Auxiliary roles of nardilysin in the early diagnosis of acute coronary syndrome: a prospective cohort study, the Nardi-ACS study.

Acute coronary syndrome (ACS) includes myocardial infarction (MI) and unstable angina (UA). MI is defined by elevated necrosis markers, preferably high-sensitivity cardiac troponins (hs-cTn). However, it takes hours for cTn to become elevated after coronary occlusion; therefore, difficulties are associated with diagnosing early post-onset MI or UA. The aim of this prospective cohort study was to examine the diagnostic ability of serum nardilysin (NRDC) for the early detection of ACS. This study consisted of two sequential cohorts, the Phase I cohort, 435 patients presenting to the emergency room (ER) with chest pain, and the Phase II cohort, 486 patients with chest pain who underwent coronary angiography. The final diagnosis was ACS in 155 out of 435 patients (35.6%) in the phase I and 418 out of 486 (86.0%) in the phase II cohort. Among 680 patients who presented within 24 h of onset, 466 patients (68.5%) were diagnosed with ACS. Serum NRDC levels were significantly higher in patients with ACS than in those without ACS. The sensitivity of NRDC in patients who presented within 6 h after the onset was higher than that of hsTnI, and the AUC of NRDC within 1 h of the onset was higher than that of hsTnI (0.718 versus 0.633). Among hsTnI-negative patients (300 of 680 patients: 44.1%), 136 of whom (45.3%) were diagnosed with ACS, the sensitivity and the NPV of NRDC were 73.5 and 65.7%, respectively. When measured in combination with hsTnI, NRDC plays auxiliary roles in the early diagnosis of ACS.

Meprin A in Patients with Acute Decompensation of Heart Failure.

Plasma levels of meprin A, IL-6, and IL-18 were measured in 68 patients with acute decompensated heart failure at the time of admission to the hospital and after 1 year. The patients were assigned to groups depending on renal function disorder which was assessed by glomerular filtration rate (GFR). During hospital stay, the plasma levels of meprin A in patients with normal GFR (≥90 ml/min/1.73 m2) were considerably higher than in patients with reduced GFR (<90 ml/min/1.73 m2): 1.80 (0.86; 2.65) and 1.04 (0.56; 1.60) ng/ml, respectively. The levels of IL-6 and IL-18 did not differ significantly. After 1 year, plasma levels of meprin A and interleukins markedly decreased in patients with normal GFR (0.33 (0.20; 0.86) ng/ml) and remained high in patients with reduced GFR (0.92 (0.39; 1.33) ng/ml). Thus, the dynamics of meprin A levels in patients with acute decompensated heart failure depends on functional state of the kidneys, which may affect the course of heart failure.

Predictive value of MEP1A in cancer prognosis: A protocol for systematic review and meta-analysis.

BACKGROUND: Meprin is a member of the astaxanthin family; it performs many functions through a wide range of proteolytic enzyme activities during health and disease, including tumors and inflammatory conditions. The purpose of this systematic review was to evaluate the predictive value of MEP1A in tumor prognosis. METHODS: A comprehensive search was conducted on PubMed, Cochrane library, and Web of Science Database using a developed search strategy. The Newcastle-Ottawa Scale (NOS) or the Cochrane Collaboration's tool for assessing risk of bias will be used to access the methodological quality of included studies, and GRADE will be applied to evaluate evidence quality of outcomes. All analyses were performed by Stata 15.0. RESULTS: The results will systematically summarize and display the currently collected evidence on the predictive value of MEP1A in different tumor prognosis. CONCLUSION: This study may play a certain role in predicting the prognosis of cancer patients in the future, and may prompt clinicians to make necessary treatment or prevention plans as soon as possible. ETHICS AND COMMUNICATION: It is not necessary because the present systematic review is based on published studies. INPLASY REGISTRATION NUMBER: INPLASY2020100005.

Poly(I:C) causes failure of immunoprophylaxis to red blood cells expressing the KEL glycoprotein in mice.

