Activation of Factor XII in Plasma from Rats Pretreated with Tranexamic Acid. Inhibition of a Plasmin-induced Loss of the Functional Activity of High Molecular Weight Kininogen

Abstract: Incubation of plasma from rats pretreated with tranexamic acid (40 mg/l 00 g) with acetone (23 % v/v) yielded enzyme preparations in which all the plasminogen present was recovered as plasmin and a plasmin-like substance without affinity for lysine-Sepharose. This substance, designated “plasmin”, was separated from plasmin and kallikrein in a three-step procedure using columns of lysine-Sepharose, DEAE-Sephadex A-50, and arginine-Sepharose. The ratios of fibrinolytic, caseinolytic, LEe esterase, BAEe esterase and kininogenase activities of “plasmin” corresponded well with those of rat plasmin and human plasmin. Both rat plasmin and “plasmin” destroyed the capacity of high molecular weight kininogen (HMWK) to function as a cofactor in the activation of factor XII in rat plasma, without causing a corresponding release of the kinin part of the molecule. Rat plasma kallikrein induced full release of kinin from HMWK, but the functional capacity was retained. It is suggested that the reduced extent of activation of factor XII observed in plasma from rats injected intravenously with dextran, or rat plasma that has been passed through a column with lysine-Sepharose, is due to the loss of functional HMWK caused by plasmin activated in vivo or on the column.     

Assessment of skin absorption and irritation potential of arachidonic acid and glyceryl arachidonate using in vitro diffusion cell techniques.

Arachidonic acid (AA), a precursor of pro-inflammatory mediators, and its glycerin ester, glyceryl arachidonate (GA), are reportedly used in cosmetic products. In vitro skin penetration of AA and GA and GA's ester hydrolysis was determined in flow-through diffusion cells. AA penetration with human and rat skin was 19.5% and 52.3% of the applied dose respectively, a substantial amount of which remained in the skin at 24h. Similar penetration results were obtained with GA in human skin. However, GA penetration through cultured skin (EpiDerm) was 51% of the applied dose, almost all of which appeared in the receptor fluid. At least 27.8% of GA penetrating skin was hydrolyzed to AA. In vitro methods were used to assess skin irritation in diffusion cells. Skin irritation of AA, sodium lauryl sulfate (SLS), and Tween 80 was determined by changes in transepidermal water loss (TEWL), skin viability (3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide, MTT, formation), and cytokine release (IL-1alpha). SLS irritation was much less pronounced in an emulsion versus an aqueous vehicle. No significant irritation was observed in vitro from AA in an emulsion. This work predicts that AA would penetrate human skin in vivo and that it could be formed in skin from topically applied GA.     

Plasma biomarkers of liver injury and inflammation demonstrate a lack of apoptosis during obstructive cholestasis in mice

Cholestasis is a pathological common component of numerous liver diseases that results in hepatotoxicity, inflammation, and cirrhosis when untreated. While the predominant hypothesis in cholestatic liver injury remains hepatocyte apoptosis due to direct toxicity of hydrophobic bile acid exposure, recent work suggests that the injury occurs through inflammatory necrosis. In order to resolve this controversy, we used novel plasma biomarkers to assess the mechanisms of cell death during early cholestatic liver injury. C57Bl/6 mice underwent bile duct ligation (BDL) for 6–72 h, or sham operation. Another group of mice were given d-galactosamine and endotoxin as a positive control for apoptosis and inflammatory necrosis. Plasma levels of full length cytokeratin-18 (FL-K18), microRNA-122 (miR-122) and high mobility group box-1 protein (HMGB1) increased progressively after BDL with peak levels observed after 48 h. These results indicate extensive cell necrosis after BDL, which is supported by the time course of plasma alanine aminotransferase activities and histology. In contrast, plasma caspase-3 activity, cleaved caspase-3 protein and caspase-cleaved cytokeratin-18 fragments (cK18) were not elevated at any time during BDL suggesting the absence of apoptosis. In contrast, all plasma biomarkers of necrosis and apoptosis were elevated 6 h after Gal/End treatment. In addition, acetylated HMGB1, a marker for macrophage and monocyte activation, was increased as early as 12 h but mainly at 48–72 h. However, progressive neutrophil accumulation in the area of necrosis started at 6 h after BDL. In conclusion, these data indicate that early cholestatic liver injury in mice is an inflammatory event, and occurs through necrosis with little evidence for apoptosis.     

