosomes had decreased renal function compared to WT1-negative patients. ROC analysis shows that WT-1 effectively predict GFR<60 ml. min-1/1.73 m².

Conclusion

The predominant presence of WT1 protein in urinary exosomes of diabetic patients and increase in its expression level with decline in renal function suggest that it could be useful as early non-invasive marker for diabetic nephropathy.     

The Continuum of Physiological Impairment during Treadmill Walking in Patients with Mild-to-Moderate COPD: Patient Characterization Phase of a Randomized Clinical Trial

Background

To have a better understanding of the mechanisms of exercise limitation in mild-to-moderate chronic obstructive pulmonary disease (COPD), we compared detailed respiratory physiology in patients with COPD and healthy age- and sex-matched controls.

Methods

Data were collected during the pre-treatment, patient characterization phase of a multicenter, randomized, double-blind, crossover study. Patients with COPD met Global Initiative for Chronic Obstructive Lung Disease (GOLD) 1 or 2 spirometric criteria, were symptomatic, and had evidence of gas trapping during exercise. All participants completed pulmonary function and symptom-limited incremental treadmill exercise tests.

Results

Chronic activity-related dyspnea measured by Baseline Dyspnea Index was similarly increased in patients with GOLD 1 (n = 41) and 2 (n = 63) COPD compared with controls (n = 104). Plethysmographic lung volumes were increased and lung diffusing capacity was decreased in both GOLD groups. Peak oxygen uptake and work rate were reduced in both GOLD groups compared with controls (p<0.001). Submaximal ventilation, dyspnea, and leg discomfort ratings were higher for a given work rate in both GOLD groups compared with controls. Resting inspiratory capacity, peak ventilation, and tidal volume were reduced in patients with GOLD 2 COPD compared with patients with GOLD 1 COPD and controls (p<0.001).

Conclusions

Lower exercise tolerance in patients with GOLD 1 and 2 COPD compared with controls was explained by greater mechanical abnormalities, greater ventilatory requirements, and increased subjective discomfort. Lower resting inspiratory capacity in patients with GOLD 2 COPD was associated with greater mechanical constraints and lower peak ventilation compared with patients with GOLD 1 COPD and controls.

Trial Registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT01072396","term_id":"NCT01072396"}}NCT01072396     

Substrate Utilization by the Failing Human Heart by Direct Quantification Using Arterio-Venous Blood Sampling

Metabolic substrate utilization of the human failing heart is an area of controversy. The purpose of this study is to directly quantify myocardial substrate utilization in moderately severe heart failure, type 2 diabetes and healthy controls using simultaneous coronary sinus and arterial blood sampling. Patients with heart failure (n = 9, mean NYHA 2.7±0.5), with type 2 diabetes (n = 5) and with normal heart function (n = 10) were studied after an overnight fast in connection with electrophysiological investigations/treatments.A systemic infusion of [²H₂]palmitate allowed for the calculation of absolute palmitate extraction across the heart. Blood samples were analysed for non-esterified fatty acids, triacylglycerol, glycerol, glucose, pyruvate, lactate, 3-hydroxybutyrate, and blood gases after simultaneous sampling of arterial and coronary sinus blood. Arterio-coronary sinus metabolite concentration differences and fractional extractions for all substrates were similar between the groups. The absolute NEFA uptakes assessed by [²H₂]palmitate extraction were also similar between the groups. Using direct measurements of metabolic substrate uptake by arterio-venous difference technique, the compensated human failing heart does not appear to have reduced myocardial fatty acid uptake.     

