Serum Biomarkers of Disease Activity in Longitudinal Assessment of Patients with ANCA-Associated Vasculitis.

OBJECTIVE: Improved biomarkers of current disease activity and prediction of relapse are needed in antineutrophil cytoplasmic antibody-associated vasculitis (AAV). For clinical relevance, biomarkers must perform well longitudinally in patients on treatment and in patients with nonsevere flares. METHODS: Twenty-two proteins were measured in 347 serum samples from 74 patients with AAV enrolled in a clinical trial. Samples were collected at Month 6 after remission induction, then every 3 months until Month 18, or at the time of flare. Associations of protein concentrations with concurrent disease activity and with future flare were analyzed using mixed-effects models, Cox proportional hazards models, and conditional logistic regression. RESULTS: Forty-two patients had flares during the 12-month follow-up period, and 32 remained in remission. Twenty-two patients had severe flares. Six experimental markers (CXCL13, IL-6, IL-8, IL-15, IL-18BP, and matrix metalloproteinase-3 [MMP-3]) and ESR were associated with disease activity using all three methods (P < 0.05, with P < 0.01 in at least one method). A rise in IL-8, IL-15, or IL-18BP was associated temporally with flare. Combining C-reactive protein (CRP), IL-18BP, neutrophil gelatinase-associated lipocalin (NGAL), and sIL-2Rα improved association with active AAV. CXCL13 and MMP-3 were increased during treatment with prednisone, independent of disease activity. Marker concentrations during remission were not predictive of future flare. CONCLUSION: Serum biomarkers of inflammation and tissue damage and repair have been previously shown to be strongly associated with severe active AAV were less strongly associated with active AAV in a longitudinal study that included mild flares and varying treatment. Markers rising contemporaneously with flare or with an improved association in combination merit further study.

Evaluating the Sensitivity and Specificity of Promising Circulating Biomarkers to Diagnose Liver Injury in Humans.

Early diagnosis of drug-induced liver injury (DILI) continues to be a major hurdle during drug development and postmarketing. The objective of this study was to evaluate the diagnostic performance of promising biomarkers of liver injury-glutamate dehydrogenase (GLDH), cytokeratin-18 (K18), caspase-cleaved K18 (ccK18), osteopontin (OPN), macrophage colony-stimulating factor (MCSF), MCSF receptor (MCSFR), and microRNA-122 (miR-122) in comparison to the traditional biomarker alanine aminotransferase (ALT). Biomarkers were evaluated individually and as a multivariate model in a cohort of acetaminophen overdose (n = 175) subjects and were further tested in cohorts of healthy adults (n = 135), patients with liver damage from various causes (n = 104), and patients with damage to the muscle (n = 74), kidney (n = 40), gastrointestinal tract (n = 37), and pancreas (n = 34). In the acetaminophen cohort, a multivariate model with GLDH, K18, and miR-122 was able to detect DILI more accurately than individual biomarkers alone. Furthermore, the three-biomarker model could accurately predict patients with liver injury compared with healthy volunteers or patients with damage to muscle, pancreas, gastrointestinal tract, and kidney. Expression of K18, GLDH, and miR-122 was evaluated using a database of transcriptomic profiles across multiple tissues/organs in humans and rats. K18 mRNA (Krt18) and MiR-122 were highly expressed in liver whereas GLDH mRNA (Glud1) was widely expressed. We performed a comprehensive, comparative performance assessment of 7 promising biomarkers and demonstrated that a 3-biomarker multivariate model can accurately detect liver injury.

Serum glutamate dehydrogenase activity enables early detection of liver injury in subjects with underlying muscle impairments.