In vitro and in vivo safety studies of a proprietary whey extract.

A proprietary whey growth factor extract (WGFE) or Lactermin (Lact milk; ermin growth factors) is a whey fraction of milk containing the major proteins lactoperoxidase and lactoferrin, together with a variety of minor proteins and peptides such as the growth factors IGF-I, IGF-II, PDGF, FGF, TGF-ss and betacellulin. This growth factor component of milk has been suggested to possess biological properties such as the promotion of tissue repair and anti-inflammatory activity. In this study the safety of Lactermin has been evaluated using genotoxicity assays (Ames, mouse lymphoma and micronucleus assay) and in a subchronic (13 week) rat oral toxicity study. In vitro Lactermin did not show any mutagenic properties in the Ames or mouse lymphoma assay and in vivo did not show any adverse clinical effects or in the bone marrow of male or female mice. In the subchronic oral toxicity study in which 10 rats per sex were fed Lactermin mixed with rat diet to deliver doses of 300, 1000 and 3000 mg/kg/day for 13 weeks, male and female rats did not show any test article-related clinical observations or effects on body weight, food consumption, ophthalmic effects, functional observational battery, organ weights, locomotor activity, hematology, serum chemistry, urinalysis or macroscopic or microscopic pathology. The results from the genotoxicity studies and the subchronic oral toxicity study suggest Lactermin is safe for consumption with a no-observed-adverse-effect level (NOAEL) of 3000 mg/kg/day.     

Some flavonoids and DHEA-S prevent the cis-effect of expanded CTG repeats in a stable PC12 cell transformant

Expanded CUG triplet repeats carrying mRNA seem to be responsible for myotonic dystrophy type 1 (DM1). To study the pathogenesis of DM1, we constructed a DM1 cell culture model using a PC12 neuronal cell line and screened flavonoids that ameliorate this mRNA gain of function. The expanded 250 CTG repeat was subcloned into the 3'-untranslated region of the luciferase gene yielding a stable transformant of PC12 (CTG-250). The cytotoxicity of CTG-250 was evaluated by intracellular LDH activity, and the cis-effect by luciferase activity. To find agents that alter CTG-250 toxic effects, 235 bioflavonoids were screened. An increased cis-effect and cytotoxicity were found when CTG-250 was treated with nerve growth factor to induce differentiation. Western blotting with anti-caspase-3 antibody suggested that cell death was caused by apoptosis. Screening analysis confirmed that a flavone (toringin), an isoflavones (genistein and formononetin), a flavanone (isosakuranetin), and DHEA-S prevent both the cytotoxicity and cis-effect of CTG-250 and that a flavanone (naringenin), isoflavone (ononin), and xanthylatin strongly inhibit the cis-effect of CTG repeats. In conclusion, we found that this neuronal cell line, which expresses the CUG repeat-bearing mRNA, showed cis-effects through the reporter gene and neuronal death after cell differentiation in vitro. However, some flavonoids and DHEA-S inhibit both the cis-effect and cytotoxicity, indicating that their chemical structures work to ameliorate both these toxic effects. This system makes it easy to evaluate the toxic effects of expanded CTG repeats and therefore should be useful for screening other DM1 treatments for their efficacies.     

