Performance evaluation of the high-throughput quantitative Alinity m BK virus assay.

BK virus (BKV) infection or reactivation in immunocompromised individuals can lead to adverse health consequences including BKV-associated nephropathy (BKVAN) in kidney transplant patients and BKV-associated hemorrhagic cystitis (BKV-HC) in allogeneic hematopoietic stem cell transplant recipients. Monitoring BKV viral load plays an important role in post-transplant patient care. This study evaluates the performance of the Alinity m BKV Investigational Use Only (IUO) assay. The linearity of the Alinity m BKV IUO assay had a correlation coefficient of 1.000 and precision of SD ≤ 0.25 Log IU/mL for all panel members tested (2.0-7.3 Log IU/mL). Detection rate at 50 IU/mL was 100%. Clinical plasma specimens tested comparing Alinity m BKV IUO to ELITech MGB Alert BKV lab-developed test (LDT) on the Abbott m2000 platform using specimen extraction protocols for DNA or total nucleic acid (TNA) resulted in coefficient of correlation of 0.900 and 0.963, respectively, and mean bias of 0.03 and -0.54 Log IU/mL, respectively. Alinity m BKV IUO compared with Altona RealStar BKV and Roche cobas BKV assays demonstrated coefficient of correlation of 0.941 and 0.980, respectively, and mean bias of -0.47 and -0.31 Log IU/mL, respectively. Urine specimens tested on Alintiy m BKV IUO and ELITech BKV LDT using TNA specimen extraction had a coefficient of correlation of 0.917 and mean bias of 0.29 Log IU/mL. The Alinity m BKV IUO assay was performed with high precision across the dynamic range and correlated well with other available BKV assays. IMPORTANCE: BK virus (BKV) in transplant patients can lead to adverse health consequences. Viral load monitoring is important in post-transplant patient care. This study evaluates the Alinity m BKV assay with currently available assays.

Effect of Metarhizium anisopliae (MetA1) on growth enhancement and antioxidative defense mechanism against Rhizoctonia root rot in okra.

Rhizoctonia solani is an important necrotrophic pathogenic fungus that causes okra root disease and results in severe yield reduction. Many biocontrol agents are being studied with the intent of improving plant growth and defense systems and reducing crop loss by preventing fungal infections. Recently, a member of the Hypocrealean family, Metarhizium anisopliae, has been reported for insect pathogenicity, endophytism, plant growth promotion, and antifungal potentialities. This research investigated the role of M. anisopliae (MetA1) in growth promotion and root disease suppression in okra. The antagonism against R. solani and the plant growth promotion traits of MetA1 were tested in vitro. The effects of endophytic MetA1 on promoting plant growth and disease suppression were assessed in planta. Dual culture and cell-free culture filtrate assays showed antagonistic activity against R. solani by MetA1. Some plant growth promotion traits, such as phosphate solubilization and catalase activity were also exhibited by MetA1. Seed primed with MetA1 increased the shoot, root, leaves, chlorophyll content, and biomass content compared to control okra plants. The plants challenged with R. solani showed the highest hydrogen peroxide (H2O2) and lipid peroxidation (MDA) contents in the leaves of okra. Whereas MetA1 applied plants showed a reduction of H2O2 and MDA by 5.21 and 14.96%, respectively, under pathogen-inoculated conditions by increasing antioxidant enzyme activities, including catalase (CAT), peroxidase (POD), glutathione S-transferase (GST), and ascorbate peroxidase (APX), by 30.11, 10.19, 5.62, and 5.06%, respectively. Moreover, MetA1 increased soluble sugars, carbohydrates, proline, and secondary metabolites, viz., phenol and flavonoid contents in okra resulting in a better osmotic adjustment of diseases infecting plants. MetA1 reduced disease incidence by 58.33% at 15 DAI compared to the R. solani inoculated plant. The results revealed that MetA1 improved plant growth, elevated the plant defense system, and suppressed root diseases caused by R. solani. Thus, MetA1 was found to be an effective candidate for the biological control program.