Serum activities of alanine and aspartate aminotransferases (ALT and AST) are used as gold standard biomarkers for the diagnosis of hepatocellular injury. Since ALT and AST lack liver specificity, the diagnosis of the onset of hepatocellular injury in patients with underlying muscle impairments is severely limited. Thus, we evaluated the potential of glutamate dehydrogenase (GLDH) as a liver specific alternative biomarker of hepatocellular injury. In our study, serum GLDH in subjects with Duchene muscular dystrophy (DMD) was equivalent to serum GLDH in age matched healthy subjects, while serum ALT was increased 20-fold in DMD subjects. Furthermore, serum GLDH in 131 subjects with variety of muscle impairments was similar to serum GLDH of healthy subjects while serum ALT corelated with serum creatine kinase, a widely accepted biomarker of muscle impairment. In addition, significant elevations of ALT, AST, and CK were observed in a case of a patient with rhabdomyolysis, while serum GLDH stayed within the normal range until the onset of hypoxia-induced liver injury. In a mouse model of DMD (DMDmdx), serum GLDH but not serum ALT clearly correlated with the degree of acetaminophen-induced liver injury. Taken together, our data support the utility of serum GLDH as a liver-specific biomarker of liver injury that has a potential to improve diagnosis of hepatocellular injury in patients with underlying muscle impairments. In drug development, GLDH may have utility as a biomarker of drug induced liver injury in clinical trials of new therapies to treat muscle diseases such as DMD.

The MicroRNA-based Liquid Biopsy Improves Early Assessment of Lethal Acetaminophen Poisoning: A Case Report.

BACKGROUND Acetaminophen overdose is the most common cause of acute liver failure. Nevertheless, new biomarker approaches enabling early prediction of the outcome of the acetaminophen overdose are needed. Recently, using next-generation sequencing analysis of serum from human study participants we uncovered injury-specific signatures of circulating microRNAs (miRNAs) that represented underlying molecular mechanisms of toxicity. This case study is first to show the application of miRNA profiling to assess prognosis of acetaminophen poisoning. CASE REPORT The patient was admitted to the hospital following supra therapeutic acetaminophen ingestion. The patient showed elevated levels of biomarkers of hepatocellular injury alanine aminotransferase, aspartate transaminase, and glutamate dehydrogenase. Even though treatment with N-acetyl cysteine was initiated 24 hours post-ingestion, levels of alanine-aminotransferase and aspartate transaminase peaked at about 40 hours post ingestion of acetaminophen. We analyzed global circulating miRNA levels from 24 consecutive serum samples from this study participant covering the period from admission to time of death. CONCLUSIONS The resulting global miRNA profiles were compared with profiles from study participants with non-lethal acetaminophen poisoning and healthy controls. At the admission, the miRNA profiles of both lethal and non-lethal acetaminophen poisoning showed induction of cellular stress and oxidative damage. Later, the miRNA profiles of the lethal poisoning featured fibrosis and coagulation pathways while profiles of non-lethal cases resembled those of healthy study participants. Although additional confirmatory studies are needed, our case study is first to indicate that global miRNA profiles to be used as liquid biopsies have potential to facilitate the assessment of acetaminophen poisoning.

Evaluation of Potential Serum Biomarkers of Disease Activity in Diverse Forms of Vasculitis.

OBJECTIVE: We evaluated potential circulating biomarkers of disease activity in giant cell arteritis (GCA), Takayasu arteritis (TA), polyarteritis nodosa (PAN), and eosinophilic granulomatosis with polyangiitis (EGPA). METHODS: A panel of 22 serum proteins was tested in patients enrolled in the Vasculitis Clinical Research Consortium Longitudinal Studies of GCA, TA, PAN, or EGPA. Mixed models were used for most analyses. A J48 classification tree method was used to find the most relevant markers to differentiate between active and inactive GCA. RESULTS: Tests were done on 418 samples from 152 patients (60 GCA, 29 TA, 26 PAN, 37 EGPA), during both active vasculitis and remission. In GCA, these showed significant (p < 0.05) differences between disease states: B cell-attracting chemokine 1 (BCA)-1/CXC motif ligand 13 (CXCL13), erythrocyte sedimentation rate (ESR), interferon-γ-induced protein 10/CXC motif chemokine 10, soluble interleukin 2 receptor α (sIL-2Rα), and tissue inhibitor of metalloproteinase-1 (TIMP-1). In EGPA, these showed significant increases during active disease: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF, interleukin (IL)-6, IL-15, and sIL-2Rα. BCA-1/CXCL13 also showed such increases, but only after adjustment for treatment. In PAN, ESR and matrix metalloprotease (MMP)-3 showed significant differences between disease states. Differences in biomarker levels between diseases were significant for 11 markers and were more striking (all p < 0.01) than differences related to disease activity. A combination of lower values of TIMP-1, IL-6, interferon-γ, and MMP-3 correctly classified 87% of samples with inactive GCA. CONCLUSION: We identified novel biomarkers of disease activity in GCA and EGPA. Differences of biomarker levels between diseases, independent of disease activity, were more apparent than differences related to disease activity. Further studies are needed to determine whether these serum proteins have potential for clinical use in distinguishing active disease from remission or in predicting longer-term outcomes.