Toll-like receptors in inflammation of the central nervous system

Toll-like receptors (TLRs) belong to pattern-recognition receptor family that could recognize exogenous pathogen-associated molecular patterns and endogenous damage-associated molecular patterns. TLRs play pivotal roles in innate and adaptive immune responses. In this review we summarize the ligands and signal transduction pathways of TLRs and highlight recent progress of the involvement of TLRs in neuroinflammation related disorders, including cerebral ischemia/stroke, brain trauma and hemorrhage, pathogen infection and autoimmune diseases, and explore the potential of TLR signaling as therapeutic targets against these disorders.     

The methanolic fraction of Centratherum anthelminticum seed downregulates pro-inflammatory cytokines,oxidative stress,and hyperglycemia in STZ-nicotinamide-induced type 2 diabetic rats

This study aimed to ascertain the potential of Centratherum anthelminticum seeds methanolic fraction (CAMFs) for the management of type 2 diabetes and its associated complications. CAMFs was initially tested on β-TC6 cells for H₂O₂-induced nuclear factor-κB (NF-κB) translocation effects. The result displayed that CAMFs significantly inhibited NF-κB translocation from cytoplasm into the nucleus, dose-dependently. Furthermore, a 12-week sub-chronic CAMFs study was carried out on streptozotocin (STZ)-nicotinamide–induced type 2 diabetic rat model to evaluate glycemia, essential biochemical parameters, lipid levels, oxidative stress markers, and pro-inflammatory cytokines level. Our study result showed that CAMFs reduced hyperglycemia by increasing serum insulin, C-peptide, total protein, and albumin levels, significantly. Whereas, elevated blood glucose, glycated hemoglobin, lipids and enzyme activities were restored to near normal. CAMFs confirmed antioxidant potential by elevating glutathione (GSH) and reducing malondialdehyde (MDA) levels in diabetic rats. Interestingly, CAMFs down-regulated elevated tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 in the tissues and serum of the diabetic rats. We conclude that CAMFs exerted apparent antidiabetic effects and demonstrated as a valuable candidate nutraceutical for insulin-resistant type 2 diabetes and its associated complications such as dyslipidemia, oxidative stress, and inflammation.     

Evaluating the role of Toll-like receptors in diseases of the central nervous system

A key part of the innate immune system is a network of pattern recognition receptors (PRRs) and their associated intracellular signalling pathways. Toll-like receptors (TLRs) are one such group of PRRs that detect pathogen associated molecular patterns (PAMPs). Activation of the TLRs with their respective agonists results in the activation of intracellular signalling pathways leading to the expression of proinflammatory mediators and anti-microbial effector molecules. Activation of the innate immune system through TLRs also triggers the adaptive immune response, resulting in a comprehensive immune program to eradicate invading pathogens. It is now known that immune surveillance and inflammatory responses occur in the central nervous system (CNS). Furthermore it is becoming increasingly clear that TLRs have a role in such CNS responses and are also implicated in the pathogenesis of a number of conditions in the CNS, such as Alzheimer's, stroke and multiple sclerosis. This is likely due to the generation of endogenous TLR agonists in these conditions which amplifies a detrimental neurotoxic inflammatory response. However TLRs in some situations can be neuroprotective, if triggered in a favourable context. This review aims to examine the recent literature on TLRs in the CNS thus demonstrating their importance in a range of infectious and non-infectious diseases of the brain.     

Soy isoflavone phyto-pharmaceuticals in interleukin-6 affections. Multi-purpose nutraceuticals at the crossroad of hormone replacement, anti-cancer and anti-inflammatory therapy

