Suppression of fibrin(ogen)-driven pathologies through controlled knockdown by lipid nanoparticle delivery of siRNA

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated siRNA targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga mRNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16% and 4% of normal within one-week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with a 0.5, 1, and 2 mg/kg dose, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumour cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provide the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

The Natural Combination Medicine Traumeel (Tr14) Improves Resolution of Inflammation by Promoting the Biosynthesis of Specialized Pro-Resolving Mediators

The resolution of inflammation is an integral part of the acute inflammatory response and eventually leads to the return to homeostasis. It is supported by specialized pro-resolving mediators (SPMs) that act as immunoresolvents via specific G-protein-coupled receptors. In contrast to classical non-steroidal anti-inflammatory drugs (NSAIDs) that suppress the formation of pro-inflammatory lipid mediators such as prostaglandins, novel pharmacotherapeutic concepts propose to foster the biosynthesis of beneficial SPMs. Here, we demonstrate that the natural combination medicine Traumeel (Tr14) improves resolution of inflammation by promoting SPM formation. Tr14 enhanced the biosynthesis of 12-/15-lipoxygenase (LOX) products and of SPMs in zymosan-induced mouse peritonitis as well as in human monocyte-derived macrophages challenged with Staphylococcus aureus. Importantly, in the peritonitis model, Tr14 supported the recruitment of innate leukocytes and the efferocytotic capacity of macrophages, and positively influenced the inflammation resolution index. Taken together, we suggest that based on these properties Tr14 may possess therapeutic potential as an enhancer for the resolution of inflammatory processes.

724 Evaluation of an anti-human IL-1β antibody in monosodium urate crystals-induced peritonitis model in hIL-1β HuGEMM™ mice

BackgroundGout is a chronic inflammatory disease featuring the deposition of monosodium urate (MSU) crystals in the synovial fluid of patients, followed by NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome activation and bioactive IL-1β release, which recruits neutrophils to the local inflammation sites. Blocking IL-1β function is becoming a a potent therapeutic approach for gout and gouty arthritis. Conventional MSU-induced peritonitis in C57BL/6 mice provides a simple and rapid evaluation of therapeutics targeting inflammasome activation. However, this murine model has limitations when it comes to the evaluation of human-specific antibodies, for example, anti-human IL-1β (anti-hIL-1β) monoclonal antibodies (mAb). Thus, a murine model to assess the efficacy of anti-hIL-1β mAb is needed. We have developed a hIL-1β knock-in mouse model (hIL-1β HuGEMM™), which is able to facilitate the pre-clinical evaluation of drugs targeting specific human biological molecules especially when mouse ortholog is not available. Therefore, an MSU crystals induced peritonitis model using hIL-1β HuGEMM™ mice provides a robust model to evaluate therapies targeting hIL-1β.MethodsMSU crystals were injected intraperitoneally into human IL-1β (hIL-1β) knock-in mice, where the coding sequence of mouse IL-1β was replaced by hIL-1β. Prior to MSU crystal administration, mice received treatment of either vehicle or anti-hIL-1β antibody. Six hours facilitate post MSU crystal injection, serum and lavage flushed with PBS were collected. Subsequently, cytokine protein levels in the serum were determined by MSD, and the population of polymorphonuclear leukocytes (PMNs) (live CD11b+ Ly-6GHi cells) in the lavage was analysed by flow cytometry.ResultsThe vehicle treatment group showed a dramatic increase in hIL-1β secretion and PMN leukocytes, in comparison to the group that did not receive MSU, which suggests a successful induction of acute inflammatory response in the peritoneal cavity. In contrast, mice that received a single administration of anti-hIL-1β antibody 24 hours prior to MSU injection exhibited a significantly lower level of hIL-1β when compared to the vehicle treatment group, which implies that the anti-hIL-1β mAb efficaciously neutralized hIL-1β secretion. In addition, TNF-α and IL-6, two further cytokines downstream of IL-1β, were significantly reduced in the anti-hIL-1β mAb treatment group. However, the PMN leukocyte infiltration in the anti-hIL-1β mAb treatment group did not change in comparison to the vehicle group.ConclusionsIn this study, an MSU crystals-induced peritonitis model was successfully established in hIL-1β HuGEMM mice, which has the potential to evaluate immune therapeutics with anti-hIL-1β blockades.

1002. Activation of the Enterococcus faecalis Cell Envelope Stress Response through the Novel MadRS System Is Associated With Increased Size of Cardiac Microlesions