Persistent Elevation of Troponin I in a Pediatric Patient Resulting From Macrotroponin Complex.

ABSTRACT: Although chest pain is a common chief complaint among pediatric patients, cardiac pathology historically has accounted for a small percentage of cases. However, the emergence of COVID-19 and particularly its potential for leading to multisystem inflammatory syndrome has changed the threshold for the evaluation of cardiac etiologies of chest pain. This evaluation often includes measurement of the serum cardiac troponin I level. We present a case of a 16-year-old male athlete who presented to an outside emergency department with chest pain and was found to have elevated serum troponin I levels. Despite sports restriction, his troponin level remained elevated for months in the absence of other clinical findings and he was subsequently referred to our outpatient pediatric cardiology clinic. Further laboratory evaluation revealed that, in addition to troponin I, the assay measured an immune complex of uncertain significance formed by anti-troponin I antibodies bound to troponin I, known as macrotroponin. Delayed clearance of this complex from the bloodstream can result in overestimation of troponin I levels that can affect clinical management and create anxiety for our patients and their families. Macrotroponin complex deserves increased recognition among the research and clinical communities, especially in the pediatric realm.

Dabigatran and Wet AMD, Results From Retinal Pigment Epithelial Cell Monolayers, the Mouse Model of Choroidal Neovascularization, and Patients From the Medicare Data Base.

Background: Age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Caucasian populations, includes destruction of the blood-retina barrier (BRB) generated by the retinal pigment epithelium-Bruch's membrane complex (RPE/BrM), and complement activation. Thrombin is likely to get access to those structures upon BRB integrity loss. Here we investigate the potential role of thrombin in AMD by analyzing effects of the thrombin inhibitor dabigatran. Material and Methods: MarketScan data for patients aged ≥65 years on Medicare was used to identify association between AMD and dabigatran use. ARPE-19 cells grown as mature monolayers were analyzed for thrombin effects on barrier function (transepithelial resistance; TER) and downstream signaling (complement activation, expression of connective tissue growth factor (CTGF), and secretion of vascular endothelial growth factor (VEGF)). Laser-induced choroidal neovascularization (CNV) in mouse is used to test the identified downstream signaling. Results: Risk of new wet AMD diagnosis was reduced in dabigatran users. In RPE monolayers, thrombin reduced TER, generated unique complement C3 and C5 cleavage products, led to C3d/MAC deposition on cell surfaces, and increased CTGF expression via PAR1-receptor activation and VEGF secretion. CNV lesion repair was accelerated by dabigatran, and molecular readouts suggest that downstream effects of thrombin include CTGF and VEGF, but not the complement system. Conclusions: This study provides evidence of association between dabigatran use and reduced exudative AMD diagnosis. Based on the cell- and animal-based studies, we suggest that thrombin modulates wound healing and CTGF and VEGF expression, making dabigatran a potential novel treatment option in AMD.

Antifibrotic efficacy of nintedanib in a cellular model of systemic sclerosis-associated interstitial lung disease.

OBJECTIVES: Nintedanib is approved for the treatment of idiopathic pulmonary fibrosis (IPF) and was demonstrated to slow disease progression in patients with IPF by reducing decline in forced vital capacity by 50%. Recently, nintedanib has been reported to exert anti-fibrotic activity on systemic sclerosis (scleroderma, SSc) skin fibroblasts and to diminish skin and lung fibrosis in mouse models. The goal of the present study was to determine the effects of nintedanib on a cellular model of SSc-associated interstitial lung disease (ILD). METHODS: Study was performed using lung fibroblasts (LF) isolated from five patients with SSc-ILD and from three control subjects. RESULTS: Nintedanib inhibited LF proliferation and migration in a concentration- and time-dependent manner. The proliferation rate of LF stimulated with PDGF in the presence of nintedanib was reduced 1.9-fold within 24 h as compared to cells stimulated with PDGF alone. Migration of SSc-ILD LF incubated with 100 nM nintedanib was reduced from 62.8±12.5% to 39.1±9.0% in the presence of PDGF and from 38.2±7.9% to 26.6±7.2% in serum-free medium. Nintedanib attenuated PDGF-induced Ca2+ efflux, reduced α-SMA promoter activity and α-SMA protein expression. Furthermore, nintedanib blocked PDGF-induced differentiation of normal LF to myofibroblasts, reduced production of collagen and fibronectin, and decreased contractility of SSc-ILD LF in both floating and fixed collagen gels. CONCLUSIONS: Our data demonstrate significant antifibrotic efficacy of nintedanib in SSc-ILD LF suggesting that nintedanib has the potential not only to prevent but also to reverse the increased activity of LF consequently attenuating excessive lung fibrosis observed in SSc-ILD.

