Lysate Assay Research Articles

Development of pre-activated α-cyclodextrin as a mucoadhesive excipient for intra-vesical drug delivery

The study was designed to synthesize and characterize pre-activated α-cyclodextrin (α-CD) derivatives as mucus adhering excipients for intra-vesical drug delivery. Sodium periodate (NaIO4) was used to oxidize α-CD and subsequently cysteamine was covalently attached to carbonyl groups of oxidized α-CD via reductive amination to produce thiolated α-CD. l-cysteine-2-mercaptonicotinic acid conjugate (Cys-MNA) was covalently attached to carbonyl groups of oxidized α-CD to produce pre-activated α-CD having enhance stability against oxidation at higher pH. Thiolated and pre-activated α-CD derivatives were quantitatively assayed for the attached thiol groups and MNA groups, respectively. Cell viability and tolerability was evaluated via resazurin assay and via red blood cells (RBC) lysis assay, respectively. Mucoadhesive properties were evaluated on porcine bladder mucosa. Trimethoprim (TMP) was encapsulated into thiolated and pre-activated α-CD derivatives and the dissolution behavior was evaluated in vitro. Thiol groups attached to thiolated α-CD derivatives α-CD-SH780 and α-CD-SH1426 were 780±68μmol/g and 1426±66μmol/g, respectively. For the entirely pre-activated α-CD derivatives, α-CD-MNA3609 and α-CD-MNA4285 number of attached MNA groups were 3609±19μmol/g and 4285±43μmol/g, respectively. Thiolated and pre-activated derivatives of α-CD did not show adverse effects to cells determined via resazurin and RBC lysis assays. Mucoadhesion on porcine bladder mucosa was significantly improved for thiolated and pre-activated α-CD derivatives. Thiolated α-CD-SH1426 showed 15-fold and pre-activated α-CD-MNA4285 showed 25-fold improved mucoadhesion compared to unmodified α-CD. Further, pre-activated α-CD-MNA4285 showed 2-fold enhanced dissolution of encapsulated TMP compared to free TMP over 3 h. The study shows that pre-activated α-CD could be an excipient of the choice for the formulations of mucoadhesive intra-vesical drug delivery systems.

Lupinifolin from Albizia myriophylla wood: A study on its antibacterial mechanisms against cariogenic Streptococcus mutans

ObjectiveTo determine the anti-Streptococcus mutans mechanisms of action of lupinifolin from Albizia myriophylla Benth. (Fabaceae) wood and provide scientific evidence to support the traditional use of the plant against dental caries. MethodsThe minimum inhibitory concentration (MIC) was evaluated using the broth micro-dilution method. The effects of lupinifolin on bactericidal activity, bacterial cell walls, and membranes were investigated by time-kill, lysis, and leakage assays, respectively. Electron microscopy was utilized to observe any cell morphological changes caused by the compound. Localization of lupinifolin in S. mutans was detected using the thin layer chromatography technique. ResultsThe MIC range of lupinifolin against S. mutans (n=6) was 2–4 μg/ml. This compound displayed bactericidal effects on S. mutans ATCC 25175 by 90–99.9% killing at 4MIC-16MIC after 8–24 hours. Lupinifolin-treated cells demonstrated no lysis. However, significant cytoplasmic leakage through the bacterial membrane was observed after treatment with lupinifolin at 4MIC-16MIC. As revealed by ultrastructural analysis, lupinifolin produced some changes in bacterial cell walls and membranes. Moreover, the compound was observed in the cytoplasmic fraction of the lupinifolin-treated cells. These results suggest that lupinifolin can enter the cell of bacteria but does not accumulate in the cell envelope and subsequently disrupts the integrity of the cytoplasmic membrane, leading to cell death. ConclusionThe scientific evidence from this study offers valuable insights into the potential role of lupinifolin in pharmaceutical and antibiotic applications and supports the therapeutic effects of A. myriophylla, which has traditionally been used as an alternative treatment for dental caries.

Strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1

Screening of isolates for their potency to produce streptokinase was an important criterion of this research. The current study emphasizes the strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1 isolated from bovine milk. The study was carried out on samples collected from milk sample. Primary screening and characterization is used as an excellent source for the isolation of β-hemolytic organisms. Strain improvement was done by both physical & chemical mutagenesis. The enzyme activity was checked by clot lysis assay and confirmed by fibrin plate method. The partially purified and crude enzyme were analysed by high-performance liquid chromatography. Molecular weight & enzyme purity was checked by SDS–PAGE, further confirmed by fibrin zymography. Out of the 3 isolated strains, only one isolate expressed β-haemolysis with streptokinase (SK) activity. Based on the results of radial caseinolytic assay and blood clot dissolving assay, isolate TNA-M1 demonstrated the highest streptokinase activity. Based on morphological, biochemical and molecular characterization, it was identified as Streptococcus uberis and the strain was named as Streptococcus uberis TNA-M1. The results indicated that ultra-violet (UV) and ethyl methane sulfonate (EMS) were effective mutagenic agents for strain improvement of Streptococcus uberis TNA-M1 and enhanced SK productivity. HPLC analysis was performed in order to confirm the presence of streptokinase with the similar retention time (0.875 min) with its standard (0.854) min. SDS-PAGE of the enzyme showed protein band of approximately 47 kDa and confirmed by fibrin zymography. It exhibited fibrinolytic activity, which was more potent than other fibrinolytic enzymes. Glucose and peptone were recorded to be the optimum carbon and nitrogen sources respectively. Thus this study presents its novelty by highlighting the potential of Streptococcus uberis TNA-M1 as a significant source for the production of fibrinolytic enzymes.

1017P - Phenotypic and functional characterization of tumor infiltrating lymphocytes (TIL) grown from non-hodgkin lymphoma tumors: Implications for the development of novel therapies for lymphoma

Background: Cell therapy with TIL is an effective treatment with acceptable safety profile for advanced metastatic melanoma patients. TIL products can be centrally manufactured for broad clinical application. Adoptive cell therapy with TIL involves collection of autologous lymphocytes from the tumor via surgical resection, ex vivo expansion of TIL, lymphodepletion of the patient prior to infusion of TIL, followed by infusion of TIL and treatment with IL-2. Here, we present findings related to expanding TIL directly from lymphoma. Methods: Using methods for TIL isolation and growth developed at Lion, we expanded TIL from 5/5 lymphoma (1 MCL, 3 follicular, 1 DLBCL) with Interleukin 2 for 11-14 days and subsequent rapid expansion for 14 days using mitogenic anti-CD3 antibody and irradiated allogeneic PBMC. Results: TIL were generated from all 5 lymphoma tumors with a maximum expansion index of 680-fold, significantly higher than previously reported. Mean CD3+ T cell population was 95% (vs 75% previously reported). As with TIL expansion from melanoma, we observed a marked relative increase in effector memory cells in lymphoma TIL. A significant increase in TEMRA (p = 0.0013; CD4, CD8) and CD28+CD4 + (p = 0.008) subsets was observed in lymphoma compared with melanoma TIL cultures. Bioluminescent Redirected Lysis Assay (BRLA) to assess TIL cytolytic activity at 4 hrs ranged from <1-6 LU50 in lymphoma TIL compared to melanoma TIL (11-75 LU50, 4hrs). ELISpot and ELISA analysis revealed IFN-γ production by lymphoma TIL comparable to that of melanoma TIL. Consistent with the observation that lymphoma-reactive T-cells are primarily TH2 and TH17, nanostring analysis revealed that lymphoma TIL expressed higher levels of RORC and IL17A than melanoma TIL. Conclusions: We demonstrate here the feasibility of growing TIL from lymphoma that have effector functions comparable to that of melanoma TIL. These findings serve as a rationale for considering TIL cell therapy for patients with lymphoma. Legal entity responsible for the study: Lion Biotechnologies, Inc Funding: Lion Biotechnologies Inc Disclosure: L. Karyampudi, A. Gokuldass, M. Blaskovich, M.T. Lotze: Employee of Lion Biotechnologies, Inc, receive compensation from Lion. Hold stock and/or stock options in Lion Biotechnologies, Inc. M. Fardis: Employee and director of Lion Biotechnologies, Inc, receive compensation from Lion. Hold stock and/or stock options in Lion Biotechnologies, Inc. Member of the Board of Directors of Lion Biotechnologies, Inc.

