Recombinant Human HPS Protects Mice and Nonhuman Primates from Acute Liver Injury.

Acute liver injury shares a common feature of hepatocytes death, immune system disorders, and cellular stress. Hepassocin (HPS) is a hepatokine that has ability to promote hepatocytes proliferation and to protect rats from D-galactose (D-Gal)- or carbon tetrachloride (CCl4)-induced liver injury by stimulating hepatocytes proliferation and preventing the high mortality rate, hepatocyte death, and hepatic inflammation. In this paper, we generated a pharmaceutical-grade recombinant human HPS using mammalian cells expression system and evaluated the effects of HPS administration on the pathogenesis of acute liver injury in monkey and mice. In the model mice of D-galactosamine (D-GalN) plus lipopolysaccharide (LPS)-induced liver injury, HPS treatment significantly reduced hepatocyte death and inflammation response, and consequently attenuated the development of acute liver failure. In the model monkey of D-GalN-induced liver injury, HPS administration promoted hepatocytes proliferation, prevented hepatocyte apoptosis and oxidation stress, and resulted in amelioration of liver injury. Furthermore, the primary pharmacokinetic study showed natural HPS possesses favorable pharmacokinetics; the acute toxicity study indicated no significant changes in behavioral, clinical, or histopathological parameters of HPS-treated mice, implying the clinical potential of HPS. Our results suggest that exogenous HPS has protective effects on acute liver injury in both mice and monkeys. HPS or HPS analogues and mimetics may provide novel drugs for the treatment of acute liver injury.

Citrullinated human fibrinogen triggers arthritis through an inflammatory response mediated by IL-23/IL-17 immune axis.

Rheumatoid arthritis (RA) is an autoimmune disease that causes joint destruction. Although its etiology remains unknown, citrullinated proteins have been considered as an auto-antigen able to trigger an inflammatory response in RA. Herein, we modified the classical antigen-induced arthritis (AIA) model by using citrullinated human plasma fibrinogen (hFIB) as an immunogen to investigate the mechanism of inflammation-driven joint damage by citrullinated hFIB in C57BL/6 mice. We found that hFIB-immunized mice showed high serum levels of anti-citrullinated peptides antibodies (ACPAs). Moreover, hFIB immunized mice showed increased mechanical hyperalgesia, massive leukocyte infiltration, high levels of inflammatory mediators, and progressive joint damage after the intra-articular challenge with citrullinated hFIB. Interestingly, hFIB-induced arthritis was dependent on IL-23/IL-17 immune axis-mediated inflammatory responses since leukocyte infiltration and mechanical hyperalgesia were abrogated in Il17ra-/- and Il23a-/- mice. Thus, we have characterized a novel model of experimental arthritis suitable to investigate the contribution of ACPAs and Th17 cell-mediated immune response in the pathogenesis of RA.

Focal adhesion kinase and Src mediate microvascular hyperpermeability caused by fibrinogen- γC- terminal fragments.

OBJECTIVES: We previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvß3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation. METHODS AND RESULTS: Using intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition. CONCLUSION: The FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.

Histologic changes associated with the use of fibrinogen- and thrombin-impregnated collagen in the prevention of pulmonary air leakage.

OBJECTIVE: Although fibrinogen- and thrombin-impregnated collagen (TachoSil; Takeda GmbH, Linz, Austria) can be applied to prevent air leakage, the impact of its use on lung healing is unknown. Therefore, we histologically evaluated the long-term healing process associated with the use of TachoSil to prevent air leakage in a canine model. METHODS: Via left thoracotomy, visceral pleural defects of 10 × 10 mm were created on each lung lobe of female beagles. After air leakage was confirmed, each pleural defect was covered with TachoSil. The repair sites were histologically evaluated on postoperative days 0, 4, 7, 14, 28, and 56. RESULTS: All animals survived, and none developed pneumothorax. Histologically, inflammatory cells infiltrated the TachoSil from the pleural defect, and pleural mesothelium comprised the regenerated outermost layer of the TachoSil soon after the surgery. Inflammatory cells, myofibroblasts, and neovascular vessels subsequently spread over the entire TachoSil. The number of inflammatory cells decreased, and myofibroblast and neovascular vessels replaced the entire TachoSil. In addition, the elastic layer started to regenerate from both edges and completely repaired the pleural defect. The lung parenchyma around the pleural defects was not influenced throughout the observational period, because these healing processes occurred only inside the TachoSil. CONCLUSIONS: TachoSil provided a mechanical scaffold on which healing could proceed, followed by biodegradation over the long term. TachoSil safely repaired the pleural defects without affecting lung parenchyma.