Metabolic signatures of esophageal cancer: NMR-based metabolomics and UHPLC-based focused metabolomics of blood serum

Focused metabolic profiling is a powerful tool for the determination of biomarkers. Here, a more global proton nuclear magnetic resonance (¹H NMR)-based metabolomic approach coupled with a relative simple ultra high performance liquid chromatography (UHPLC)-based focused metabolomic approach was developed and compared to characterize the systemic metabolic disturbances underlying esophageal cancer (EC) and identify possible early biomarkers for clinical prognosis. Serum metabolic profiling of patients with EC (n = 25) and healthy controls (n = 25) was performed by using both ¹H NMR and UHPLC, and metabolite identification was achieved by multivariate statistical analysis. Using orthogonal projection to least squares discriminant analysis (OPLS-DA), we could distinguish EC patients from healthy controls. The predictive power of the model derived from the UHPLC-based focused metabolomics performed better in both sensitivity and specificity than the results from the NMR-based metabolomics, suggesting that the focused metabolomic technique may be of advantage in the future for the determination of biomarkers. Moreover, focused metabolic profiling is highly simple, accurate and specific, and should prove equally valuable in metabolomic research applications. A total of nineteen significantly altered metabolites were identified as the potential disease associated biomarkers. Significant changes in lipid metabolism, amino acid metabolism, glycolysis, ketogenesis, tricarboxylic acid (TCA) cycle and energy metabolism were observed in EC patients compared with the healthy controls. These results demonstrated that metabolic profiling of serum could be useful as a screening tool for early EC diagnosis and prognosis, and might enhance our understanding of the mechanisms involved in the tumor progression.     

Metabolome-Wide Association Study of Neovascular Age-Related Macular Degeneration

Purpose

To determine if plasma metabolic profiles can detect differences between patients with neovascular age-related macular degeneration (NVAMD) and similarly-aged controls.

Methods

Metabolomic analysis using liquid chromatography with Fourier-transform mass spectrometry (LC-FTMS) was performed on plasma samples from 26 NVAMD patients and 19 controls. Data were collected from mass/charge ratio (m/z) 85 to 850 on a Thermo LTQ-FT mass spectrometer, and metabolic features were extracted using an adaptive processing software package. Both non-transformed and log2 transformed data were corrected using Benjamini and Hochberg False Discovery Rate (FDR) to account for multiple testing. Orthogonal Partial Least Squares-Discriminant Analysis was performed to determine metabolic features that distinguished NVAMD patients from controls. Individual m/z features were matched to the Kyoto Encyclopedia of Genes and Genomes database and the Metlin metabolomics database, and metabolic pathways associated with NVAMD were identified using MetScape.

Results

Of the 1680 total m/z features detected by LC-FTMS, 94 unique m/z features were significantly different between NVAMD patients and controls using FDR (q = 0.05). A comparison of these features to those found with log2 transformed data (n = 132, q = 0.2) revealed 40 features in common, reaffirming the involvement of certain metabolites. Such metabolites included di- and tripeptides, covalently modified amino acids, bile acids, and vitamin D-related metabolites. Correlation analysis revealed associations among certain significant features, and pathway analysis demonstrated broader changes in tyrosine metabolism, sulfur amino acid metabolism, and amino acids related to urea metabolism.

Conclusions

These data suggest that metabolomic analysis can identify a panel of individual metabolites that differ between NVAMD cases and controls. Pathway analysis can assess the involvement of certain metabolic pathways, such as tyrosine and urea metabolism, and can provide further insight into the pathophysiology of AMD.     

t-10, c-12 CLA Dietary Supplementation Inhibits Atherosclerotic Lesion Development Despite Adverse Cardiovascular and Hepatic Metabolic Marker Profiles