EVALUATION OF EX VIVO THROMBOLYTIC ACTIVITY AND IN VITRO ANTI-INFLAMMATORY ACTIVITY OF THESPESIA POPULNEA LEAF EXTRACT

Thrombotic disorders like myocardial and cerebral infarction are fatal blood clotting related diseases. Synthetic therapeutics used in such disorders has serious adverse effects, so there is a need to investigate some more safe natural thrombolytic agents. Present study is a preliminary work towards such endeavors. During this study analysis of thrombolytic activity and anti-inflammatory activity of Thespesia populnea leaf extract using a simple and quick in vitro clot lysis assay was performed. Various concentrations of leaf extract i.e. 200µg/ml; 400µg/ml and 600µg/ml were tested at various time intervals including; 24, 48 and 72 hours duration of incubation at 37°C for observing maximum clot lysis. The result findings indicated that concentrations of leaf extract enhanced the percentage of clot lysis in dose dependent manner along with the incubation time factor. However; streptokinase SK a reference standard and water were used as a positive and negative control showed clot lysis maximum 96.35% and 35.22% in 72 hours of incubation respectively. Alcohol extract of whole plant of Thespesia populnea (Family: Malvaceae) was assessed for its anti-inflammatory activity by in vitro methods. In vitro anti-inflammatory activity was evaluated using albumin denaturation assay at different concentrations. Diclofenac sodium was used as standard drug. The results showed that Thespesia populnea alcohol extract at a concentration range of 400-1600µl significantly protects from protein denaturation.

Effects of wogonin on the mechanism of melanin synthesis in A375 cells

The present study aimed to investigate the effect of wogonin on the mechanism of melanin synthesis in the A375 melanoma cell line. A375 cells, cultured in vitro, were treated with wogonin and the activity of tyrosinase (TYR) and melanin synthesis were examined via MTT assay, L-dopa oxidation assay and an NaOH lysis assay. Protein expression levels of TYR and c-Jun N-terminal kinase (JNK) were examined via western blotting. mRNA expression levels of TYR, tyrosinase related protein (TRP)-1, TRP-2, extracellular signal-regulated kinase (ERK)-1, ERK-2 and JNK-2 were analyzed by reverse transcription-quantitative polymerase chain reaction. Furthermore, the effect of wogonin on estrogen receptor inhibitor (ICI182780) and ERK pathway inhibitor (U0126) was investigated. Safe doses of wogonin (10, 1, 10-1, 10-2 or 10-3 µmol/l) significantly inhibited melanin synthesis and TYR activity (P<0.05). Wogonin (10 µmol/l) inhibited the protein expression levels of TYR, JNK and mRNA expression levels of TYR, TRP-1, TRP-2, ERK-1, ERK-2, JNK-2 in A375 cells (P<0.01). The estrogen receptor inhibitor, ICI182780, and MEK inhibitor, U0126, significantly reversed the effects of wogonin on protein and mRNA expression levels of TYR, TRP-1, TRP-2, ERK-1, ERK-2 and JNK-2 (all P<0.01). To conclude, the present study identified that wogonin is able to inhibit the synthesis of melanin in A375 cells, through inhibiting protein and mRNA expression levels of TYR, TRP-1, and TRP-2, and ERK1, ERK2 and JNK2, respectively.

Prevention of Ophthalmia Neonatorum Caused by Neisseria gonorrhoeae Using a Fatty Acid-Based Formulation.

ABSTRACTOphthalmia neonatorum, also called neonatal conjunctivitis, acquired during delivery can occur in the first 28 days of life. Commonly caused by the bacterial pathogen Neisseria gonorrhoeae, infection can lead to corneal scarring, perforation of the eye, and blindness. One approach that can be taken to prevent the disease is the use of an ophthalmic prophylaxis, which kills the bacteria on the surface of the eye shortly after birth. Current prophylaxes are based on antibiotic ointments. However, N. gonorrhoeae is resistant to many antibiotics and alternative treatments must be developed before the condition becomes untreatable. This study focused on developing a fatty acid-based prophylaxis. For this, 37 fatty acids or fatty acid derivatives were screened in vitro for fast antigonococcal activity. Seven candidates were identified as bactericidal at 1 mM. These seven were subjected to irritation testing using three separate methods: the bovine corneal opacity and permeability (BCOP) test; the hen’s egg test—chorioallantoic membrane (HET-CAM); and the red blood cell (RBC) lysis assay. The candidates were also tested in artificial tear fluid to determine whether they were effective in this environment. Four of the candidates remained effective. Among these, two lead candidates, monocaprin and myristoleic acid, displayed the best potential as active compounds in the development of a fatty acid-based prophylaxis for prevention of ophthalmia neonatorum.

Stretching single fibrin fibers hampers their lysis.

Fibrin fibers are the major structural component of a blood clot. We developed a highly stretchable substrate made from fugitive glue and a suspended fibrin fiber lysis assay to investigate the effect of stretching on single fibrin fibers lysis. The key findings from our experiments are: 1) Fibers thicken and elongate upon lysis; 2) stretching strongly reduces lysis; 3) this effect is more pronounced for uncrosslinked fibers; and 4) stretching fibers has a similar effect on reducing lysis as crosslinking fibers. At the molecular level, strain may distort plasmin cleavage sites, or restrict access to those sites. Our results suggest that strain may be a novel mechanobiological factor that regulates fibrinolysis.