Background Enterococci are opportunistic pathogens that can present a therapeutic challenge due to the acquisition of antibiotic resistance. Our previous work has shown the MadRS stress response system plays an important role in defending the enterococcal membrane against daptomycin and antimicrobial peptides (AMP) made by the innate immune system. Strains lacking the MadR response regulator show increased susceptibility to the cathelicidn LL-37 in vitro. A change from alanine to glutamate in the sensor kinase MadS (madSA202E) leads to activation of the system and impaired killing by AMPs. In this study, we evaluated the impact of MadRS function in vivo using a mouse peritonitis model of E. faecalis (Efs) infection. Methods A laboratory strain Efs OG1RF and two derivatives, OG1RFΔmadR and OG1RFmadSA202E were included. Six mice per strain were inoculated via intraperitoneal injection of ~5x108 CFU/mL of bacteria in 50% sterile rat fecal extract, and followed for 96 hours post infection. Difference in survival between strains was determined by Mantel-Cox test. At the time of death, hearts were aseptically removed, fixed in formalin, and embedded in paraffin. Organs were bisected and sectioned, with every 4th section stained with hematoxylin and eosin (8 total sections per animal). Sections were imaged at 40x magnification, the number of lesions for each section was recorded, and lesion size was determined using imageJ. Results There was no difference in median survival between animals infected with OG1RF and OG1RFΔmadR (22.5 v 21 hours, p=0.31), OG1RF and OG1RFmadSA202E (22.5 v 24 hours, p=0.29), or OG1RFΔmadR and OG1RFmadSA202E (21 v 24 hours, p=0.13). There was a significant difference in the number and size of cardiac lesions between the strains. Mice infected with OG1RFmadSA202E had a significantly higher number of cardiac microlesions as compared to those infected with OG1RFΔmadR (Fig 1). The size of the lesions in mice infected with OG1RFmadSA202E was also significantly larger than those in OG1RF wild type (Fig 1). Figure 1. Cardiac microlesions in a mouse peritonitis model of Enterococcus faecalis infection. Mouse hearts were removed at time of animal death, placed in formalin, and embedded in paraffin. Organs were bisected, then sectioned with every 4th section stained with hematoxylin and eosin (H&E, 8 sections per animal). H&E stained sections were imaged at 40x magnification, the number and size of lesions was determined for 48 sections per strain. Representative cardiac microlesions (arrow) are shown for A) E. faecalis OG1RF, B) OG1RFΔmadR and C) OG1RFmadSA202E, scale bar 20 μm. D) The number of microlesions observed in each section, and E) the area of the lesions for each strain are shown above, differences in means were determined with one way ANOVA using Tukey’s test for multiple comparisons. ns, not significant. Conclusion Changes in MadRS did not impact overall survival in mice, but did alter the number and size of cardiac microlesions. Further experiments are needed to determine if these changes could adversely affect therapy or rates of relapse. Disclosures William R. Miller, MD , Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support) William R. Miller, MD , Entasis (Individual(s) Involved: Self): Scientific Research Study Investigator; Merck (Individual(s) Involved: Self): Grant/Research Support Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

Efficacy of Omadacycline Against Multiresistant Enterococcus faecium Strains in a Mouse Peritonitis Model

Omadacycline (OMC) showed better in vitro potency than daptomycin (DAP) or vancomycin (VAN) against Van R , AMP R , DAP non-susceptible, linezolid R , cfr (B) + Enterococcus faecium strains. In a mouse peritonitis model, OMC also showed significantly better animal survival during and at the end of the study than DAP or VAN against these E. faecium strains. However, OMC, DAP and VAN showed comparable in vitro and in vivo efficacy against a non-VRE, tetracycline-resistant, DAP-susceptible, E. faecium strain.

Thiosulfinate-Enriched Allium sativum Extract as an Adjunct to Antibiotic Treatment of Sepsis in a Rat Peritonitis Model

Up to now, there are no studies that have shown a decrease in morbidity and mortality in the context of sepsis and septic shock, except for antibiotic therapy and the objective-guided resuscitation strategy. The goal was to evaluate the use of thiosulfinate-enriched Allium sativum extract (TASE) as an adjuvant in the management of sepsis. An experimental in vivo study was carried out with male Sprague Dawley® rats. Animals were randomized in three treatment groups: the control group (I), antibiotic (ceftriaxone) treatment group (II) and ceftriaxone plus TASE treatment group (III). All animals were housed and inoculated with 1 × 1010 CFU/15 mL of intraperitoneal Escherichia coli ATCC 25922. Subsequently, they received a daily treatment according to each group for 7 days. Clinical, analytical, microbiological, and histopathological parameters were evaluated. Statistically significant clinical improvement was observed in the ceftriaxone plus TASE vs. ceftriaxone group in weight, ocular secretions, whiskers separation and physical activity level (p ≤ 0.05). When comparing interleukins on the third day of treatment between II and III, we found statistically significant differences in IL-1 levels (p < 0.05). Blood and peritoneal liquid cultures of group I were positive for multisensitive E. coli. Group II and III cultures were negative for E. coli, although an overgrowth of Enterococcus faecalis was found. In conclusion, TASE used as an adjuvant to antibiotic treatment in the management of sepsis could improve response profiles with sepsis attenuation, thus reducing overall mortality after an animal peritonitis model.

A novel mast cell-dependent allergic peritonitis model

Background: Typical murine models of allergic inflammation are induced by the combination of ovalbumin and aluminum hydroxide. However, accumulating evidence indicates that, in models of asthma and atopic dermatitis, allergic inflammation can be generated in the absence of aluminum hydroxide. Moreover, co-administration of S. aureus enterotoxin B with ovalbumin can enhance inflammation. Objective: The objective of this study was to establish a rapid and mast cell-dependent murine model of allergic inflammation by inducing allergic peritonitis using ovalbumin and S. aureus enterotoxin B. Methods: Allergic peritonitis was induced in C57BL/6 mice by subcutaneous sensitization and intraperitoneal challenge with ovalbumin and S. aureus enterotoxin B. Disease characteristics were assessed by flow cytometry, ELISA, Trypan Blue exclusion and colorimetric assays. Results: Time course of the allergic peritonitis revealed a peak of peritoneal inflammation 48h after challenge, as assessed by total cells and eosinophil counts. Decrease of cell numbers started 96h post challenge with complete clearance within 168h. Moreover, significantly higher levels of tryptase and increased vascular permeability were found 30 min following challenge. Allergic inflammation induction by ovalbumin and S. aureus enterotoxin B was impaired in mast cells deficient mice and partially restored by mice reconstitution with bone marrow derived mast cells, indicating the mast cell role in this model. Conclusion: We present a novel model of allergic peritonitis that is mast cell-dependent, simple and robust. Moreover, the use of S. aureus enterotoxin B better resembles human allergic inflammation, which is known to be characterized by the colonization of Staphylococcus aureus.