Progression of Interstitial Lung Disease in Systemic Sclerosis: The Importance of Pneumoproteins Krebs von den Lungen 6 and CCL18.

OBJECTIVE: To investigate the relationship between Krebs von den Lungen 6 (KL-6) and CCL18 levels and the severity and progression of systemic sclerosis (SSc)-related interstitial lung disease (ILD). METHODS: Patients enrolled in the Scleroderma Lung Study II (cyclophosphamide [CYC] versus mycophenolate mofetil [MMF]) were included. Baseline and 12-month plasma samples were analyzed by enzyme-linked immunosorbent assay to assess CCL18 and KL-6 levels. The forced vital capacity (FVC) and the diffusing capacity for carbon monoxide (DLco) were measured every 3 months. Joint models were created to investigate the relationship between baseline CCL18 and KL-6 levels and the course of the FVC and DLco over 1 year according to treatment arm. RESULTS: Baseline KL-6 and CCL18 levels each correlated with the extent of radiographic fibrosis. Levels of both CCL18 and KL-6 declined significantly at 1 year. In both treatment arms (n = 71 for CYC, n = 62 for MMF), a higher baseline KL-6 level predicted progression of ILD based on the course of FVC (P = 0.024 for CYC; P = 0.005 for MMF) and DLco (P < 0.001 for CYC; P = 0.004 for MMF) over 1 year. A higher baseline CCL18 level predicted progression of ILD based on the course of the FVC (P < 0.001 for CYC; P = 0.007 for MMF) and DLco (P = 0.001 for CYC; P < 0.001 for MMF) over 1 year, as well as mortality (P = 0.0008 for CYC arm only). CONCLUSION: In a rigorously conducted clinical trial for SSc-related ILD, KL-6 and CCL18 levels correlated with ILD severity and declined with immunosuppression. Patients with higher baseline KL-6 and CCL18 levels were more likely to experience disease progression despite treatment. KL-6 and CCL18 levels could be used to identify patients with a progressive ILD phenotype who may benefit from a more aggressive initial treatment approach.

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10.

OBJECTIVE: M10 is a ten amino acid peptide generated from the intracellular cytoplasmic tail of the hepatocyte growth factor (HGF) receptor c-Met following cleavage by caspase-3. Recently we reported that M10 interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo and can be advanced into a novel antifibrotic remedy. The current study was undertaken to develop an immunoassay to measure M10 concentration in biological specimens. EXPERIMENTAL DESIGN: An Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for detection of M10 in biological fluids was developed using pharmaceutical grade synthetic M10 as a calibrator and commercially available anti-c-Met C12 antibody. RESULTS: M10 ELISA specifically detected in plasma M10, but not a scrambled peptide, following a single intraperitoneal administration of M10 (1mg/kg) to mice. The detection limit was 9.6 ng/ml, and the measuring limit was between 15 ng/ml and 200 ng/ml. The recovery limits of M10 were between 80% and 120%; intra-assay coefficient of variation was between 5.3% and 6.3%; inter-assay coefficient of variation was between 5.0% and 8.0% over the buffer concentration tested in the range from 15 ng /ml to 250 ng /ml. The peak of M10 concentration following a single intraperitoneal injection (1mg/kg) was achieved within 6 hours and declined to minimal levels by 48 hours. The experimentally obtained half-life for M10 was comparable to the theoretically predicted half-life for M10. CONCLUSIONS: We have established a highly sensitive ELISA to detect the antifibrotic peptide M10 in plasma samples, which should prove to be a novel tool to study the pharmacokinetics and efficacy of M10 in the treatment of fibroproliferative disorders.

