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.

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.

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.

Generation of C5a in the absence of C3: a new complement activation pathway.

Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.

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.

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.

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.

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.

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.

Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss.

Aprotinin has been used widely in surgery as an anti-bleeding agent but is associated with a number of side effects. We report that textilinin-1, a serine protease inhibitor from Pseudonaja textilis venom with sequence relatedness to aprotinin, is a potent but reversible plasmin inhibitor and has a narrower range of protease inhibition compared to aprotinin. Like aprotinin, textilinin-1 at 5 micromol/l gave almost complete inhibition of tissue plasminogen activator-induced fibrinolysis of whole blood clots. The activated partial thromboplastin time for plasma was markedly increased by aprotinin but unaffected by textilinin-1. In a mouse tail-vein bleeding model, intravenous textilinin-1 and aprotinin caused similar decreases in blood loss but time to haemostasis in the textilinin-treated animals was significantly shorter than in aprotinin-treated mice. Based on these data, textilinin-1 merits further investigation as a therapeutic alternative to aprotinin.

A combination of curcumin and ginger extract improves abrasion wound healing in corticosteroid-impaired hairless rat skin.

Hairless rats were topically treated with a combination of 10% curcumin and 3% ginger extract (or with each agent alone) for a 21-day period. Following this, the rats were treated topically with Temovate (corticosteroid) for an additional 15 days. At the end of the treatment period, superficial abrasion wounds were induced in the treated skin. Abrasion wounds healed more slowly in the skin of Temovate-treated rats than in skin of control animals. Healing was more rapid in skin of rats that had been pretreated with either curcumin or ginger extract alone or with the combination of curcumin-ginger extract (along with Temovate) than in the skin of rats treated with Temovate and vehicle alone. Skin samples were obtained at the time of wound closure. Collagen production was increased and matrix metalloproteinase-9 production was decreased in the recently healed skin from rats treated with the botanical preparation relative to rats treated with Temovate plus vehicle. In none of the rats was there any indication of skin irritation during the treatment phase or during wounding and repair. Taken together, these data suggest that a combination of curcumin and ginger extract might provide a novel approach to improving structure and function in skin and, concomitantly, reducing formation of nonhealing wounds in "at-risk" skin.

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.

MDI 301, a nonirritating retinoid, improves abrasion wound healing in damaged/atrophic skin.

MDI 301 is a picolinic acid-substituted ester of 9-cis retinoic acid. It has been shown in the past that MDI 301 increases epidermal thickness, decreases matrix metalloproteinase (MMP) activity, and increases procollagen synthesis in organ-cultured human skin. Unlike all-trans retinoic acid (RA), MDI 301 does not induce expression of proinflammatory cytokines or induce expression of leukocyte adhesion molecules in human skin. In the present study we examined topical MDI 301 treatment for ability to improve the structure and function of skin in three models of skin damage in rodents and for ability to improve abrasion wound healing in these models. MDI 301 was applied daily to the skin of rats treated with the potent corticosteroid, clobetasol propionate, to the skin of diabetic rats (8 weeks posttreatment with streptozotocin) and to the skin of aged (14-16-month-old) rats. In all three models, subsequently induced abrasion wounds healed more rapidly in the retinoid-treated animals than in vehicle-treated controls. Immediately after complete wound closure, tissue from the wound site (as well as from a control site) was put into organ culture and maintained for 3 days. At the end of the incubation period, culture fluids were assessed for soluble type I collagen and for MMPs-2 and -9. In all three models, the level of type I collagen was increased and MMP levels were decreased by MDI 301. In all three models, skin irritation during the retinoid-treatment phase was virtually nonexistent.

Inhibition of retinoic acid-induced skin irritation in calorie-restricted mice.