Analysis of the safety and pharmacodynamics of human fibrinogen concentrate in animals.

Fibrinogen, a soluble 340kDa plasma glycoprotein, is critical in achieving and maintaining hemostasis. Reduced fibrinogen levels are associated with an increased risk of bleeding and recent research has investigated the efficacy of fibrinogen concentrate for controlling perioperative bleeding. European guidelines on the management of perioperative bleeding recommend the use of fibrinogen concentrate if significant bleeding is accompanied by plasma fibrinogen levels less than 1.5-2.0g/l. Plasma-derived human fibrinogen concentrate has been available for therapeutic use since 1956. The overall aim of the comprehensive series of non-clinical investigations presented was to evaluate i) the pharmacodynamic and pharmacokinetic characteristics and ii) the safety and tolerability profile of human fibrinogen concentrate Haemocomplettan P® (RiaSTAP®). Pharmacodynamic characteristics were assessed in rabbits, pharmacokinetic parameters were determined in rabbits and rats and a safety pharmacology study was performed in beagle dogs. Additional toxicology tests included: single-dose toxicity tests in mice and rats; local tolerance tests in rabbits; and neoantigenicity tests in rabbits and guinea pigs following the introduction of pasteurization in the manufacturing process. Human fibrinogen concentrate was shown to be pharmacodynamically active in rabbits and dogs and well tolerated, with no adverse events and no influence on circulation, respiration or hematological parameters in rabbits, mice, rats and dogs. In these non-clinical investigations, human fibrinogen concentrate showed a good safety profile. This data adds to the safety information available to date, strengthening the current body of knowledge regarding this hemostatic agent.

Positive allosteric modulation of metabotropic glutamate receptor 5 down-regulates fibrinogen-activated microglia providing neuronal protection.

Microglial activation and blood brain barrier dysfunction are significant hallmarks in an array of neurodegenerative disorders. A leaky blood brain barrier potentially allows infiltration of blood-borne proteins into the CNS parenchyma, and previous studies have shown that the blood borne protein fibrinogen (FG) can activate microglia to produce a neurotoxic phenotype. Here we show that FG-mediated neurotoxicity and ERK1/2 phosphorylation in neuronal cultures is significantly attenuated by activation of metabotropic glutamate receptor 5 (mGluR5) but not mGluR2. Furthermore, FG-mediated microglial activation was down-regulated by direct mGluR5 activation on these cells but not by mGluR2, suggesting that targeting microglial mGluR5 provides neuronal protection against blood protein-triggered innate inflammatory responses.

A fibrinogen-based precision microporous scaffold for tissue engineering.

Fibrin has been long used as an effective scaffolding material to grow a variety of cells and tissue constructs. It has been utilized mainly as a hydrogel in varying concentrations to provide an environment in which suspended cells work to rearrange the fibers and lay down their own extracellular matrix. For these fibrin hydrogels to be useful in many tissue-engineering applications, the gels must be cultured for long periods of time in order to increase their mechanical strength to the levels of native tissues. High concentrations of fibrinogen increase the mechanical strength of fibrin hydrogels, but at the same time reduce the ability of cells within the scaffold to spread and survive. We present a method to create a microporous, nanofibriliar fibrin scaffold that has controllable pore size, porosity, and microstructure for applications in tissue engineering. Fibrin has numerous advantages as a scaffolding material as it is normally used by the body as temporary scaffolding for tissue regeneration and healing, and can be autologously sourced. We present here a scaffolding process which enhances the mechanical properties of the fibrin hydrogel by forming it surrounding poly(methyl-methacrylate) beads, then removing the beads with acetone to form an interconnected microporous network. The acetone serves the dual purpose of precipitating and fixing the fibrinogen-based scaffolds as well as adding strength to the network during polymer bead removal. Effects of fibrinogen concentration and time in acetone were examined as well as polymerization with thrombin. A natural crosslinker, genipin, was also used to add strength to the scaffolds, producing a Young's modulus of up to 184+/-5 kPa after 36 h of reaction. Using these methods we were able to produce microporous fibrin scaffolds that support cell growth and have mechanical properties similar to many native tissues.