Polyclonal anti-D (Rh immune globulin [RhIg]) therapy has mitigated hemolytic disease of the newborn over the past half century, although breakthrough anti-D alloimmunization still occurs in some treated females. We hypothesized that antiviral responses may impact the efficacy of immunoprophylaxis therapy in a type 1 interferon (IFN)-dependent manner and tested this hypothesis in a murine model of KEL alloimmunization. Polyclonal anti-KEL immunoprophylaxis (KELIg) was administered to wild-type or knockout mice in the presence or absence of polyinosinic-polycytidilic acid (poly[I:C]), followed by the transfusion of murine red blood cells (RBCs) expressing the human KEL glycoprotein. Anti-KEL alloimmunization, serum cytokines, and consumption of the transfused RBCs were evaluated longitudinally. In some experiments, recipients were treated with type 1 IFN (IFN-α/ß). Recipient treatment with poly(I:C) led to breakthrough anti-KEL alloimmunization despite KELIg administration. Recipient CD4+ T cells were not required for immunoprophylaxis efficacy at baseline, and modulation of the KEL glycoprotein antigen occurred to the same extent in the presence or absence of recipient inflammation. Under conditions where breakthrough anti-KEL alloimmunization occurred, KEL RBC consumption by inflammatory monocytes and serum monocyte chemoattractant protein-1 and interleukin-6 were significantly increased. Poly(I:C) or type I IFN administration was sufficient to cause breakthrough alloimmunization, with poly(I:C) inducing alloimmunization even in the absence of recipient type I IFN receptors. A better understanding of how recipient antiviral responses lead to breakthrough alloimmunization despite immunoprophylaxis may have translational relevance to instances of RhIg failure that occur in humans.

Targeted proteomic response to coffee consumption.

PURPOSE: Coffee is widely consumed and implicated in numerous health outcomes but the mechanisms by which coffee contributes to health is unclear. The purpose of this study was to test the effect of coffee drinking on candidate proteins involved in cardiovascular, immuno-oncological and neurological pathways. METHODS: We examined fasting serum samples collected from a previously reported single blinded, three-stage clinical trial. Forty-seven habitual coffee consumers refrained from drinking coffee for 1 month, consumed 4 cups of coffee/day in the second month and 8 cups/day in the third month. Samples collected after each coffee stage were analyzed using three multiplex proximity extension assays that, after quality control, measured a total of 247 proteins implicated in cardiovascular, immuno-oncological and neurological pathways and of which 59 were previously linked to coffee exposure. Repeated measures ANOVA was used to test the relationship between coffee treatment and each protein. RESULTS: Two neurology-related proteins including carboxypeptidase M (CPM) and neutral ceramidase (N-CDase or ASAH2), significantly increased after coffee intake (P < 0.05 and Q < 0.05). An additional 46 proteins were nominally associated with coffee intake (P < 0.05 and Q > 0.05); 9, 8 and 29 of these proteins related to cardiovascular, immuno-oncological and neurological pathways, respectively, and the levels of 41 increased with coffee intake. CONCLUSIONS: CPM and N-CDase levels increased in response to coffee intake. These proteins have not previously been linked to coffee and are thus novel markers of coffee response worthy of further study. CLINICAL TRIAL REGISTRY: http://www.isrctn.com/ISRCTN12547806.

Clinical determination of serum nardilysin levels in predicting 30-day mortality among adults with malignant cerebral infarction.

BACKGROUND: Nardilysin, a kind of metalloendopeptidase, plays an important role in numerous inflammatory diseases. Malignant cerebral infarction (Glasgow coma scale score of <9) is associated with a high mortality risk. Here, we intended to investigate the relationship between serum nardilysin levels and prognosis of patients with malignant cerebral infarction. METHODS: Serum nardilysin concentrations were quantified at malignant cerebral infarction diagnosis moment in 105 patients and at study entrance in 105 healthy controls. Association of nardilysin concentrations with 30-day mortality and overall survival was estimated using multivariate analyses. RESULTS: The patients exhibited substantially increased serum nardilysin concentrations, as compared to the controls. Nardilysin concentrations were in pronounced correlation with Glasgow coma scale scores and serum C-reactive protein concentrations. Serum nardilysin was independently predictive of 30-day mortality and overall survival. Under receiver operating characteristic curve, its high discriminatory ability was found. CONCLUSIONS: Rising serum nardilysin concentrations following malignant cerebral infarction are strongly related to stroke severity, inflammatory extent and a higher risk of mortality, substantializing serum nardilysin as a potential prognostic biomarker for malignant cerebral infarction.

Serum Nardilysin, a Surrogate Marker for Epithelial-Mesenchymal Transition, Predicts Prognosis of Intrahepatic Cholangiocarcinoma after Surgical Resection.