Animal and human studies have indicated that fatty acids such as the conjugated linoleic acids (CLA) found in milk could potentially alter the risk of developing metabolic disorders including diabetes and cardiovascular disease (CVD). Using susceptible rodent models (apoE^−/− and LDLr^−/− mice) we investigated the interrelationship between mouse strain, dietary conjugated linoleic acids and metabolic markers of CVD. Despite an adverse metabolic risk profile, atherosclerosis (measured directly by lesion area), was significantly reduced with t-10, c-12 CLA and mixed isomer CLA (Mix) supplementation in both apoE^−/− (p<0.05, n = 11) and LDLr^−/− mice (p<0.01, n = 10). Principal component analysis was utilized to delineate the influence of multiple plasma and tissue metabolites on the development of atherosclerosis. Group clustering by dietary supplementation was evident, with the t-10, c-12 CLA supplemented animals having distinct patterns, suggestive of hepatic insulin resistance, regardless of mouse strain. The effect of CLA supplementation on hepatic lipid and fatty acid composition was explored in the LDLr^−/− strain. Dietary supplementation with t-10, c-12 CLA significantly increased liver weight (p<0.05, n = 10), triglyceride (p<0.01, n = 10) and cholesterol ester content (p<0.01, n = 10). Furthermore, t-10, c-12 CLA also increased the ratio of 18∶1 to 18∶0 fatty acid in the liver suggesting an increase in the activity of stearoyl-CoA desaturase. Changes in plasma adiponectin and liver weight with t-10, c-12 CLA supplementation were evident within 3 weeks of initiation of the diet. These observations provide evidence that the individual CLA isomers have divergent mechanisms of action and that t-10, c-12 CLA rapidly changes plasma and liver markers of metabolic syndrome, despite evidence of reduction in atherosclerosis.     

Increased Erythrocytes By-Products of Arginine Catabolism Are Associated with Hyperglycemia and Could Be Involved in the Pathogenesis of Type 2 Diabetes Mellitus

Diabetes mellitus (DM) is a worldwide disease characterized by metabolic disturbances, frequently associated with high risk of atherosclerosis and renal and nervous system damage. Here, we assessed whether metabolites reflecting oxidative redox state, arginine and nitric oxide metabolism, are differentially distributed between serum and red blood cells (RBC), and whether significant metabolism of arginine exists in RBC. In 90 patients with type 2 DM without regular treatment for diabetes and 90 healthy controls, paired by age and gender, we measured serum and RBC levels of malondialdehyde (MDA), nitrites, ornithine, citrulline, and urea. In isolated RBC, metabolism of L-[¹⁴C]-arginine was also determined. In both groups, nitrites were equally distributed in serum and RBC; citrulline predominated in serum, whereas urea, arginine, and ornithine were found mainly in RBC. DM patients showed hyperglycemia and increased blood HbA_1C, and increased levels of these metabolites, except for arginine, significantly correlating with blood glucose levels. RBC were observed to be capable of catabolizing arginine to ornithine, citrulline and urea, which was increased in RBC from DM patients, and correlated with an increased affinity for arginine in the activities of putative RBC arginase (Km = 0.23±0.06 vs. 0.50±0.13 mM, in controls) and nitric oxide synthase (Km = 0.28±0.06 vs. 0.43±0.09 mM, in controls). In conclusion, our results suggest that DM alters metabolite distribution between serum and RBC, demonstrating that RBC regulate serum levels of metabolites which affect nitrogen metabolism, not only by transporting them but also by metabolizing amino acids such as arginine. Moreover, we confirmed that urea can be produced also by human RBC besides hepatocytes, being much more evident in RBC from patients with type 2 DM. These events are probably involved in the specific physiopathology of this disease, i.e., endothelial damage and dysfunction.     

The Urinary Cytokine/Chemokine Signature of Renal Hyperfiltration in Adolescents with Type 1 Diabetes

Objective

Urinary cytokine/chemokine levels are elevated in adults with type 1 diabetes (T1D) exhibiting renal hyperfiltration. Whether this observation extends to adolescents with T1D remains unknown. Our first objective was to determine the relationship between hyperfiltration and urinary cytokines/chemokines in normotensive, normoalbuminuric adolescents with T1D using GFR_cystatin. Our second aim was to determine the relationship between urine and plasma levels of inflammatory biomarkers, to clarify the origin of these factors.

Methods

Urine and serum cytokines/chemokines (Luminex platform) and GFR_cystatin were measured in normofiltering (n = 111, T1D-N, GFR<135 ml/min/1.73 m²) and hyperfiltering (n = 31, T1D-H, GFR≥135 ml/min/1.73 m²) adolescents with T1D (ages 10–16), and in age and sex matched healthy control subjects (HC, n = 59).