The association of serum interleukin-6 levels with clinical outcomes in antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: To investigate serum IL-6 (sIL-6) levels during active disease, complete remission (CR), and relapse in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), and to explore the association of changes in sIL-6 with clinical outcomes. METHODS: sIL-6 levels were measured at baseline and longitudinally over 18 months, in 78 patients with AAV enrolled in a randomized controlled trial comparing treatment with either rituximab (RTX) or cyclophosphamide (CYC)/azathioprine (AZA). Outcome variables included baseline clinical features, ANCA specificity, disease activity (active disease versus CR), time to relapse events, B cell repopulation, and ANCA titer increases. RESULTS: At baseline, sIL6 levels were detectable in 81% of patients; 73% (n = 57) of subjects were proteinase 3 (PR3)-ANCA positive, sIL-6 levels were higher in subjects with PR3-ANCAs and positively correlated with their levels (rs = 0.36,p < 0.01), but not with levels of myeloperoxidase (MPO)-ANCA (rs = -0.17,p = 0.47). Higher baseline sIL-6 levels were associated with PR3-ANCA positivity, fever, pulmonary nodules/cavities, conductive deafness, and absence of urinary red blood cell casts (p < 0.05). Baseline sIL6 levels did not predict CR at month 6 (p = 0.71), and the median sIL-6 level declined from baseline with induction therapy, regardless of CR achievement. An increase in sIL-6 during CR was a predictor for subsequent severe relapse in RTX-treated patients (hazard ratio (HR):7.24,p = 0.01), but not in CYC/AZA-treated patients (HR:0.62,p = 0.50). In contrast, a sIL-6 increase did not predict B cell repopulation or ANCA titer increase in either treatment arm (p > 0.05). CONCLUSION: At baseline, sIL-6 concentrations correlate with PR3-ANCA titers and are associated with specific clinical manifestations of AAV. Baseline sIL6 concentrations do not predict CR at 6 months, but the increase in sIL-6 concentrations during CR is associated with subsequent severe relapse among RTX-treated patients. Further investigation into the mechanistic role of IL6 in AAV might lead to identifying this pathway as a potential therapeutic target in this disease.

Brief Report: Circulating Cytokine Profiles and Antineutrophil Cytoplasmic Antibody Specificity in Patients With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: To evaluate circulating cytokine profiles in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV), classified by antineutrophil cytoplasmic antibody (ANCA) specificity (proteinase 3 ANCA [PR3-ANCA] versus myeloperoxidase ANCA [MPO-ANCA]) or by clinical diagnosis (granulomatosis with polyangiitis [GPA] versus microscopic polyangiitis [MPA]). METHODS: A panel of 29 cytokines was tested in 186 patients with active AAV at inclusion into the Rituximab in AAV trial. Cytokine concentrations were compared between groups within each classification system. Multivariable analyses adjusted for age, sex, and renal insufficiency were performed, with each biomarker as a dependent variable and ANCA specificity and clinical diagnosis as explanatory variables of interest. RESULTS: Levels of 9 circulating cytokines (interleukin-6 [IL-6], granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-15, IL-18, CXCL8/IL-8, CCL-17/thymus and activation-regulated chemokine [TARC], IL-18 binding protein [IL-18 BP], soluble IL-2 receptor α [sIL-2Rα], and nerve growth factor ß [NGFß]) were significantly higher in PR3-AAV than MPO-AAV, 4 cytokines (sIL6R, soluble tumor necrosis factor receptor type II [sTNFRII], neutrophil gelatinase-associated lipocalin [NGAL], and soluble intercellular adhesion molecule 1 [sICAM-1]) were higher in MPO-AAV than in PR3-AAV, 6 cytokines (IL-6, GM-CSF, IL-15, IL-18, sIL-2Rα, and NGFß) were higher in GPA than in MPA, and 3 cytokines (osteopontin, sTNFRII, and NGAL) were higher in MPA than in GPA (all P < 0.05). For nearly all cytokines, the difference between PR3-AAV and MPO-AAV was larger than that between GPA and MPA. The multivariate analysis showed that 8 cytokines (IL-15, IL-8, IL-18 BP, NGF-ß, sICAM-1, TARC, osteopontin, and kidney injury molecule 1 (P < 0.05) distinguished patients with AAV better (lower P values and larger effect sizes) when grouped by ANCA specificity than by clinical diagnosis. CONCLUSION: Distinct cytokine profiles were identified for PR3-AAV versus MPO-AAV and for GPA versus MPA. Differences in these circulating immune mediators are more strongly associated with ANCA specificity than with clinical diagnosis, suggesting that heterogeneity in the AAV subtypes extends beyond clinical phenotypes.