Cytotoxic and antitumor effect of fibrinogen-methotrexate conjugate.

In this paper we describe the chemical procedure of fibrinogen-methotrexate (F-MTX) conjugate preparation and its in vitro and in vivo antitumor activity. F-MTX conjugates were synthesized in reaction of fibrinogen with MTX N-hydroxysuccynimide ester. The conjugates were not cross-linked and were soluble in water. The results of the in vitro and in vivo studies have shown: (1) a lower in vitro cytotoxicity of the F-MTX conjugate as compared with MTX alone; (2) a significantly higher in vivo antitumor activity of the F-MTX conjugate in mice with P388 leukemia as compared with MTX alone; (3) a significantly increased in vivo lethal toxicity of F-MTX as compared with MTX. The results suggest the therapeutic utility of the fibrinogen-methotrexate conjugate and the usefulness of fibrinogen as a chemotherapeutic drug carrier. However, a new effort in the preparation of F-MTX conjugate should be made to decrease its in vivo toxicity.

Mediastinal fibrin glue: hemostatic effect and tissue response in calves.

There is continued controversy regarding the effectiveness and potential adverse effects of fibrin glue. Thus, we chose to evaluate it in a model of experimental calf aortic valve replacement that has been previously well established. Concentrated fibrinogen and topical thrombin were sprayed to form a thin layer of fibrin glue over the mediastinal tissues of 20 consecutive calves undergoing aortic valve replacement. Chest tube outputs of these animals were compared with those of the preceding 20 consecutive calves undergoing aortic valve replacement without fibrin glue. All procedures were performed by the same surgeon, and no other technical changes were made between the two series. Total postoperative chest tube output (mean +/- standard error) was 553 +/- 50 mL for the calves treated with fibrin glue and 1,155 +/- 103 mL for the control calves (p less than 0.001). On histological examination of mediastinal tissues from 5 treated calves killed 6 weeks after operation, there was no evidence of inflammation, fibrosis, or residual fibrin. To our knowledge, this is the first controlled laboratory study to show that fibrin glue spray is an effective hemostatic agent and that it produces no long-term tissue reaction.

A screening method using tissue culture for evaluation of potential retinal adhesives.

Several adhesives have been tested for their potentially toxic effects on embryonic retinal tissue. The authors have characterized the effects of the adhesives on neurofilament extension and also on surgical-wound "re-knitting." While none of the adhesives in the sample (including those in current surgical use) seem to be ideal, the model advanced has application for the continuing development of better 'bio-adhesives'. The most immediate application is within the field of vitreoretinal surgery in situations where conventional procedures currently seem inadequate.

Autologous fibrin tissue adhesive for cerebrospinal fluid leaks: a controlled study of neurotoxicity.

Postoperative cerebrospinal fluid (CSF) leakage continues to be one of the most common and potentially serious complications following translabyrinthine surgery despite numerous strategies aimed at its prevention. Fibrinogen-based tissue adhesives may be helpful in decreasing this complication rate. Although commercial glues have been used widely in Europe (especially for dural repairs), they are not approved for use in the United States. Recent investigation has provided a relatively simple technique for producing a comparable autologous glue that obviates the risks of the commercial product. Since this glue will bind fascia, fat, and dura in the watertight fashion, it is potentially ideal for preventing CSF leaks. Experimental studies in rabbits reveal that autologous tissue adhesive can be used safely around intact nerves, suggesting it can be used safely to supplement fat, fascia, or muscle plugs for closing translabyrinthine defects. Clinical trials to test the efficacy of tissue adhesives in this application are currently under way.

Autologous fibrin tissue adhesive biodegration and systemic effects.

A series of experiments was conducted to investigate the rate of Autologous Fibrin Tissue Adhesive (AFTA) degradation by the fibrinolysis inhibitor, epsilon amino caproic acid (EACA). The duration of AFTA clots in vitro, subcutaneous, and in the middle ear was prolonged for a time interval that was proportional to the concentration of EACA in Component II of the adhesive. No toxic reactions were observed in the middle or inner ear. Systemic pathology (thrombosis or emboli) could not be related to the presence of EACA applied in the middle ear or directly into the blood stream at concentrations (mg/kg body weight) up to 1,500 times that expected to occur during surgery on humans.