PURPOSE: Few studies have investigated prognostic biomarkers in patients with intrahepatic cholangiocarcinoma (ICC). Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has been suggested to play important roles in inflammation and several cancer types. We herein examined the clinical significance and biological function of NRDC in ICC.Experimental Design: We measured serum NRDC levels in 98 patients with ICC who underwent surgical resection in two independent cohorts to assess its prognostic impact. We also analyzed NRDC mRNA levels in cancerous tissue specimens from 43 patients with ICC. We investigated the roles of NRDC in cell proliferation, migration, gemcitabine sensitivity, and gene expression in ICC cell lines using gene silencing. RESULTS: High serum NRDC levels were associated with shorter overall survival and disease-free survival in the primary (n = 79) and validation (n = 19) cohorts. A correlation was observed between serum protein levels and cancerous tissue mRNA levels of NRDC (Spearman ρ = 0.413; P = 0.006). The gene knockdown of NRDC in ICC cell lines attenuated cell proliferation, migration, and tumor growth in xenografts, and increased sensitivity to gemcitabine. The gene knockdown of NRDC was also accompanied by significant changes in the expression of several epithelial-mesenchymal transition (EMT)-related genes. Strong correlations were observed between the mRNA levels of NRDC and EMT-inducing transcription factors, ZEB1 and SNAI1, in surgical specimens from patients with ICC. CONCLUSIONS: Serum NRDC, a possible surrogate marker reflecting the EMT state in primary tumors, predicts the outcome of ICC after surgical resection.

Increased serum nardilysin is associated with worse long-term outcome of ST-elevation myocardial infarction.

OBJECTIVE: Nardilysin (N-arginine dibasic convertase, NRDC) is a kind of metalloendopeptidase associated with several inflammatory diseases. NRDC is reported to be eligible for early detection of acute coronary syndrome. However, the availability and effectiveness of NRDC in predicting the prognosis of patients with ST-elevation myocardial infarction has never been investigated. PATIENTS AND METHODS: From January 2010 to January 2012, the prospective observational cohort study enrolled a total of 396 STEMI patients, who were sampled with blood within 24 hours after admission. A long-term follow-up was conducted to record the primary endpoint of all-cause mortality, as well as secondary endpoint of myocardial infarction, stroke, emergent revascularization and admission due to heart failure. Hazard ratio (HR) related to the serum NRDC level was estimated by Cox regression model. RESULTS: The enrolled patients completed the follow-up with an average of 4.6 ± 0.5 years, of whom 24 patients died (primary endpoint, 6.1%), while 89 episodes of secondary endpoints occurred (22.5%). Patients with highest quartile level of NRDC (Q4 level, > 2041 pg/ml) were subjected to higher risk of all-cause death [HR 3.973, 95% CI (1.648-9.575), p = 0.002] compared to patients with lower three quartiles level of NRDC (Q1 to Q3, < 2041 pg/ml), while there was no difference in adverse events (p = 0.403). CONCLUSIONS: The increased serum NRDC level at admission is associated with a higher risk of all-cause mortality for ST-elevation myocardial infarction patients.

Conjugation with Eight-Arm PEG Markedly Improves the In Vitro Activity and Prolongs the Blood Circulation of Staphylokinase.

Staphylokinase (SAK) is a profibrinolytic protein and can be used for therapy of acute myocardial infarction and coronary thrombosis. However, SAK suffers from a short serum half-life time (∼6 min) that limits its clinical application. PEGylation prolongs the half-life time of SAK, whereas it significantly decreases the bioactivity of SAK for the steric shielding effect of PEG. To improve the bioactivity and prolong the half-life time of SAK, 8-arm PEG maleimide (8-arm PEG) was used for conjugation of multiple SAK molecules in one entity. C terminus of SAK was engineered with cysteine residue, followed by reaction with the maleimide moieties of 8-arm PEG to obtain the conjugate (SAKp-PEG). Conjugation with 8-arm PEG retained the secondary structure of SAK, slightly perturbed the tertiary structure of SAK, and essentially maintained its in vitro bioactivity by the multivalence of SAK. Conjugation with 8-arm PEG increased the hydrodynamic volume and thus significantly prolonged the half-life time of SAK. SAKp-PEG elicited a 1.4-fold increase in the SAK-specific IgG titers as compared with SAK, and rendered no apparent toxicity to the cardiac, liver and renal functions of mice. Thus, multiple conjugation of a protein with 8-arm PEG was an effective strategy to develop a long-acting protein drug with improved bioactivity and prolonged blood circulation.