Assessment of serum bile acid profiles as biomarkers of liver injury and liver disease in humans.

To assess the potential of individual bile acids (IBA) and their profiles as mechanistic biomarkers of liver injury for humans in real world situations, we interrogated samples collected under minimum controlled conditions (ie subjects were not fasted). Total bile acids (TBA) have been considered to be biomarkers of liver injury for decades, and more recently, monitoring of IBA has been proposed for differentiation of variety of etiologies of liver injury. We established a LC-MS/MS methodology to analyze nine IBA, generated reference ranges, and examined effects of age, gender, and ethnicity for each IBA. Furthermore, we evaluated the ability of IBA and their profiles to detect hepatic injury in subjects with a broad range of liver impairments. To date, our study utilized the largest total cohort of samples (N = 645) that were divided into 2 groups, healthy or liver impaired, to evaluate IBA as biomarkers. The TBA serum levels in the Asian ethnic group trended higher when compared to other ethnic groups, and the serum concentrations of IBA, such as glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), chenodeoxycholic acid (CDCA), and taurochenoxycholic acid (TCDCA) were significantly increased. To our knowledge, this report is the first to describe ethnic differences in serum concentrations of IBAs. In patients with hepatic impairments, with the exception of deoxycholic acid (DCA), the concentrations of IBAs were significantly elevated when compared with healthy subjects. The conjugated bile acids displayed greater differences between healthy subjects and subjects with hepatic impairments than non-conjugated bile acids. Furthermore, the subjects with hepatic impairments exhibited distinct profiles (signatures) of IBAs that clustered subjects according the nature of their liver impairments. Although additional studies are needed, our data suggested that the analysis of IBA has the potential to become useful for differentiation of various forms of liver injury.

Serum microRNA signatures as "liquid biopsies" for interrogating hepatotoxic mechanisms and liver pathogenesis in human.

MicroRNAs (miRNAs) released into the peripheral circulation upon cellular injury have shown a promise as a new class of tissue-specific biomarkers. We were first to demonstrate that next-generation sequencing analysis of serum from human subjects with acetaminophen-induced liver injury revealed a specific signature of circulating miRNAs. We consequently hypothesized that different types of hepatic liver impairments might feature distinct signatures of circulating miRNAs and that this approach might be useful as minimally invasive diagnostic "liquid biopsies" enabling the interrogation of underlying molecular mechanisms of injury in distant tissues. Therefore we examined serum circulating miRNAs in a total of 72 serum samples from a group of 53 subjects that included patients with accidental acetaminophen overdose, hepatitis B infection, liver cirrhosis and type 2 diabetes as well as gender- and age-matched healthy subjects with no evidence of liver disease. The miRNA signatures were identified using next-generation sequencing that provided analysis for the whole miRNome, including miRNA isoforms. Compared to the healthy subjects, a total of 179 miRNAs showed altered serum levels across the diseased subjects. Although many subjects have elevated alanine aminotransferase suggesting liver impairments, we identified distinct miRNA signatures for different impairments with minimum overlap. Furthermore, the bioinformatics analysis of miRNA signatures revealed relevant molecular pathways associated with the mechanisms of toxicity and or pathogenesis of disease. Interestingly, the high proportion of miRNA isoforms present in the respective signatures indicated a new level of complexity in cellular response to stress or disease. Our study demonstrates for the first time that signatures of circulating miRNAs show specificity for liver injury phenotypes and, once validated, might become useful for diagnosis of organ pathologies as "liquid biopsies".

