Biodistribution of the recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in rats

AbstractIntroductionThe recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) is undergoing clinical trials for prophylaxis and on-demand treatment of haemophilia B patients. The aim of this study was to investigate the pharmacokinetics, whole-body and knee joint distribution of rIX-FP following intravenous administration to rats, compared with a marketed, non-fused rFIX and recombinant human albumin.Material and Methods[3H]-rIX-FP, [3H]-rFIX or [3H]-albumin were administered to rats followed by quantitative whole-body autoradiography over 24 or 240hours, and the tissue distribution as well as elimination of radioactivity were measured.ResultsElimination of all radioactivity derived from the three proteins was shown to occur primarily via the urine. The tissue distribution of [3H]-rIX-FP and [3H]-rFIX (but not of [3H]-albumin) was comparable, both penetrating predominantly into bone, and well-perfused tissues, suggesting that the rIX moiety determines the distribution pattern of rIX-FP, while the albumin moity is responsible for the prolonged plasma and tissue retention. Detailed knee-joint analysis indicated rapid presence of [3H]-rIX-FP and [3H]-rFIX in synovial and mineralised bone tissue, mostly localised to the zone of calcified cartilage. Longest retention times were observed in the bone marrow and the endosteum of long bones. Intriguingly, [3H]-rIX-FP- and [3H]-albumin-derived radioactive signals were detectable up to 240hours, while [3H]-rFIX-derived radioactivity rapidly declined after 1hour post-dosing correlating to the extended plasma half-life of [3H]-rIX-FP.ConclusionThe prolonged plasma and tissue retention of rIX-FP achieved by albumin fusion may allow a reduction in dosing frequency leading to increased therapeutic compliance and convenience

Defective glycosylation of coagulation factor XII underlies hereditary angioedema type III

Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12–/– mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes

Monocyte-Directed RNAi Targeting CCR2 Improves Infarct Healing in Atherosclerosis-Prone Mice

Background—Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates left ventricular remodeling. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. However, the appropriate magnitude and timing of interventions are largely unknown, in part because noninvasive monitoring tools are lacking. Here, we used nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6Chigh monocyte subset traffic, to reduce infarct inflammation in apolipoprotein E–deficient (apoE−/−) mice after MI. We used dual-target positron emission tomography/magnetic resonance imaging of transglutaminase factor XIII (FXIII) and myeloperoxidase (MPO) activity to monitor how monocyte subset–targeted RNAi altered infarct inflammation and healing. Methods and Results—Flow cytometry, gene expression analysis, and histology revealed reduced monocyte numbers and enhanced resolution of inflammation in infarcted hearts of apoE−/− mice that were treated with nanoparticle-encapsulated siRNA. To follow extracellular matrix cross-linking noninvasively, we developed a fluorine-18–labeled positron emission tomography agent (18F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged with a molecular magnetic resonance imaging sensor of MPO activity (MPO-Gd). Positron emission tomography/magnetic resonance imaging detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal; P<0.05), whereas 18F-FXIII positron emission tomography reflected unimpeded matrix cross-linking in the infarct. Silencing of CCR2 during the first week after MI improved ejection fraction on day 21 after MI from 29% to 35% (P<0.05). Conclusion—CCR2-targeted RNAi reduced recruitment of Ly-6Chigh monocytes, attenuated infarct inflammation, and curbed post-MI left ventricular remodeling.National Heart, Lung, and Blood InstituteUnited States. Dept. of Health and Human Services (contract No. HHSN268201000044C)National Institutes of Health (U.S.) (grant R01-HL096576)National Institutes of Health (U.S.) (grant R01-HL095629)National Institutes of Health (U.S.) (grant T32-HL094301)Deutsche Forschungsgemeinschaft (HE-6382/1-1

Special considerations for studies of extracellular vesicles from parasitic helminths: A community‐led roadmap to increase rigour and reproducibility

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved

Estrogen receptor transcription and transactivation: Structure-function relationship in DNA- and ligand-binding domains of estrogen receptors

Estrogen receptors are members of the nuclear receptor steroid family that exhibit specific structural features, ligand-binding domain sequence identity and dimeric interactions, that single them out. The crystal structures of their DNA-binding domains give some insight into how nuclear receptors discriminate between DNA response elements. The various ligand-binding domain crystal structures of the two known estrogen receptor isotypes (α and β) allow one to interpret ligand specificity and reveal the interactions responsible for stabilizing the activation helix H12 in the agonist and antagonist positions

Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-Binding Motif

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR

New World Hantaviruses Activate IFNλ Production in Type I IFN-Deficient Vero E6 Cells

Hantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS). These viruses induce a strong interferon-stimulated gene (ISG) response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN), rendering them unable to mount an efficient innate immune response to virus infection. Interferon lambda, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner.We show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNlambda. Three New World hantaviruses were similarly able to induce IFNlambda expression in this cell line. The IFNlambda contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7) without inducing ISGs.Herein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNlambda. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNlambda production in these cells might increase their utility for virus propagation

Synthesis Paper: Targeted Livestock Grazing: Prescription for Healthy Rangelands

Targeted livestock grazing is a proven tool for manipulating rangeland vegetation, and current knowledge about targeted livestock grazing is extensive and expanding rapidly. Targeted grazing prescriptions optimize the timing, frequency, intensity, and selectivity of grazing (or browsing) in combinations that purposely exert grazing/browsing pressure on specific plant species or portions of the landscape. Targeted grazing differs from traditional grazing management in that the goal of targeted grazing is to apply defoliation or trampling to achieve specific vegetation management objectives, whereas the goal of traditional livestock grazing management is generally the production of livestock commodities. A shared aim of targeted livestock grazing and traditional grazing management is to sustain healthy soils, flora, fauna, and water resources that, in turn, can sustain natural ecological processes (e.g., nutrient cycle, water cycle, energy flow). Targeted grazing prescriptions integrate knowledge of plant ecology, livestock nutrition, and livestock foraging behavior. Livestock can be focused on target areas through fencing, herding, or supplement placement. Although practices can be developed to minimize the impact of toxins contained in target plants, the welfare of the animals used in targeted grazing must be a priority. Monitoring is needed to determine if targeted grazing is successful and to refine techniques to improve efficacy and efficiency. Examples of previous research studies and approaches are presented to highlight the ecological benefits that can be achieved when targeted grazing is applied properly. These cases include ways to suppress invasive plants and ways to enhance wildlife habitat and biodiversity. Future research should address the potential to select more adapted and effective livestock for targeted grazing and the associated animal welfare concerns with this practice. Targeted livestock grazing provides land managers a viable alternative to mechanical, chemical, and prescribed fire treatments to manipulate rangeland vegetation

Genome-wide analysis identifies 12 loci influencing human reproductive behavior.

The genetic architecture of human reproductive behavior-age at first birth (AFB) and number of children ever born (NEB)-has a strong relationship with fitness, human development, infertility and risk of neuropsychiatric disorders. However, very few genetic loci have been identified, and the underlying mechanisms of AFB and NEB are poorly understood. We report a large genome-wide association study of both sexes including 251,151 individuals for AFB and 343,072 individuals for NEB. We identified 12 independent loci that are significantly associated with AFB and/or NEB in a SNP-based genome-wide association study and 4 additional loci associated in a gene-based effort. These loci harbor genes that are likely to have a role, either directly or by affecting non-local gene expression, in human reproduction and infertility, thereby increasing understanding of these complex traits