The use of molecular biological methods to assess the effects of endocrine disrupting chemicals and natural hormones on growth in the sheepshead minnow (Cyprinodon variegatus)

The work presented in this dissertation examines possible modes of action for growth inhibition by anthropogenic endocrine disrupting chemicals (EDCs) as well as endogenous hormones associated with growth in fish. Using the sheepshead minnow (SHM) (Cyprinodon variegatus) as a model, I developed methods to examine perturbations in the endocrine axis controlling fish growth, and also examined effects of EDCs on the whole fish. I used two relatively new techniques to study the endocrine growth axis, quantitative real-time PCR (TaqMan) and differential display analysis. TaqMan analysis is a highly sensitive method to measure specific sequences from a small amount of total RNA using a fluorescent probe and specific primer pairs. I optimized a TaqMan assay for SHM IGF-I to measure hepatic IGF-I mRNA concentrations. in fish injected with hormones known to influence fish growth (GH, T₃, E₂, insulin, or a carrier control). IGF-I mRNA levels increased in fish injected with GH, T₃ and insulin, peaking at 12 h post-injection. IGF-I mRNA levels decreased significantly at 8 h and 12 h post-injection in fish injected with E₂, suggesting that pharmacological levels of E₂ may affect the GH/IGF-I axis and could have consequences for fish living in waters polluted by EDCs. Differences in growth were observed in fish exposed for 18 weeks to E₂ or chlorpyrifos (an organophsophate). Fish exposed to the highest dose of E₂ grew larger than controls only during the last week of the experiment. Fish exposed to the lower dose of E₂ were not significantly different from controls. The fish exposed to all doses of chloryprifos grew significantly less than controls in a dose-dependent manner. No significant differences were found in hepatic IGF-I mRNA levels in any treatments. To establish patterns of gene up- or down-regulation, I performed differential display analysis on livers of several fish from the previous two experiments. Several genes were identified as being similar to fish including a microsatellite sequence, a choriogenin (vitelline envelope) protein mRNA sequence, a transferrin mRNA sequence and several ribosomal RNA sequences. This technique to evaluate gene expression will become more useful when more fish genes are added to the data bases

Concizumab restores thrombin generation potential in patients with haemophilia: Pharmacokinetic/pharmacodynamic modelling results of concizumab phase 1/1b data

Introduction: Concizumab enhances thrombin generation (TG) potential in haemophilia patients by inhibiting tissue factor pathway inhibitor (TFPI). In EXPLORER3 (phase 1b), a dose‐dependent pharmacokinetic/pharmacodynamic (PK/PD) relationship was confirmed between concizumab dose, free TFPI and TG potential. Aim: Determine the association between concizumab exposure, PD markers (free TFPI; peak TG) and bleeding episodes to establish the minimum concizumab concentration for achieving sufficient efficacy. Methods: Free TFPI predictions were generated using an estimated concizumab‐free TFPI exposure‐response (Emax) model based on concizumab phase 1/1b data for which simultaneously collected concizumab and free TFPI samples were available. Concizumab concentration at the time of a bleed was predicted using a PK model, based on available data for concizumab doses >50 μg/kg to ≤9 mg/kg. Peak TG vs concizumab concentration analyses and an Emax model were constructed based on EXPLORER3 observations. Results: The Emax model showed a tight PK/PD relationship between concizumab exposure and free TFPI; free TFPI decreased with increasing concizumab concentration. A strong correlation between concizumab concentration and peak TG was observed; concizumab >100 ng/mL re‐established TG potential to within the normal reference range. Estimated EC50 values for the identified concizumab‐free TFPI and concizumab‐TG potential models were very similar, supporting free TFPI as an important biomarker. A correlation between bleeding episode frequency and concizumab concentration was indicated; patients with a concizumab concentration >100 ng/mL experienced less frequent bleeding. The PK model predicted that once‐daily dosing would minimize within‐patient concizumab PK variability.Novo Nordis

Expert Statements on the Standard of Care in Critically Ill Adult Patients With Atypical Hemolytic Uremic Syndrome.

A typical hemolytic uremic syndrome (aHUS) presents similarly to thrombotic thrombocytopenic purpura (TTP) and other causes or conditions with thrombotic microangiopathy (TMA), such as disseminated intravascular coagulation or sepsis. Similarity in clinical presentation may hinder diagnosis and optimal treatment selection in the urgent setting in the ICU. However, there is currently no consensus on the diagnosis or treatment of aHUS for ICU specialists. This review aims to summarize available data on the diagnosis and treatment strategies of aHUS in the ICU to enhance the understanding of aHUS diagnosis and outcomes in patients managed in the ICU. To this end, a review of the recent literature (January 2009-March 2016) was performed to select the most relevant articles for ICU physicians. Based on the paucity of adult aHUS cases overall and within the ICU, no specific recommendations could be formally graded for the critical care setting. However, we recognize a core set of skills required by intensivists for diagnosing and managing patients with aHUS: recognizing thrombotic microangiopathies, differentiating aHUS from related conditions, recognizing involvement of other organ systems, understanding the pathophysiology of aHUS, knowing the diagnostic workup and relevant outcomes in critically ill patients with aHUS, and knowing the standard of care for patients with aHUS based on available data and guidelines. In conclusion, managing critically ill patients with aHUS requires basic skills that, in the absence of sufficient data from patients treated within the ICU, can be gleaned from an increasingly relevant literature outside the ICU. More data on critically ill patients with aHUS are needed to validate these conclusions within the ICU setting