Monoclonal antibodies specific for oncofetal antigen--immature laminin receptor protein: Effects on tumor growth and spread in two murine models.

The oncofetal antigen - immature laminin receptor protein (OFA/iLRP) has been linked to metastatic tumor spread for several years. The present study, in which 2 highly-specific, high-affinity OFA/iLRP-reactive mouse monoclonal antibodies were examined for ability to suppress tumor cell growth and metastatic spread in the A20 B-cell leukemia model and the B16 melanoma model, provides the first direct evidence that targeting OFA/iLRP with exogenous antibodies can have therapeutic benefit. While the antibodies were modestly effective at preventing tumor growth at the primary injection site, both antibodies strongly suppressed end-organ tumor formation following intravenous tumor cell injection. Capacity of anti-OFA/iLRP antibodies to suppress tumor spread through the blood in the leukemia model suggests their use as a therapy for individuals with leukemic disease (either for patients in remission or even as part of an induction therapy). The results also suggest use against metastatic spread with solid tumors.

PF-1355, a mechanism-based myeloperoxidase inhibitor, prevents immune complex vasculitis and anti-glomerular basement membrane glomerulonephritis.

Small vessel vasculitis is a life-threatening condition and patients typically present with renal and pulmonary injury. Disease pathogenesis is associated with neutrophil accumulation, activation, and oxidative damage, the latter being driven in large part by myeloperoxidase (MPO), which generates hypochlorous acid among other oxidants. MPO has been associated with vasculitis, disseminated vascular inflammation typically involving pulmonary and renal microvasculature and often resulting in critical consequences. MPO contributes to vascular injury by 1) catabolizing nitric oxide, impairing vasomotor function; 2) causing oxidative damage to lipoproteins and endothelial cells, leading to atherosclerosis; and 3) stimulating formation of neutrophil extracellular traps, resulting in vessel occlusion and thrombosis. Here we report a selective 2-thiouracil mechanism-based MPO inhibitor (PF-1355 [2-(6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide) and demonstrate that MPO is a critical mediator of vasculitis in mouse disease models. A pharmacokinetic/pharmacodynamic response model of PF-1355 exposure in relation with MPO activity was derived from mouse peritonitis. The contribution of MPO activity to vasculitis was then examined in an immune complex model of pulmonary disease. Oral administration of PF-1355 reduced plasma MPO activity, vascular edema, neutrophil recruitment, and elevated circulating cytokines. In a model of anti-glomerular basement membrane disease, formerly known as Goodpasture disease, albuminuria and chronic renal dysfunction were completely suppressed by PF-1355 treatment. This study shows that MPO activity is critical in driving immune complex vasculitis and provides confidence in testing the hypothesis that MPO inhibition will provide benefit in treating human vasculitic diseases.

Application of high-throughput sequencing to circulating microRNAs reveals novel biomarkers for drug-induced liver injury.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and the major reason for withdrawal of drugs from the market. Preclinical evaluation of drug candidates has failed to detect about 40% of potentially hepatotoxic compounds in humans. At the onset of liver injury in humans, currently used biomarkers have difficulty differentiating severe DILI from mild, and/or predict the outcome of injury for individual subjects. Therefore, new biomarker approaches for predicting and diagnosing DILI in humans are urgently needed. Recently, circulating microRNAs (miRNAs) such as miR-122 and miR-192 have emerged as promising biomarkers of liver injury in preclinical species and in DILI patients. In this study, we focused on examining global circulating miRNA profiles in serum samples from subjects with liver injury caused by accidental acetaminophen (APAP) overdose. Upon applying next generation high-throughput sequencing of small RNA libraries, we identified 36 miRNAs, including 3 novel miRNA-like small nuclear RNAs, which were enriched in the serum of APAP overdosed subjects. The set comprised miRNAs that are functionally associated with liver-specific biological processes and relevant to APAP toxic mechanisms. Although more patients need to be investigated, our study suggests that profiles of circulating miRNAs in human serum might provide additional biomarker candidates and possibly mechanistic information relevant to liver injury.