Results

We noted significant step-wise increases in urinary cytokine/chemokine excretion according to filtration status with highest levels in T1D-H, with parallel trends in serum analyte concentrations. After adjusting for serum glucose at the time of sampling, differences in urinary cytokine excretion were not statistically significant. Only serum IL-2 significantly differed between HC and T1D (p = 0.0076).

Conclusions

Hyperfiltration is associated with increased urinary cytokine/chemokine excretion in T1D adolescents, and parallel trends in serum cytokine concentration. The GFR-associated trends in cytokine excretion may be driven by the effects of ambient hyperglycemia. The relationship between hyperfiltration, glycemia, and variations in serum and urine cytokine expression and their impact on future renal and systemic vascular complications requires further study.     

Longer Telomere Length in COPD Patients with α1-Antitrypsin Deficiency Independent of Lung Function

Oxidative stress is involved in the pathogenesis of airway obstruction in α₁-antitrypsin deficient patients. This may result in a shortening of telomere length, resulting in cellular senescence. To test whether telomere length differs in α₁-antitrypsin deficient patients compared with controls, we measured telomere length in DNA from peripheral blood cells of 217 α₁-antitrypsin deficient patients and 217 control COPD patients. We also tested for differences in telomere length between DNA from blood and DNA from lung tissue in a subset of 51 controls. We found that telomere length in the blood was significantly longer in α₁-antitrypsin deficient COPD patients compared with control COPD patients (p = 1×10⁻²⁹). Telomere length was not related to lung function in α₁-antitrypsin deficient patients (p = 0.3122) or in COPD controls (p = 0.1430). Although mean telomere length was significantly shorter in the blood when compared with the lungs (p = 0.0078), telomere length was correlated between the two tissue types (p = 0.0122). Our results indicate that telomere length is better preserved in α₁-antitrypsin deficient COPD patients than in non-deficient patients. In addition, measurement of telomere length in the blood may be a suitable surrogate for measurement in the lung.     

Metabolite Profiling Identifies Candidate Markers Reflecting the Clinical Adaptations Associated with Roux-en-Y Gastric Bypass Surgery

Background

Roux-en-Y gastric bypass (RYGB) surgery is associated with weight loss, improved insulin sensitivity and glucose homeostasis, and a reduction in co-morbidities such as diabetes and coronary heart disease. To generate further insight into the numerous metabolic adaptations associated with RYGB surgery, we profiled serum metabolites before and after gastric bypass surgery and integrated metabolite changes with clinical data.

Methodology and Principal Findings

Serum metabolites were detected by gas and liquid chromatography-coupled mass spectrometry before, and 3 and 6 months after RYGB in morbidly obese female subjects (n = 14; BMI = 46.2±1.7). Subjects showed decreases in weight-related parameters and improvements in insulin sensitivity post surgery. The abundance of 48% (83 of 172) of the measured metabolites changed significantly within the first 3 months post RYGB (p<0.05), including sphingosines, unsaturated fatty acids, and branched chain amino acids. Dividing subjects into obese (n = 9) and obese/diabetic (n = 5) groups identified 8 metabolites that differed consistently at all time points and whose serum levels changed following RYGB: asparagine, lysophosphatidylcholine (C18:2), nervonic (C24:1) acid, p-Cresol sulfate, lactate, lycopene, glucose, and mannose. Changes in the aforementioned metabolites were integrated with clinical data for body mass index (BMI) and estimates for insulin resistance (HOMA-IR). Of these, nervonic acid was significantly and negatively correlated with HOMA-IR (p = 0.001, R = −0.55).

Conclusions

Global metabolite profiling in morbidly obese subjects after RYGB has provided new information regarding the considerable metabolic alterations associated with this surgical procedure. Integrating clinical measurements with metabolomics data is capable of identifying markers that reflect the metabolic adaptations following RYGB.     

Distinct Patterns of IgG and IgA against Food and Microbial Antigens in Serum and Feces of Patients with Inflammatory Bowel Diseases

Background

Inflammatory bowel disease (IBD) is associated with a defective intestinal barrier and enhanced adaptive immune responses against commensal microbiota. Immune responses against food antigens in IBD patients remain poorly defined.