Interleukin-6 is a pleiotropic cytokine which plays a crucial role in immune physiology and is tightly controlled by hormonal feedback mechanisms. After menopause or andropause, loss of the normally inhibiting sex steroids (estrogen, testosterone) results in elevated IL6 levels that are further progressively increasing with age. Interestingly, excessive IL6 production promotes tumorigenesis (breast, prostate, lung, colon, ovarian), and accounts for several disease-associated pathologies and phenotypical changes of advanced age, such as osteoporosis, rheumatoid arthritis, multiple myeloma, neurodegenerative diseases and frailty. In this respect, pharmacological modulation of IL6 gene expression levels may have therapeutical benefit in preventing cancer progression, ageing discomforts and restoring immune homeostasis. Although "plant extracts" are used in folk medicine within living memory, it is only since the 20th century that numerous scientific investigations have been performed to discover potential health-protective food compounds or "nutraceuticals" which might prevent cancer and ageing diseases. About 2000 years ago, Hippocrates already highlighted "Let food be your medicine and medicine be your food". Various nutrients in the diet play a crucial role in maintaining an "optimal" immune response, such that deficient or excessive intakes can have negative consequences on the organism's immune status and susceptibility to a variety of pathologies. Over the last few decades, various immune-modulating nutrients have been identified, which interfere with IL6 gene expression. Currently, a broad range of phyto-pharmaceuticals with a claimed hormonal activity, called "phyto-estrogens", is recommended for prevention of various diseases related to a disturbed hormonal balance (i.e. menopausal ailments and/or prostate/breast cancer). In this respect, there is a renewed interest in soy isoflavones (genistein, daidzein, biochanin) as potential superior alternatives to the synthetic selective estrogen receptor modulators (SERMs), which are currently applied in hormone replacement therapy (HRT). As phyto-chemicals integrate hormonal ligand activities and interference with signaling cascades, therapeutic use may not be restricted to hormonal ailments only, but may have applications in cancer chemoprevention and/or NF-kappaB-related inflammatory disorders as well.     

Neutrophils: Cinderella of innate immune system

Neutrophils are the first line of innate immune defense against infectious diseases. However, since their discovery by Elie Metchnikoff, they have always been considered tissue-destructive cells responsible for inflammatory tissue damage occurring during acute infections. Now, extensive research in the field of neutrophil cell biology and their role skewing the immune response in various infections or inflammatory disorders revealed their importance in the regulation of immune response. Along with releasing various antimicrobial molecules, neutrophils also release neutrophil extracellular traps (NETs) for the containment of infection and inflammation. Activated neutrophils provide signals for the activation and maturation of macrophages as well as dendritic cells. Neutrophils are also involved in the regulation of T-cell immune response against various pathogens and tumor antigens. Thus, the present review is intended to highlight the emerging role of neutrophils in the regulation of both innate and adaptive immunity during acute infectious or inflammatory conditions.     

Effects of carbachol on lead-induced impairment of the long-term potentiation/depotentiation in rat dentate gyrus in vivo.

The present study aims at evaluating the impairment of LTP and depotentiation (DP) of LTP induced by acute lead exposure, and the effects of peripheral carbachol (CCh) application on LTP/DP of acute and chronic lead-exposed rats in dentate gyrus in vivo. Rats (80-100 days) were acutely exposed to lead by intraperitoneal injection of 0.2% lead acetate (PbAc) solution (1.5mg/100g) and/or CCh (1 micro g/100g). Rats were chronically exposed to lead from parturition through adulthood (80-100 days) by the drinking of 0.2% PbAc solution and/or CCh (1 micro g/100g) chronic intraperitoneal injection one week. The input-output (I/O) function, paired-pulse reaction (PPR), excitatory postsynaptic potential (EPSP) and population spike (PS) amplitude were measured in response to stimulation applied to the lateral perforant path. Results showed that: first, acute lead exposure significantly depressed the amplitudes of LTP/DP of both EPSP slope and PS amplitude. Second, CCh significantly increased the amplitudes of both EPSP LTP/DP and PS LTP of acute Pb-exposed rats. After CCh treatment, the magnitudes of EPSP LTP/DP and PS LTP of acute Pb-exposed rats showed no significant difference with controls. Third, Chronic CCh application also reversed chronic Pb-induced impairment of PS LTP and EPSP DP of LTP. As CCh does not cross blood-brain barrier in healthy animals, the data suggest that CCh may traverse BBB in Pb-exposed animals and cure Pb-induced dysfunction of learning and memory.     