Identification of a protein signature in renal allograft rejection.

PURPOSE: Serum creatinine functions as a poor surrogate marker of renal allograft dysfunction and long-term graft survival. By measuring multiple proteins simultaneously in the serum of transplant patients, we can identify unique protein signatures of graft dysfunction. EXPERIMENTAL DESIGN: We utilized training and validation cohorts composed of healthy and volunteer subjects, stable renal transplant patients, and renal transplant patients experiencing acute allograft rejection. Utilizing our antibody microarray, we measured 108 proteins simultaneously in these groups. RESULTS: Using Mann-Whitney tests with Bonferroni correction, we identified ten serum proteins from 19 renal transplant patients with stable renal function, which are differentially expressed, compared to healthy control subjects. In addition, we identified 17 proteins that differentiate rejecting renal transplant recipients from stable renal transplant. Validation cohorts substantiated these findings. CONCLUSION AND CLINICAL RELEVANCE: Our preliminary results support that a specific pattern of protein expression or "protein signature" may be able to differentiate between stable transplant patients from those with rejection. Future studies will focus on other etiologies of renal allograft dysfunction and the effect of treatment on protein expression and long-term outcome.

Performance of Biopsy Needle With Therapeutic Injection System to Prevent Bleeding Complications.

Renal disease is epidemic in the United States with approximately 8 × 106 people having chronic kidney disease. Renal biopsies are widely used to provide essential diagnostic information to physicians. However, the risk of bleeding complications possibly leading to life-threatening situations results in the contra-indication of biopsy in certain patient populations. Safer renal biopsies will allow more accurate diagnosis and better management of this epidemic health problem. We report the preclinical testing of a novel biopsy device called the therapeutic injection system (TIS). The device introduces a third stage to the standard two-stage side-cut percutaneous biopsy process. The third stage is designed to reduce bleeding complications by injecting a hemostatic plug at the time of biopsy. Laboratory evaluation and preliminary in vivo animal testing using an anticoagulated porcine model of the TIS and Bard Monopty® (Bard Medical, Covington, GA) control device were performed. The hemostatic material Gelfoam® (Pfizer, Brussels, Belgium) was selected as the active material comprising the hemostatic plugs. The performance of two composite plugs, one composed of polyvinyl alcohol (PVA) combined in 2:1 and 12:1 ratios with the hemostatic material, and one plug composed of 100[Formula: see text] hemostatic material were tested. Stroke sequence and hemostatic plug deployment were verified by sequential firing of the TIS biopsy needle into clear gelatin and ex vivo bovine kidney specimens. In vivo trials with porcine specimens revealed a significant reduction in blood loss (8.1 [Formula: see text] 3.9 ml, control versus 1.9 [Formula: see text] 1.6 ml, 12:1 PVA/hemostatic, TIS, [Formula: see text] = 0.01, [Formula: see text] = 6). The 100[Formula: see text] hemostatic plug showed a substantial and immediate reduction in blood loss (9.2 ml, control versus 0.0 ml, TIS, [Formula: see text] = 1). The prototype device was shown to work repeatedly and reliably in laboratory trials. Initial results show promise in this approach to control post biopsy bleeding. This solution maintains the simplicity and directness of the percutaneous approach, while not significantly changing the standard percutaneous biopsy procedure.

Assessment of emerging biomarkers of liver injury in human subjects.