Suppression of active, but not total MMP-3, is associated with treatment response in a phase III clinical study of rheumatoid arthritis.

OBJECTIVE: Biologics for rheumatoid arthritis (RA) patients with moderate to severe disease may preserve joint function. Matrix metalloproteinase 3 (MMP-3), a key tissue degrading protease, is highly elevated in RA. MMP-3, which measures the total pool of circulating MMP-3 species (cMMP3), is a commonly measured biomarker in rheumatology. The aim was to investigate the association of activated MMP-3 (actMMP3) species with treatment response compared to cMMP-3. METHODS: The LITHE biomarker study (n=741) was a 1-year phase III, double-blind, placebo-controlled, parallel group study of TCZ in RA patients on stable methotrexate. cMMP-3 and actMMP-3 were assessed in fasting serum at baseline, week 4, 16, 24 and 52. Patients not achieving ACR20 remission at week 16 or 28 received rescue treatment (escapers). Spearman's correlation was analysed between biomarker baseline level or biomarker delta and clinical measures. Changes in biomarker levels were studied as a function of time and treatment. RESULTS: ActMMP-3 16-week change in treatment groups was predictive of 1-year radiographic progression; a small change in actMMP3 was equal to worsening radiographics. Baseline cMMP-3 was associated with 52-weeks' radiographic status and cMMP3 16-weeks' change was predictive of 1-year change in disease activity. ActMMP-3 was dose-dependently decreased by TCZ, and escapers decreased in actMMP-3 upon treatment. CONCLUSIONS: ActMMP-3 and cMMP-3 were found to be efficacy biomarkers of TCZ and actMMP-3 were able to differentiated doses. Moreover, the suppression of actMMP3, but not cMMP3 was associated with treatment response. This study illustrates that two biomarkers of the same protein may have different predictive capacities.

Nardilysin is a promising biomarker for the early diagnosis of acute coronary syndrome.

BACKGROUND: Biomarkers for detection of transient myocardial ischemia in patients with unstable angina (UA) or for very early diagnosis of acute myocardial infarction (AMI) are not currently available. METHODS AND RESULTS: We performed two sequential screenings of autoantibodies elevated shortly after the onset of acute coronary syndrome (ACS), and focused on metalloendopeptidase nardilysin (NRDC) among 19 identified candidate antigens. In a retrospective analysis among 93 ACS and 117 non-ACS patients, the serum level of NRDC was significantly increased in patients with ACS compared with that in patients with non-ACS (2073.5±189.8pg/ml versus 775.7±63.4pg/ml, P<0.0001). The area under the curve of NRDC for the diagnosis of ACS was 0.822 by the receiver operating characteristic curves analysis. In the time course analysis in 43 consecutive ACS patients (AMI: N=35 and UA: N=8), serum concentration of NRDC was significantly increased even in UA patients with a peak serum NRDC levels reached at admission both in AMI and UA patients. In a mouse model of AMI, we found an acute increase in serum NRDC and reduced NRDC expression in ischemic regions shortly after coronary artery ligation. NRDC expression was also reduced in infarcted regions in human autopsy samples from AMI patients. Moreover, the short treatment of primary culture of rat cardiomyocytes with H2O2 or A23187 induced NRDC secretion without cell toxicity. CONCLUSION: NRDC is a promising biomarker for the early detection of ACS, even in UA patients without elevation of necrosis markers.

Levels of Matrix Metalloproteinases in Arthroplasty Patients and Their Correlation With Inflammatory and Thrombotic Activation Processes.

An imbalance of matrix metalloproteinases (MMPs) and their inhibitors is thought to play a major role in the pathophysiology of joint diseases. The aim of this study is to provide additional insights into the relevance of MMP levels in arthroplasty patients in relation to inflammation and thrombosis. Deidentified plasma samples from 100 patients undergoing total hip arthroplasty or total knee arthroplasty were collected preoperatively, on postoperative day 1, and on postoperative day 3. Tissue inhibitor of MMP 4, tumor necrosis factor α (TNF-α), pro-MMP1, MMP3, MMP9, MMP13, and d-dimer were measured using enzyme-linked immunosorbent assay kits. A biochip array was used to profile interleukin (IL) 2, IL-4, IL-6, IL-8, IL-10, vascular endothelial growth factor (VEGF), interferon gamma, TNF-α, IL-1α, IL-1ß, monocyte chemoattractant protein 1, and endothelial growth factor (EGF) levels. The levels of MMP1, MMP9, MMP13, and TNF-α were elevated preoperatively in arthroplasty patients when compared to healthy individuals. The concentrations of MMP1 and MMP9 increased slightly in postsurgical samples. d-Dimer levels were elevated preoperatively, increased postoperatively, and started decreasing on postoperative day 3. Significant correlations between MMP9 with TNF-α, IL-6, IL-8, VEGF, and EGF were identified. Elevated preoperative MMP1, MMP9, and MMP13 concentrations suggest that they may play a role in the pathogenesis of arthritis. There is also evidence of increased coagulation activity and possible upregulation of several MMPs postsurgically. Correlation analysis indicates that MMP9 levels may potentially be related to inflammation and thrombosis in arthroplasty patients.