D1398G Variant of MET Is Associated with Impaired Signaling of Hepatocyte Growth Factor in Alveolar Epithelial Cells and Lung Fibroblasts.

Pulmonary fibrosis represents the terminal stage of a diverse group of lung diseases including scleroderma associated interstitial lung disease. The molecular mechanisms underlying the pathogenesis of lung fibrosis are not well understood and there is a great need for more effective treatment for this lethal disease. We recently discovered a small fragment of hepatocyte growth factor (HGF) receptor MET as a peptide designated "M10," with strong antifibrotic properties. Furthermore, we showed that aspartic acid at position 1398 of MET is essential for M10 generation. The current study was undertaken to investigate the D1398G variant of MET in which aspartic acid at position 1398 was mutated to glycine resulting in loss of M10. We demonstrate that lung fibroblasts, A549, and primary alveolar epithelial cells (AEC) expressing D1398G MET exhibit reduced auto-phosphorylation on tyrosine residues and reduced activation of Ras and MAPK. HGF treatment of scleroderma lung fibroblasts as well as HGF treatment of TGFß-treated normal lung fibroblasts transfected with wild type MET is associated with decreased collagen, connective tissue growth factor (CTGF, CCN2) and smooth muscle α-actin (SMA). However, HGF has no such effects in cells transfected with MET D1398G. Cisplatin- and FasL-induced apoptosis is significantly reduced in AEC transfected with MET wild type, but not in AEC transfected with MET D1398G. We conclude that the D1398G variant of MET is associated with compromised phosphorylation and impaired HGF signaling in lung fibroblasts and AEC, two cell types implicated in the pathogenesis of pulmonary fibrosis associated with scleroderma. Ongoing studies will explore the frequency of this variant and its relationship to pulmonary outcomes in scleroderma patients.

M10, a caspase cleavage product of the hepatocyte growth factor receptor, interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo.

Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif "DEVD-T" that on cleavage by caspase-3 generates a 10-amino acid peptide, TRPASFWETS, designated as "M10". M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFß-stimulated normal lung and skin fibroblasts. M10 interacts with the Mad Homology 2 domain of Smad2 and inhibits TGFß-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases.

Epidermal growth factor-induced proliferation of collecting duct cells from Oak Ridge polycystic kidney mice involves activation of Na+/H+ exchanger.

Epidermal growth factor (EGF) is linked to the pathogenesis of polycystic kidney disease (PKD). We explored signaling pathways activated by EGF in orpk cilia (-) collecting duct cell line derived from a mouse model of PKD (hypomorph of the Tg737/Ift88 gene) with severely stunted cilia, and in a control orpk cilia (+) cell line with normal cilia. RT-PCR demonstrated mRNAs for EGF receptor subunits ErbB1, ErbB2, ErbB3, ErbB4, and mRNAs for Na(+)/H(+) exchangers (NHE), NHE-1, NHE-2, NHE-3, NHE-4, and NHE-5 in both cell lines. EGF stimulated proton efflux in both cell lines. This effect was significantly attenuated by MIA, 5-(n-methyl-N-isobutyl) amiloride, a selective inhibitor of NHE-1 and NHE-2, and orpk cilia (-) cells were more sensitive to MIA than control cells (P < 0.01). EGF significantly induced extracellular signal-regulated kinase (ERK) phosphorylation in both cilia (+) and cilia (-) cells (63.3 and 123.6%, respectively), but the effect was more pronounced in orpk cilia (-) cells (P < 0.01). MIA significantly attenuated EGF-induced ERK phosphorylation only in orpk cilia (-) cells (P < 0.01). EGF increased proliferation of orpk cilia (+) cells and orpk cilia (-) cells, respectively, and MIA at 1-5 µM attenuated EGF-induced proliferation in orpk cilia (-) cells without affecting proliferation of orpk cilia (+) cells. EGF-induced proliferation of both cell lines was significantly decreased by the EGFR tyrosine kinase inhibitor AG1478 and MEK inhibitor PD98059. These results suggest that EGF exerts mitogenic effects in the orpk cilia (-) cells via activation of growth-associated amiloride-sensitive NHEs and ERK.