The role of damage associated molecular pattern molecules in acetaminophen-induced liver injury in mice

The idiosyncratic nature, severity and poor diagnosis of drug-induced liver injury (DILI) make these reactions a major safety issue during drug development, as well as the most common cause for the withdrawal of drugs from the pharmaceutical market. Elucidation of the underlying mechanism(s) is necessary for identifying predisposing factors and developing strategies in the treatment and prevention of DILI. Acetaminophen (APAP) is a widely used over the counter therapeutic that is known to be effective and safe at therapeutic doses. However, in overdose situations fatal and non-fatal hepatic necrosis can result. Evidence suggests that the chemically reactive metabolite of the drug initiates hepatocyte damage and that inflammatory innate immune responses also occur within the liver, leading to the exacerbation and progression of tissue injury. Here we investigate whether following APAP-induced liver injury (AILI) damaged hepatocytes release “danger” signals or damage associated molecular pattern (DAMP) molecules, which induce pro-inflammatory activation of hepatic macrophages, further contributing to the progression of liver injury. Our study demonstrated a clear activation of Kupffer cells following early exposure to APAP (1 h). Activation of a murine macrophage cell line, RAW cells, was also observed following treatment with liver perfusate from APAP-treated mice, or with culture supernatant of APAP-challenged hepatocytes. Moreover, in these media, the DAMP molecules, heat-shock protein-70 (HSP-70) and high mobility group box-1 (HMGB1) were detected. Overall, these findings reveal that DAMP molecules released from damaged and necrotic hepatocytes may serve as a crucial link between the initial hepatocyte damage and the activation of innate immune cells following APAP-exposure, and that DAMPs may represent a potential therapeutic target for AILI.     

New molecular targets for the treatment of osteoarthritis

Osteoarthritis (OA) is a chronic degenerative joint disorder characterized by destruction of the articular cartilage, subchondral bone alterations and synovitis. Current treatments are focused on symptomatic relief but they lack efficacy to control the progression of this disease which is a leading cause of disability. Therefore, the development of effective disease-modifying drugs is urgently needed. Different initiatives are in progress to define the molecular mechanisms involved in the initiation and progression of OA. These studies support the therapeutic potential of pathways relevant in joint metabolism such as Wnt/β-catenin, discoidin domain receptor 2 or proteinase-activated receptor 2. The dysregulation in cartilage catabolism and subchondral bone remodeling could be improved by selective inhibitors of matrix metalloproteinases, aggrecanases and other proteases. Another approach would favor the activity of anabolic processes by using growth factors or regulatory molecules. Recent studies have also revealed the role of oxidative stress and synovitis in the progression of this disease, supporting the development of a number of inhibitory strategies. Novel targets in OA are represented by genes involved in OA pathophysiology discovered using gene network, epigenetic and microRNA approaches. Further insights into the molecular mechanisms involved in OA initiation and progression may lead to the development of new therapies able to control joint destruction and repair.     

Inhibition of leukocyte function and interleukin-2 gene expression by 2-methylarachidonyl-(2'-fluoroethyl)amide, a stable congener of the endogenous cannabinoid receptor ligand anandamide