International recommendations on the diagnosis and treatment of acquired hemophilia A

Acquired hemophilia A (AHA), a rare bleeding disorder caused by neutralizing autoantibodies against coagulation factor VIII (FVIII), occurs in both men and women without a previous history of bleeding. Patients typically present with an isolated prolonged activated partial thromboplastin time due to FVIII deficiency. Neutralizing antibodies (inhibitors) are detected using the Nijmegen-modified Bethesda assay. Approximately 10% of patients do not present with bleeding and, therefore, a prolonged activated partial thromboplastin time should never be ignored prior to invasive procedures. Control of acute bleeding and prevention of injuries that may provoke bleeding are top priorities in patients with AHA. We recommend treatment with bypassing agents, including recombinant activated factor VII, activated prothrombin complex concentrate, or recombinant porcine FVIII in bleeding patients. Autoantibody eradication can be achieved with immunosuppressive therapy, including corticosteroids, cyclophosphamide and rituximab, or combinations thereof. The median time to remission is 5 weeks, with considerable interindividual variation. FVIII activity at presentation, inhibitor titer and autoantibody isotype are prognostic markers for remission and survival. Comparative clinical studies to support treatment recommendations for AHA do not exist; therefore, we provide practical consensus guidance based on recent registry findings and the authors’ clinical experience in treating patients with AHA

Peritoneal cavity phagocytes from the teleost sea bass express a glucocorticoid receptor (cloned and sequenced) involved in genomic modulation of the in vitro chemiluminescence response to zymosan

Abstract To gain further insight into the role of cortisol in Wsh innate immune responses, we cloned and sequenced a 2592 bp cDNA from sea bass (Dicentrarchus labrax) peritoneal leukocytes (PCLs) encoding a glucocorticoid receptor (DlGR1). The deduced aminoacid sequence displayed that DlGR1 belong to a multigenic family of steroid hormone receptors, and exhibited high homology (80%) to the Burton’s mouth breeder (Haplochromis burtoni) HbGR1. The DlGR1 functional domains presented homologies with those of several vertebrate species. In situ hybridization assay revealed that DlGR1 was expressed in macrophages and neutrophils from the peritoneal cavity. Since in a previous paper, sea bass PCL chemiluminescence response (CL) has been related to increased respiratory burst of phagocytes stimulated with zymosan, PCLs, preincubated in vitro with cortisol at various concentrations, were assayed for their CL response. Dose-dependent cortisol inhibitory eVects, and signiWcant competitive activity of a low concentration of mifepristone (RU486), a glucocorticoid-receptor blocker, supported that cortisol–GR interaction was involved in modulating CL response via a genomic pathway. Results also indicated that cortisol could be eVective through an additional not-genomic way, and showed that high doses of RU486 exerted an inhibitory eVect on PCL chemiluminescence activity. © 2006 Elsevier Inc. All rights reserved

Safety and pharmacokinetics of anti-TFPI antibody (concizumab) in healthy volunteers and patients with hemophilia: a randomized first human dose trial

BACKGROUND: Prophylaxis with either intravenous (i.v.) factor VIII (FVIII) or FIX is the gold standard of care for patients with severe hemophilia. A monoclonal antibody (concizumab) targeting tissue factor pathway inhibitor (TFPI) that can be administered subcutaneously (s.c.) has the potential to alter current concepts of prophylaxis in hemophilia. OBJECTIVES: To evaluate the safety and describe the pharmacokinetics and pharmacodynamics of single-dose concizumab in healthy volunteers and patients with hemophilia A or B. METHODS: In this first human dose, phase 1, multicenter, randomized, double-blind, placebo-controlled trial escalating single i.v. (0.5-9000 μg kg(-1) ) or s.c. (50-3000 μg kg(-1) ) doses of concizumab were administered to healthy volunteers (n = 28) and hemophilia patients (n = 24). RESULTS: Concizumab had a favorable safety profile after single i.v. or s.c. administration. There were no serious adverse events and no anti-concizumab antibodies. No clinically relevant changes in platelets, prothrombin time, activated partial thromboplastin time, fibrinogen, or antithrombin were found. A dose-dependent procoagulant effect of concizumab was seen as increased levels of D-dimers and prothrombin fragment 1 + 2. Nonlinear pharmacokinetics of concizumab was observed due to target-mediated clearance. A maximum mean AUC0-∞ of 33 960 h μg mL(-1) and a maximum mean concentration of 247 μg mL(-1) was measured at the highest dose. CONCLUSIONS: Concizumab showed a favorable safety profile after i.v. or s.c. administration and nonlinear pharmacokinetics was observed due to target-mediated clearance. A concentration-dependent procoagulant effect of concizumab was observed, supporting further study into the potential use of s.c. concizumab for hemophilia treatment

Expert statement on the ICU management of patients with thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is fatal in 90% of patients if left untreated and must be diagnosed early to optimize patient outcomes. However, the very low incidence of TTP is an obstacle to the development of evidence-based clinical practice recommendations, and the very wide variability in survival rates across centers may be partly ascribable to differences in management strategies due to insufficient guidance. We therefore developed an expert statement to provide trustworthy guidance about the management of critically ill patients with TTP. As strong evidence was difficult to find in the literature, consensus building among experts could not be reported for most of the items. This expert statement is timely given the recent advances in the treatment of TTP, such as the use of rituximab and of the recently licensed drug caplacizumab, whose benefits will be maximized if the other components of the management strategy follow a standardized pattern. Finally, unanswered questions are identified as topics of future research on TTP