Development and validation of an indirect competitive ELISA for quantification of recombinant staphylokinase in rabbit plasma: Application to pharmacokinetic study.

The relatively short circulatory half-life (2-3 min) of staphylokinase is a major drawback in the development of SAK- (staphylokinase) based thrombolytic drug. A rapid and sensitive method, based on indirect competitive ELISA, was developed and validated for quantitative determination of SAK in rabbit plasma. The dynamic range of the assay varied between 0.41 ± 0.16 µg/L and 9.03 ± 0.38 µg/L (R(2) = 0.98) for SAK in rabbit plasma. There were no dilution linearity issues apparent with this assay. The precision (% CV) ranged from 4.6-9.7% for the intraassay and from 17.1-19.3% for interassay. This validated method was successfully employed for evaluation of various pharmacokinetic parameters of SAK in rabbit.

The prevalence of anti-K in Canadian prenatal patients.

BACKGROUND: Anti-KEL1(K) is a major cause of hemolytic disease of the fetus and newborn. We utilized data from prenatal testing of patients in Western Canada to determine the frequency of anti-K. In Manitoba, we evaluated the frequency of transfusion as the likely cause for alloimmunization. We reviewed international practices to prevent alloimmunization. STUDY DESIGN AND METHODS: Prenatal patients undergo antibody screening using an automated testing platform and uniform testing algorithm. Data on the frequency of antibodies, transfusion history, and donor K typing were extracted from the relevant databases at Canadian Blood Services. National standards were reviewed with the help of local experts. RESULTS: Anti-K was found in 397 of 390,193 patients from 2011 to 2013 (1.02 per 1000) and was the second most frequent antibody after anti-E. In Manitoba, 26 of 75 (35%) anti-K patients had received transfusions in the province since 2001; 14 of the 26 (54%) had received at least one K+ RBC unit and three had received all K- units, while in nine, donor K typing was incomplete. Only eight of the 26 had previous pregnancies, three with K+ partners. International practice varies; however, prophylactic use of matched or K- units is standard in many European countries. CONCLUSIONS: Anti-K was found in 0.1% of prenatal patients. Although our data on the history of transfusion are incomplete, they demonstrate that transfusion with a K+ unit is a major cause of alloimmunization. Given advances in phenotyping and genotyping technologies, prophylactic matching should be considered in Canada.

Acute extravascular hemolytic transfusion reaction due to anti-Kpa antibody missed by electronic crossmatch.

BACKGROUND: Kpa antigen is a low incidence red blood cell antigen within the Kell system. Anti-Kpa alloantibody may be associated with acute and delayed hemolytic transfusion reactions. CASE STUDY: We report a case of a clinically significant acute extravascular hemolytic transfusion reaction mediated by previously unrecognized (and undetected) anti-Kpa alloantibody. This reaction occurred in a patient who met all criteria for electronic crossmatch, resulting in the transfusion of an incompatible red cell unit. RESULTS: Post-transfusion investigation showed the transfused red cell unit was crossmatch compatible at the immediate spin phase but was 3 + incompatible at the antiglobulin phase. No evidence of intravascular hemolysis was observed upon visual comparison of the pre- and post-transfusion peripheral blood plasma. Further testing showed the presence of anti-Kpa antibody. The clinical course of the patient included acute febrile and systemic reaction. CONCLUSION: Acute extravascular hemolytic transfusion reaction may occur due to undetected anti-Kpa alloantibody. Various strategies for crossmatching are discussed in the context of antibodies to low incidence antigens.

Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and in vivo activity.

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, [(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only ∼10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ([(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

Bone turnover markers do not predict stress fracture in elite combat recruits.