Hepatotoxicity remains a major challenge in drug development. Although alanine aminotransferase (ALT) remains the gold standard biomarker of liver injury, alternative biomarker strategies to better predict the potential for severe drug-induced liver injury (DILI) are essential. In this study, we evaluated the utility of glutamate dehydrogenase (GLDH), purine nucleoside phosphorylase (PNP), malate dehydrogenase (MDH), and paraxonase 1 (PON1) as indicators of liver injury in cohorts of human subjects, including healthy subjects across age and gender, subjects with a variety of liver impairments, and several cases of acetaminophen poisoning. In the healthy subjects, levels of GLDH and MDH were not affected by age or gender. Reference ranges for GLDH and MDH in healthy subjects were 1-10 and 79-176U/L, respectively. In contrast, the levels of PON1 and PNP were not consistent across cohorts of healthy subjects. Furthermore, GLDH and MDH had a strong correlation with elevated ALT levels and possessed a high predictive power for liver injury, as determined by ROC analysis. In contrast, PON1 and PNP did not detect liver injury in our study. Finally, evaluation of patients with acetaminophen-induced liver injury provided evidence that both GLDH and MDH might have utility as biomarkers of DILI in humans. This study is the first to evaluate GLDH, MDH, PON1, and PNP in a large number of human subjects and, and it provides an impetus for prospective clinical studies to fully evaluate the diagnostic value of GLDH and MDH for detection of liver injury.

Serum proteins reflecting inflammation, injury and repair as biomarkers of disease activity in ANCA-associated vasculitis.

OBJECTIVE: To identify circulating proteins that distinguish between active anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and remission in a manner complementary to markers of systemic inflammation. METHODS: Twenty-eight serum proteins representing diverse aspects of the biology of AAV were measured before and 6 months after treatment in a large clinical trial of AAV. Subjects (n=186) enrolled in the Rituximab in ANCA-Associated Vasculitis (RAVE) trial were studied. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels were available for comparison. The primary outcome was the ability of markers to distinguish severe AAV (Birmingham Vasculitis Activity Score for Wegener's granulomatosis (BVAS/WG)≥3 at screening) from remission (BVAS/WG=0 at month 6), using areas under receiver operating characteristic (ROC) curve (AUC). RESULTS: All subjects had severe active vasculitis (median BVAS/WG=8) at screening. In the 137 subjects in remission at month 6, 24 of the 28 markers showed significant declines. ROC analysis indicated that levels of CXCL13 (BCA-1), matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) best discriminated active AAV from remission (AUC>0.8) and from healthy controls (AUC>0.9). Correlations among these markers and with ESR or CRP were low. CONCLUSIONS: Many markers are elevated in severe active AAV and decline with treatment, but CXCL13, MMP-3 and TIMP-1 distinguish active AAV from remission better than the other markers studied, including ESR and CRP. These proteins are particularly promising candidates for future studies to address unmet needs in the assessment of patients with AAV.

Maternal micronutrient supplementation suppresses T cell chemokine receptor expression and function in F1 mice.

Prenatal environmental exposures play a critical role in determining late-life chronic disease susceptibility. However, the mechanisms linking the in utero environment and disease development in the offspring are poorly understood. Recent investigations have confirmed a central pathogenic role of T cell chemokine receptors, particularly C-C chemokine receptor (CCR) 2 and CCR5, in chronic inflammatory conditions. This study was designed to determine the effect of a synthetic prenatal micronutrient supplementation (MS) diet rich in methionine pathway metabolites on the T cell chemokine system in F1 C57Bl/6 mice. Female mice were fed either an MS or control diet 3 wk prior to mating, during pregnancy, and lactation. At 4 wk of age, F1 mice were killed for experiments or were fed the standard NIH-31 diet and allowed to age. Food consumption, maternal weight gain, and litter size were similar in dams fed the control and MS diets. However, the F1 offspring of dams fed the MS diet were smaller in size (P < 0.001). T cells from the MS F1 offspring had global hypermethylation compared with control F1 offspring (P < 0.005), corresponding to lower T cell chemokine receptor expression [CCR2 (P < 0.001), CCR5 (P < 0.001), and C-x-C chemokine receptor 3 (P < 0.01)] and cytokine expression [TNFα (P < 0.05), IL-2 (P < 0.001), and IL-4 (P < 0.01)]. Reduced T cell chemokine receptor gene expression in MS F1 mice was associated with decreased chemotaxis in vitro to C-C chemokine ligand (CCL) 2 and C-X-C chemokine ligand 10 (P < 0.01) and in vivo to CCL2 (P < 0.01). Taken together, the results suggest that epigenetic alteration through prenatal diet manipulation reduces the response to proinflammatory signals in mice.