Endogenous production of cytotoxic factor in mice induced by a combination of interferon-gamma and heterologous fibrinogen.

The ability of heterologous fibrinogen in combination with interferon (IFN)-gamma to induce endogenous production of cytotoxic factor was examined. Heterologous but not homologous fibrinogen induced high production of cytotoxic factor in IFN-gamma-primed mice. The cytotoxic activity was maximal 1 h after this triggering. The LD50 value of heterologous fibrinogen in mice was greater than 250 mg/kg i.v. But heterologous fibrinogen induced antibody, causing anaphylaxis. Therefore, the effect of successive injections of fibrinogens from a different species was tested. Cytotoxic factor could be produced repeatedly by successive treatments with a combination of IFN-gamma and heterologous fibrinogen from one species for 1 week, although the cytotoxic activity induced by successive injections gradually decreased. After the decrease of the triggering effect of heterologous fibrinogen of one species, heterologous fibrinogen from a different species could induce cytotoxic activity at the same level as that after the first triggering. Thus, a combination of IFN-gamma and heterologous fibrinogen is effective for cytotoxic factor production, provided different heterologous fibrinogens are used successively. This combination should be useful for endogenous cytotoxic factor production in clinical trials.

Effect of human fibrin adhesive on the ear. An electrophysiological study of auditory function in the guinea pig correlated to light and electron microscopy of middle ear mucosa and inner ear structures.

The effect of human fibrin adhesive applied to the middle ear has been studied in guinea pig. Auditory function was measured using acoustically evoked brainstem responses. Middle and inner ear structures were studied with light, transmission and scanning electron microscopy. A transitory conductive hearing loss was observed, but after 8 weeks the auditory function appeared normal. Microscopy of the middle and inner ear failed to show any tissue damage.

A new autologous fibrinogen-based adhesive for otologic surgery.

Many middle ear reconstructive procedures would be facilitated by use of a relatively safe surgical adhesive. A fibrinogen-based adhesive (Tisseel) has been effective in otologic surgery in Europe, but because it is derived from pooled human blood, it carries a risk of transmitting hepatitis, acquired immune deficiency syndrome, and other illnesses. This report details a new procedure for creating an autologous fibrinogen-based adhesive, obviating these risks. The fibrinogen and factor XIII component of the adhesive was isolated by polyethylene glycol precipitation from human plasma within a few hours, and was used either immediately or frozen for use up to 3 weeks later. Fifteen chinchillas had either the single donor adhesive, the commercial European adhesive, or saline placed on the oval and round windows, and no evidence of cochlear, mucosal, or ossicular damage was seen by light microscopy 30 days later. Auditory brain stem response thresholds remained stable, except in three animals that developed otitis media. Based on this investigation, autologous fibrinogen-based adhesive appears promising as a relatively safe, biological bonding material for otologic surgery, and is worthy of further study.

[Role of serum fibrinogen penetrating the subarachnoid space in the pathogenesis of brief cerebral arteriospasm]. / Die Rolle des in den Subarachnoidalraum eingedrungenen Blutfibrinogens in der Pathogenese des kurzzeitigen Hirnarterienspasmus.

A mixture of fibrinogen (4 ml in a 1 per cent solutions) and thrombin (10 I.U.) were instilled into the cisterma magna of 18 dogs which led to the formation of a fibrin clot in the subarachnoid space. This fibrin clot produced a spasm of the large cerebral arteries, which resulted in a narrowing to 84.8 +/- 1.8 per cent of the initial value. The subsequent administration of 400 units of fibrinolysin into the cisterna magna led to the dissolution of the fibrin clot and the dilatation of the arterie to 116.3 +/- 2.5 per cent of the initial value. Experiments carried out in 15 cats showed that, after being brought in the subarachnoid space, the spasmogenic factor of the clot, which consisted of all blood components, passes through the arachnoid into the subdural space. The penetration of the spasmogenic factor from the blood clot located on the surface of the arachnoid from the subdural to the subrarachnoid space was not observed. The arachnoid proves to be a membrane which can only be passed in one direction by the spasmogenic blood factor.