Arachidonylethanolamide (anandamide, AEA) has been identified as an endogenous ligand for cannabinoid receptors CB1 and CB2. Characterization of the direct cannabimimetic actions of anandamide has been hampered by its short duration of action and rapid degradation in in vivo and in vitro systems to arachidonic acid, a precursor in the biosynthesis of a broad range of biologically active molecules. In the present studies, we utilized 2-methylarachidonyl-(2'-fluoroethyl)amide (F-Me-AEA), an analog of anandamide resistant to enzymatic degradation, to determine whether F-Me-AEA modulated T cell function similar to that of plant-derived cannabinoids. Indeed, F-Me-AEA at low micromolar concentrations exhibited a marked inhibition of phorbol ester plus calcium ionophore (PMA/Io)-induced IL-2 protein secretion and steady state mRNA expression. Likewise, a modest suppression of the mixed lymphocyte response was observed in the presence of F-Me-AEA indicating an alteration in T cell responsiveness to allogeneic MHC class II antigens. F-Me-AEA was also found to modestly inhibit forskolin-stimulated adenylate cyclase activity in thymocytes and splenocytes, a hallmark of cannabinoid receptor agonists. Further characterization of the influence of F-Me-AEA on the cAMP signaling cascade revealed an inhibition of CREB-1/ATF-1 phosphorylation and subsequently, an inhibition of CRE DNA binding activity. Characterization of nuclear binding proteins further revealed that NF-AT and, to a lesser extent, NF-kappaB DNA binding activities were also suppressed. These studies demonstrate that F-Me-AEA modulates T cell function in a similar manner to plant-derived and endogenous cannabinoids and therefore can be utilized as an amidase- and hydrolysis-resistant endogenous cannabinoid.     

Evaluation of a 3D Human Artificial Lymph Node as Test Model for the Assessment of Immunogenicity of Protein Aggregates

The immunogenicity of protein aggregates has been investigated in numerous studies. Nevertheless, it is still unknown which kind of protein aggregates enhance immunogenicity the most. The ability of the currently used in vitro and in vivo systems regarding their predictability of immunogenicity in humans is often questionable, and results are partially contradictive. In this study, we used a 2D in vitro assay and a complex 3D human artificial lymph node model to predict the immunogenicity of protein aggregates of bevacizumab and adalimumab. The monoclonal antibodies were exposed to different stress conditions such as light, heat, and mechanical stress to trigger the formation of protein aggregates and particles, and samples were analyzed thoroughly. Cells and culture supernatants were harvested and analyzed for dendritic cell marker and cytokines. Our study in the artificial lymph node model revealed that bevacizumab after exposure to heat triggered a TH1- and proinflammatory immune response, whereas no trend of immune responses was seen for adalimumab after exposure to different stress conditions. The human artificial lymph node model represents a new test model for testing the immunogenicity of protein aggregates combining the relevance of a 3D human system with the rather easy handling of an in vitro setup.     

Effects of flavonoid mixtures on the transport of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through Caco-2 monolayers: an in vitro and kinetic modeling approach to predict the combined effects on transporter inhibition.

This study describes and kinetically models the effect of flavonoid mixtures on PhIP transport through Caco-2 monolayers. Previously it was shown that quercetin, luteolin, naringenin and myricetin increase the apical to basolateral PhIP transport in Caco-2 monolayers. In this study, apigenin was shown to exert a similar effect with an apparent K(i) value of 10.8 microM. Additional experiments revealed that several binary flavonoid mixtures and one mixture containing all five model flavonoids increased the apical to basolateral PhIP transport through the Caco-2 monolayer. Assuming competitive inhibition of the apparent active transporter by the flavonoids and concentration-additivity for their inhibiting effect, the kinetic model previously developed to describe the effect of the individual flavonoids on PhIP transport, could be extended and adequately describes the experimental values obtained for the flavonoid mixtures. We conclude that combinations of flavonoids increase the transport of PhIP and do so by interacting in an additive way with the active transport of PhIP. This flavonoid-mediated increase in PhIP transport through Caco-2 monolayers may point at a possible increased bioavailability of PhIP in the presence of flavonoid mixtures in the in vivo situation. This would imply an adverse effect of these supposed beneficial food ingredients.     

Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid–polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene—5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.     

Identification of competitive inhibitors of the human taurine transporter TauT in a human kidney cell line

Background

The osmolyte and antioxidant taurine plays an important role in regulation of cellular volume, oxidative status and Ca²⁺-homeostasis. Taurine uptake in human cells is regulated by the Na⁺- and Cl^?-dependent taurine transporter TauT. In order to gain deeper structural insights about the substrate binding pocket of TauT, a HEK293 cell line producing a GFP-TauT fusion protein was generated.