A multi-component herbal preparation (PADMA 28) improves structure/function of corticosteroid-treated skin, leading to improved wound healing of subsequently induced abrasion wounds in rats.

PADMA 28 is a multi-component herbal mixture formulated according to an ancient Tibetan recipe. PADMA 28 is known to stimulate collagen production and reduced levels of collagen-degrading matrix metalloproteinases (MMPs). The goal of the present study was to determine whether topical treatment of rat skin with PADMA 28 would improve skin structure/function, and whether subsequently induced abrasion wounds would heal more rapidly in skin that had been pretreated with PADMA 28. Hairless rats were exposed to a potent topical corticosteroid (Temovate) in combination with either DMSO alone or with PADMA 28 given topically. At the end of the treatment period, superficial wounds were created in the skin, and time to wound closure was assessed. Collagen production and matrix-degrading MMPs were assessed. Abrasion wounds in skin that had been pretreated with PADMA 28 healed more rapidly than did wounds in Temovate plus DMSO-treated skin. Under conditions in which improved wound healing was observed, there was an increased collagen production and decreased MMP expression, but no significant epidermal hyperplasia and no evidence of skin irritation. The ability to stimulate collagen production and inhibit collagen-degrading enzymes in skin and facilitate more rapid wound closure without irritation should provide a rationale for development of the herbal preparation as a "skin-repair" agent.

Hemostatic properties of a venomic protein in rat organ trauma.

Previous in vitro work characterized the protease Q8009 isolated from the venom of the Australian brown snake Pseudonaja textilis textilis with Factor Xa-like activity and hemostatic properties. The purpose of the work described here characterizes the in vivo hemostatic properties in a rat model of parenchymatous organ injury. The key parameters of activity included reduction in time-to-hemostasis and total volume of blood loss in spleen, liver and kidney wound models in rats. The surgical protocols involved exposure of the organs via a midline abdominal laparotomy. Using a clean metal template with 6, 6.5, 9 mm holes for spleen, liver and kidney, respectively, a predetermined volume of the organ was gently extruded through the template hole and excised with a razor blade. About 50 to 75 microL of collagen matrix with the different test solutions was applied to the wounds. Blood was collected and at the end of the procedure animals were humanely sacrificed with an anesthetic overdose. Determination of blood was performed using the hematin assay using a standard curve. Blood loss per minute and total blood loss were calculated. Results from the studies demonstrated that the application of Q8009 and collagen matrix to surgical wounds significantly reduced the total amount of blood loss and the time-to-hemostasis. In the spleen wound model, Q8009 at 100, 250 and 1000 microg/ml significantly reduced (p<0.001) the total volume of blood lost relative to thrombin and reduced the time-to-hemostasis by 25-50%, as compared to 7% by thrombin. In the liver wound model, Q8009 at 250 and 1000 microg/ml significantly reduced (p<0.001) the total volume of blood lost relative to thrombin and reduced the time-to-hemostasis from 10.5 min by thrombin to 5.6 min with Q8009. In the kidney wound model, Q8009 at 250 microg/ml significantly reduced (p<0.05) the total volume of blood lost and reduced the time-to-hemostasis by 25% when compared to thrombin. The hemostasis levels were consistent with previous findings in skin wound rat models where Q8009 consistently reduced the total volume of blood lost and shortened time-to-hemostasis. Application of Q8009 plus collagen matrix significantly reduced the volume of total blood loss and time-to-hemostasis in rat surgical organ wound models induced bleeding, as compared to a commercially available hemostat device. The protein Q8009 has greater capacity to reduce blood loss and shorten time-to-hemostasis; highly desirable properties where rapid hemostasis is needed in surgical wounds in parenchymatous organs.