Methods

IgG and IgA specific for food and microfloral antigens (wheat and milk extracts; purified ovalbumin; Escherichia coli and Bacteroides fragilis lysates; mannan from Saccharomyces cerevisiae) were analyzed by ELISA in the serum and feces of patients with Crohn''s disease (CD; n = 52 for serum and n = 20 for feces), ulcerative colitis (UC; n = 29; n = 17), acute gastroenteritis/colitis (AGE; n = 12; n = 9) as well as non-inflammatory controls (n = 61; n = 39).

Results

Serum anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-B. fragilis IgG and IgA levels were increased in CD patients whereas antibody (Ab) levels against E. coli and food antigens were not significantly different within the patient groups and controls. Subgroup analysis revealed that CD patients with severe diseases defined by stricturing and penetrating lesions have slightly higher anti-food and anti-microbial IgA levels whereas CD and UC patients with arthropathy have decreased anti-food IgG levels. Treatment with anti-TNF-α Abs in CD patients was associated with significantly decreased ASCA IgG and IgA and anti-E. coli IgG. In the feces specific IgG levels against all antigens were higher in CD and AGE patients while specific IgA levels were higher in non-IBD patients. Anti-food IgG and IgA levels did not correlate with food intolerance.

Summary

In contrast to anti-microbial Abs, we found only minor changes in serum anti-food Ab levels in specific subgroups of IBD patients. Fecal Ab levels towards microbial and food antigens show distinct patterns in controls, CD and UC patients.     

Stool Microbiome and Metabolome Differences between Colorectal Cancer Patients and Healthy Adults

In this study we used stool profiling to identify intestinal bacteria and metabolites that are differentially represented in humans with colorectal cancer (CRC) compared to healthy controls to identify how microbial functions may influence CRC development. Stool samples were collected from healthy adults (n = 10) and colorectal cancer patients (n = 11) prior to colon resection surgery at the University of Colorado Health-Poudre Valley Hospital in Fort Collins, CO. The V4 region of the 16s rRNA gene was pyrosequenced and both short chain fatty acids and global stool metabolites were extracted and analyzed utilizing Gas Chromatography-Mass Spectrometry (GC-MS). There were no significant differences in the overall microbial community structure associated with the disease state, but several bacterial genera, particularly butyrate-producing species, were under-represented in the CRC samples, while a mucin-degrading species, Akkermansia muciniphila, was about 4-fold higher in CRC (p<0.01). Proportionately higher amounts of butyrate were seen in stool of healthy individuals while relative concentrations of acetate were higher in stools of CRC patients. GC-MS profiling revealed higher concentrations of amino acids in stool samples from CRC patients and higher poly and monounsaturated fatty acids and ursodeoxycholic acid, a conjugated bile acid in stool samples from healthy adults (p<0.01). Correlative analysis between the combined datasets revealed some potential relationships between stool metabolites and certain bacterial species. These associations could provide insight into microbial functions occurring in a cancer environment and will help direct future mechanistic studies. Using integrated “omics” approaches may prove a useful tool in identifying functional groups of gastrointestinal bacteria and their associated metabolites as novel therapeutic and chemopreventive targets.     

Low Serum 25-Hydroxyvitamin D Concentrations Are Associated with Increased Risk for Melanoma and Unfavourable Prognosis

Background

Low vitamin D status (serum 25(OH)D concentration) is associated with increased incidence and unfavourable outcome of various types of cancer. However, there are limited data on influence of serum 25(OH)D on risk and prognosis of malignant melanoma.

Methods

Basal serum 25(OH)D concentrations were retrospectively analyzed in a cohort of melanoma patients (n = 324) and healthy controls (n = 141). We tested the hypothesis that serum 25(OH)D concentrations are predictive of melanoma risk, thickness of primary melanomas, and overall survival (OS).