Recent advances in understanding the pathogenesis of scleroderma-interstitial lung disease.

Systemic sclerosis (scleroderma, SSc) is a heterogeneous autoimmune connective tissue disease of unknown etiology. Interstitial lung disease (ILD) is a frequent complication, and a significant contributor to morbidity and mortality among SSc patients. SSc-ILD most commonly occurs within 10 years of diagnosis, and may be seen in patients with either the limited or diffuse cutaneous subset of SSc. SSc-ILD is a multifaceted disease process in which different factors and pathways are involved. Aberrant function of a variety of lung cells, cytokines, growth factors, peptides, and bioactive proteins, in combination with genetic and epigenetic regulators, have crucial functions in the pathogenesis of this disease. Here we present our view on recent advances regarding the pathogenesis of SSc-ILD.

Overexpression of c-Met and CD44v6 receptors contributes to autocrine TGF-ß1 signaling in interstitial lung disease.

The hepatocyte growth factor (HGF) and the HGF receptor Met pathway are important in the pathogenesis of interstitial lung disease (ILD). Alternatively spliced isoforms of CD44 containing variable exon 6 (CD44v6) and its ligand hyaluronan (HA) alter cellular function in response to interaction between CD44v6 and HGF. TGF-ß1 is the crucial cytokine that induces fibrotic action in ILD fibroblasts (ILDFbs). We have identified an autocrine TGF-ß1 signaling that up-regulates both Met and CD44v6 mRNA and protein expression. Western blot analysis, flow cytometry, and immunostaining revealed that CD44v6 and Met colocalize in fibroblasts and in tissue sections from ILD patients and in lungs of bleomycin-treated mice. Interestingly, cell proliferation induced by TGF-ß1 is mediated through Met and CD44v6. Further, cell proliferation mediated by TGF-ß1/CD44v6 is ERK-dependent. In contrast, action of Met on ILDFb proliferation does not require ERK but does require p38(MAPK). ILDFbs were sorted into CD44v6(+)/Met(+) and CD44v6(-)/Met(+) subpopulations. HGF inhibited TGF-ß1-stimulated collagen-1 and α-smooth muscle cell actin expression in both of these subpopulations by interfering with TGF-ß1 signaling. HGF alone markedly stimulated CD44v6 expression, which in turn regulated collagen-1 synthesis. Our data with primary lung fibroblast cultures with respect to collagen-1, CD44v6, and Met expressions were supported by immunostaining of lung sections from bleomycin-treated mice and from ILD patients. These results define the relationships between CD44v6, Met, and autocrine TGF-ß1 signaling and the potential modulating influence of HGF on TGF-ß1-induced CD44v6-dependent fibroblast function in ILD fibrosis.

Thrombin increases lung fibroblast survival while promoting alveolar epithelial cell apoptosis via the endoplasmic reticulum stress marker, CCAAT enhancer-binding homologous protein.