Mice on a calorie-restricted (CR) diet (total calories restricted to 70% of ad libitum; AL) for periods of time ranging from 3 to 18 months were examined for response to topical treatment with all-trans retinoic acid (RA). Daily application of a 0.1% solution of RA to the shaved skin of UM-HET3 mice on an AL diet produced a severe irritation that was evident by day 4, maximal at day 7-8 and still detectable at day 14. Skin irritation was characterized by redness, dryness, flaking and failure of the hair to grow at the treated site. In CR mice, the same treatment produced little detectable irritation. Animals were sacrificed at the end of the retinoid-treatment period (day 7 or day 14) and skin from these animals was examined histologically. In both AL and CR mice, a similar degree of epidermal hyperplasia was observed. Numerous inflammatory cells (mononuclear cells and granulocytes) were present in the skin of both groups. Occasional S100-positive cells (presumably Langerhans cells) were also observed in the epidermis of skin from both groups. S100-positive cells were also observed in the dermis. When skin from CR and AL mice was incubated in organ culture for 3 days (on day 7 after initiation of RA treatment), similar levels of four different pro-inflammatory cytokines were found in the conditioned medium. Soluble type I collagen levels were also similar. In contrast, the level of matrix metalloproteinase-9 was lower in the conditioned medium of skin from CR mice than in conditioned medium from skin cultures of AL mice. Taken together, these studies suggest that CR may provide a way to mitigate the irritation that normally accompanies RA treatment without compromising the beneficial effects of retinoid use. CR appears to exert a protective effect at the target tissue level rather than by a reduction in pro-inflammatory events, per se.

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.

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.

Benzodiazepine-induced superoxide signals B cell apoptosis: mechanistic insight and potential therapeutic utility.

The properties of a proapoptotic 1,4-benzodiazepine, Bz-423, identified through combinatorial chemistry and phenotype screening are described. Bz-423 rapidly generated superoxide (O(2)(-)) in transformed Ramos B cells. This O(2)(-) response originated from mitochondria prior to mitochondrial transmembrane gradient collapse and opening of the permeability transition pore. Bz-423-induced O(2)(-) functioned as an upstream signal that initiated an apoptotic program characterized by cytochrome c release, mitochondrial depolarization, and caspase activation. Pretreatment of cells with agents that either block the formation of Bz-423-induced O(2)(-) or scavenge free radicals attenuated the death cascade, which demonstrated that cell killing by Bz-423 depends on O(2)(-). Parallels between Ramos cells and germinal center B cells prompted experiments to determine whether Bz-423 had therapeutic activity in vivo. This possibility was tested using the (NZB x NZW)F(1) murine model of lupus, in which the pathologically enhanced survival and expansion of germinal center B cells mediate disease. Administration of Bz-423 for 12 weeks specifically controlled germinal center hyperplasia and reduced the histological evidence of glomerulonephritis. Collectively, these studies define a new structure-function relationship for benzodiazepines and point to a new target and mechanism that could be of value for developing improved drugs to manage systemic lupus erythematosus and related disorders.

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".

Matrix metalloproteinases and matrix metalloproteinase inhibitors in acute lung injury.

The objective of this study was to assess matrix metalloproteinase (MMP) and MMP inhibitor expression in the airspace of patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and to determine the prognostic significance of MMP expression in this patient population. Twenty-eight patients with ALI or ARDS were prospectively enrolled in this study; bronchoalveolar lavage (BAL) fluid obtained from these patients was examined for expression of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-8 (neutrophil collagenase), and MMP-9 (gelatinase B). Levels of MMP inhibitors (ie, tissue inhibitor of metalloproteinases-1 and -2 [TIMP-1 and TIMP-2]) were examined in parallel. Expression of MMPs was correlated with relevant clinical outcomes in patients with ALI/ARDS. In nearly all specimens obtained from patients with ALI/ARDS, there were high levels of MMP-2, MMP-8, MMP-9, and TIMP-1, but in only a small subset of patients (6/28) were there detectable levels of MMP-1 and/or MMP-3. In the patients with elevated MMP-1 and/or MMP-3, the mortality rate was higher (83%) than in the group without detectable levels of these enzymes (32%). Likewise, the overall severity of disease as indicated by Acute Physiology and Chronic Health Evaluation III scores was higher in this group (98 +/- 30) than in the group without detectable MMP-1 or MMP-3 (78 +/- 28). The percentage of individuals in whom lung disease was complicated by multiorgan failure was also higher in the group with detectable MMP-1 and/or MMP-3 (83%) than in the group without (64%), as was the number of organs that failed. In contrast, there was no correlation between MMP-1 and/or MMP-3 expression and impairment in gas exchange, as determined by the ratio of partial pressure of oxygen to fraction of inspired oxygen (Pao(2)/Fio(2)) on the day of BAL sample. Based on these findings, we conclude that elevated MMP-2, MMP-8, and MMP-9 in BAL fluid is a marker of acute lung injury (and, perhaps, a contributor to ALI) but is not necessarily an indicator of a poor outcome. On the other hand, the presence of detectable MMP-1 and/or MMP-3 is an indicator of more ominous disease progression.