Results

Median serum 25(OH)D concentrations were significantly lower (p = 0.004) in melanoma patients (median = 13.6 ng/ml) as compared to controls (median = 15.6 ng/ml). Primary tumors of patients with low serum 25(OH)D concentrations (<10 ng/ml) had significantly (p = 0.006) greater Breslow thickness (median: 1.9 mm) as compared to patients with higher levels (>20 ng/ml; median: 1.00 mm). Patients with 25(OH)D serum concentrations in the lowest quartile had inferior overall survival (median: 80 months) comparing with the highest quartile (median: 195 months; p = 0.049).

Conclusions

Our data support the concept that serum 25(OH)D concentrations are associated with risk and prognosis of melanoma. Whether normalizing serum 25(OH)D concentrations in these patients improves outcomes will require testing in future clinical trials.     

Peptidomic Identification of Serum Peptides Diagnosing Preeclampsia

We sought to identify serological markers capable of diagnosing preeclampsia (PE). We performed serum peptide analysis (liquid chromatography mass spectrometry) of 62 unique samples from 31 PE patients and 31 healthy pregnant controls, with two-thirds used as a training set and the other third as a testing set. Differential serum peptide profiling identified 52 significant serum peptides, and a 19-peptide panel collectively discriminating PE in training sets (n = 21 PE, n = 21 control; specificity = 85.7% and sensitivity = 100%) and testing sets (n = 10 PE, n = 10 control; specificity = 80% and sensitivity = 100%). The panel peptides were derived from 6 different protein precursors: 13 from fibrinogen alpha (FGA), 1 from alpha-1-antitrypsin (A1AT), 1 from apolipoprotein L1 (APO-L1), 1 from inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), 2 from kininogen-1 (KNG1), and 1 from thymosin beta-4 (TMSB4). We concluded that serum peptides can accurately discriminate active PE. Measurement of a 19-peptide panel could be performed quickly and in a quantitative mass spectrometric platform available in clinical laboratories. This serum peptide panel quantification could provide clinical utility in predicting PE or differential diagnosis of PE from confounding chronic hypertension.     

High Levels of Both n-3 and n-6 Long-Chain Polyunsaturated Fatty Acids in Cord Serum Phospholipids Predict Allergy Development

Background

Long-chain polyunsaturated fatty acids (LCPUFAs) reduce T-cell activation and dampen inflammation. They might thereby counteract the neonatal immune activation and hamper normal tolerance development to harmless environmental antigens. We investigated whether fatty acid composition of cord serum phospholipids affects allergy development up to age 13 years.

Methods

From a population-based birth-cohort born in 1996/7 and followed until 13 years of age (n = 794), we selected cases with atopic eczema (n = 37) or respiratory allergy (n = 44), as well as non-allergic non-sensitized controls (n = 48) based on diagnosis at 13 years of age. Cord and maternal sera obtained at delivery from cases and controls were analysed for proportions of saturated, monounsaturated and polyunsaturated fatty acids among serum phospholipids.

Results

The cord serum phospholipids from subject who later developed either respiratory allergy or atopic eczema had significantly higher proportions of 5/8 LCPUFA species, as well as total n-3 LCPUFA, total n-6 LCPUFA and total LCPUFA compared to cord serum phospholipids from controls who did not develop allergy (P<0.001 for all comparisons). Conversely, individuals later developing allergy had lower proportion of the monounsaturated fatty acid 18∶1n-9 as well as total MUFA (p<0.001) among cord serum phospholipids. The risk of respiratory allergy at age 13 increased linearly with the proportion of n-3 LCPUFA (P_trend<0.001), n-6 LCPUFA (P_trend = 0.001), and total LCPUFA (P_trend<0.001) and decreased linearly with the proportions of total MUFA (P_trend = 0.025) in cord serum phospholipids. Furthermore, Kaplan-Meier estimates of allergy development demonstrated that total LCPUFA proportion in cord serum phospholipids was significantly associated with respiratory allergy (P = 0.008) and sensitization (P = 0.002), after control for sex and parental allergy.

Conclusion

A high proportion of long-chain PUFAs among cord serum phospholipids may predispose to allergy development. The mechanism is unknown, but may involve dampening of the physiologic immune activation in infancy needed for proper maturation of the infant''s immune system.