BACKGROUND: With bone resorption rates greater than formation, stress fracture pathogenesis plausibly involves bone remodeling imbalance. If this is the case, one would anticipate serum levels of bone turnover markers would be higher in patients with stress fractures than in those without. QUESTIONS/PURPOSES: We therefore asked whether: (1) bone turnover markers differ between soldiers who will or will not have stress fractures during basic training; (2) bone turnover markers change during basic training; and (3) serial bone formation or bone resorption markers differ between subjects with and without stress fractures during basic training? METHODS: We performed serial determinations of serum bone formation (bone alkaline phosphatase [BAP] and procollagen type I amino-terminal propeptide [PINP]), and resorption (tartrate-resistant acid phosphatase [TRAP5b] and cross-linked collagen telopeptide [CTx]) biomarkers, measured at 2- to 4-week intervals (during 18 weeks) in 69 male soldiers in the Israeli Defense Forces during elite basic training. Twenty-two soldiers (32%) were diagnosed with stress fractures. The mean training week at diagnosis was 8.0±2.0 weeks. RESULTS: We observed no differences in bone turnover markers between soldiers with and without stress fractures. During basic training, the mean values of all subjects for bone turnover markers (BAP, PINP, and CTx) changed in comparison to their mean levels at induction (43.9 versus 37.3 µg/L, 110.4 versus 78.0 µg/L, 1.4 versus 1.1 ng/mL, respectively). We found no changes in bone formation and resorption markers between subjects with and without stress fractures. CONCLUSIONS: These specific bone turnover markers cannot be considered as either diagnostic or predictive tools for stress fracture detection in young male military recruits. LEVEL OF EVIDENCE: Level II prognostic study. See the Guidelines for Authors for a complete description of levels of evidence.

Effect of small hairpin RNA targeting endothelin-converting enzyme-1 in monocrotaline-induced pulmonary hypertensive rats.

The purpose of this study was to investigate the therapeutic effects of small hairpin RNA (shRNA) targeting endothelin-converting enzyme (ECE)-1 in monocrotaline (MCT)-induced pulmonary hypertensive rats. Ninty-four Sprague-Dawley rats were divided into three groups: control (n = 24), MCT (n = 35) and shRNA (n = 35). Four-week survival rate in the shRNA group was significantly increased compared to that in the MCT group. The shRNA group showed a significant improvement of right ventricular (RV) pressure compared with the MCT group. The MCT and shRNA groups also showed an increase in RV/(left ventricle + septum) ratio and lung/body weight. Plasma endothelin (ET)-1 concentrations in the shRNA group were lower than those in the MCT group. Medial wall thickness of pulmonary arterioles were increased after MCT injection and was significantly decreased in the shRNA group. The number of intra-acinar muscular pulmonary arteries was decreased in the shRNA group. The mRNA expressions of ET-1 and ET receptor A (ET(A)) were significantly decreased in the shRNA group in week 4. The protein levels of ET(A) were decreased in the shRNA group in week 2. The protein levels of tumor necrosis factor-α and vascular endothelial growth factor were decreased in the shRNA group in week 4. In conclusion, the gene silencing with lentiviral vector targeting ECE-1 could be effective against hemodynamic, histopathological and gene expression changes in pulmonary hypertension.

A murine model to characterize the antithrombotic effect of molecules targeting human von Willebrand factor.

von Willebrand factor (VWF) is a promising target for developing antithrombotic drugs. The absence of accessible animal models impedes the study of specific human VWF (huVWF) targeting molecules in thrombosis. huVWF is not functional in the mouse because of a lack of interaction between huVWF and murine glycoprotein Ib. Using site-directed mutagenesis, we have replaced single or multiple amino acids in huVWF with their murine counterparts to eliminate species incompatibility. Using hydrodynamic injection, we have expressed the different chimeric VWF constructs into VWF(-/-) mice. Only huVWF with a complete murine A1 domain insertion was able to correct bleeding in vivo and form occlusive thrombi in mesenteric vessels after FeCl(3) treatment. Using this model, we tested the antithrombotic effect of monoclonal antibodies against huVWF, blocking its interaction with collagens (mAbs 203 and 505) or with glycoprotein IIbIIIa (mAb 9). The 3 mAbs inhibited the thrombotic process in arterioles of VWF(-/-) mice expressing huVWFmuA1. Inhibiting VWF-interaction with collagens was more potent, emphasizing the potential of such a target as an antithrombotic tool. Our results validate our murine model as a simple in vivo tool to evaluate anti-huVWF agents.