Apoptosis of alveolar epithelial cells (AECs) and survival of lung fibroblasts are critical events in the pathogenesis of pulmonary fibrosis; however, mechanisms underlying the apoptosis of AECs and the resistance of lung fibroblasts to apoptosis remain obscure. Herein, we demonstrate that the fate of these two cell types depends on the expression of CCAAT enhancer-binding homologous protein (CHOP). We observed that thrombin, which is overexpressed in scleroderma (SSc; systemic sclerosis) and other interstitial lung diseases (ILDs), increases the expression of CHOP in primary AECs and in A549 cells via an Ets1-dependent pathway. In addition, thrombin activates caspase-3 in AECs and induces apoptosis of these cells in a CHOP-dependent manner. In contrast, thrombin decreases endoplasmic reticulum stress-induced CHOP in lung fibroblasts through Myc-dependent mechanisms and protects such cells from apoptosis. Furthermore, when lung fibroblasts are transfected with recombinant CHOP, they then undergo apoptosis, even in the presence of thrombin, suggesting that CHOP signaling pathways are downstream of thrombin. In accordance with the differential effects of thrombin on AECs and lung fibroblasts, we observed strong expression of CHOP in AECs in fibrotic lung tissue isolated from patients with SSc-associated ILD (SSc-ILD), but not in lung myofibroblasts nor in normal lung tissue. Expression of CHOP in SSc lung is accompanied by positive staining for the thrombin receptor, protease-activated receptor-1, and for terminal deoxynucleotidyl transferase dUTP nick end labeling, suggesting roles for both thrombin and CHOP in AEC apoptosis in SSc-ILD. We conclude that regulation of CHOP by thrombin directs AECs toward apoptosis while promoting survival of lung fibroblasts, ultimately contributing to the persistent fibroproliferation seen in SSc-ILD and other fibrosing lung diseases.

The PPARγ Agonist Rosiglitazone Is Antifibrotic for Scleroderma Lung Fibroblasts: Mechanisms of Action and Differential Racial Effects.

We present novel data demonstrating that the expression of PPARγ is reduced in lung fibroblasts from black SSc-ILD patients as compared to white patients. Activating PPARγ with the agonist rosiglitazone increased the expression of MMP-1 and inhibited collagen type I in lung fibroblasts isolated from white, but not black, SSc-ILD patients. Blocking the c-Met receptor abolishes rosiglitazone's effects on collagen and MMP-1 in lung fibroblasts isolated from white SSc-ILD patients, while augmenting the expression of the c-Met receptor in fibroblasts from black SSc-ILD patients replicates the effects of rosiglitazone seen in whites. We conclude that PPARγ agonists warrant consideration as potential antifibrotic drugs in patients with SSc-ILD. Differential therapeutic effects might be anticipated especially relative to racial differences and the functional expression of the c-Met receptor.

Antiinflammatory and antifibrotic effects of the oral direct thrombin inhibitor dabigatran etexilate in a murine model of interstitial lung disease.

OBJECTIVE: Activation of the coagulation cascade leading to generation of thrombin has been documented extensively in various forms of lung injury, including that associated with systemic sclerosis. We previously demonstrated that the direct thrombin inhibitor dabigatran inhibits thrombin-induced profibrotic signaling in lung fibroblasts. This study was undertaken to test whether dabigatran etexilate attenuates lung injury in a murine model of interstitial lung disease. METHODS: Lung injury was induced in female C57BL/6 mice by a single intratracheal instillation of bleomycin. Dabigatran etexilate was given as supplemented chow beginning on day 1 of bleomycin instillation (early treatment, study of antiinflammatory effect) or on day 8 following bleomycin instillation (late treatment, study of antifibrotic effect). Mice were killed 2 weeks or 3 weeks after bleomycin instillation, and lung tissue, bronchoalveolar lavage (BAL) fluid, and plasma were investigated. RESULTS: Both early treatment and late treatment with dabigatran etexilate attenuated the development of bleomycin-induced pulmonary fibrosis. Dabigatran etexilate significantly reduced thrombin activity and levels of transforming growth factor ß1 in BAL fluid, while simultaneously reducing the number of inflammatory cells and protein concentrations. Histologically evident lung inflammation and fibrosis were significantly decreased in dabigatran etexilate-treated mice. Additionally, dabigatran etexilate reduced collagen, connective tissue growth factor, and α-smooth muscle actin expression in mice with bleomycin-induced lung fibrosis, whereas it had no effect on basal levels of these proteins. CONCLUSION: Inhibition of thrombin using the oral direct thrombin inhibitor dabigatran etexilate has marked antiinflammatory and antifibrotic effects in a bleomycin model of pulmonary fibrosis. Our data provide preclinical information about the feasibility and efficacy of dabigatran etexilate as a new therapeutic approach for the treatment of interstitial lung disease.