Inhibition of PAI-1 Activity by Toddalolactone as a Mechanism for Promoting Blood Circulation and Removing Stasis by Chinese Herb Zanthoxylum nitidum var. tomentosum.

Traditional Chinese medicine has been used to treat a variety of human diseases for many centuries. Zanthoxylum nitidum var. tomentosum is used as an adjuvant to promote blood circulation and remove stasis. However, the mechanisms of improving circulation and other biological activities of Z. nitidum var. tomentosum are still unclear. Plasminogen activator inhibitor-1 (PAI-1) regulates the plasminogen activation system through inhibition of tissue-type and urokinase-type plasminogen activators (tPA and uPA). PAI-1 has been linked to fibrin deposition that evolves into organ fibrosis and atherosclerosis. In the present study, we showed that ethanol extract prepared from Z. nitidum var. tomentosum exhibited PAI-1 inhibitory activity, and identified toddalolactone as the main active component that inhibited the activity of recombinant human PAI-1 with IC50 value of 37.31 ± 3.23 μM, as determined by chromogenic assay, and the effect was further confirmed by clot lysis assay. In vitro study showed that toddalolactone inhibited the binding between PAI-1 and uPA, and therefore prevented the formation of the PAI-1/uPA complex. Intraperitoneal injection of toddalolactone in mice significantly prolonged tail bleeding and reduced arterial thrombus weight in a FeCl3-induced thrombosis model. In addition, the hydroxyproline level in the plasma and the degree of liver fibrosis in mice were decreased after intraperitoneal injection of toddalolactone in CCl4-induced mouse liver fibrosis model. Taken together, PAI-1 inhibition exerted by toddalolactone may represent a novel molecular mechanism by which Z. nitidum var. tomentosum manifests its effect in the treatment of thrombosis and fibrosis.

Microvesicles released during the interaction between Trypanosoma cruzi TcI and TcII strains and host blood cells inhibit complement system and increase the infectivity of metacyclic forms of host cells in a strain-independent process.

Extracellular vesicles, whether microvesicles (MVs) or exosomes, shed by pathogens transfer virulence factors and biomolecules to host cells, thereby altering the host's susceptibility to infection. We have previously demonstrated that MV release is increased during the interaction between the infective forms of Trypanosoma cruzi and host cells. MVs confer parasite resistance to complement-mediated lysis and enhance parasite invasion. In this study, we show that differences exist in the levels of MVs released during the interaction between metacyclic trypomastigotes of different T. cruzi strains (with varied sensitivity to complement-mediated lysis, namely sensitive G strain TcI and resistant Y strain TcII) and host cells. MVs produced during the interaction between TcII parasites and host cells increased parasite resistance to complement lysis from 50% to 80% and parasite invasion was increased to over 50%. MVs purified during the interaction between TcI parasites and host cells have a stronger effect, doubling complement resistance and parasite invasion. The complement-mediated lysis assays showed that all MVs inhibit mainly the lectin pathway. Interestingly, MVs derived from parasites of one class did not alter complement resistance and the invasion process of parasites from the other class. This is the first description of MVs from T. cruzi with strain-dependent phenotypic effects.

Engineering Alkoxyphenacyl-Polycarbonate Nanoparticles for Potential Application in Near-Infrared Light-Modulated Drug Delivery via Photon Up-Conversion Process

Photoresponsive delivery systems that are activated by high energy photo-triggers have been accorded much attention because of the capability to achieve reliable photoreactions at short irradiation times. However, the application of a high energy photo-trigger (UV light) is not clinically viable. Meanwhile, the process of photon-upconversion is an effective strategy to generate a high energy photo-trigger (in-situ) through exposure to clinically relevant near-infrared (NIR) light. In this regard, we synthesized photon upconverting nanocrystals (UCNCs) that were subsequently loaded into photoresponsive nanoparticles (NPs) that were prepared using alkoxyphenacyl-based polycarbonate homopolymer (UCNC-APP-NPs). UCNC loading affected resultant NP size, size distribution, colloidal stability but not the zeta potential. The efficiency of NIR-modulated drug delivery was impacted by the heterogenetic nature of the resultant UCNC-APP-NPs which was plausibly formed through a combination of UCNC entrapment within the polymeric NP matrix and nucleation of polymer coating on the surface of the UCNCs. The biocompatibility of UCNC-APP-NPs was demonstrated through cytotoxicity, macrophage activation, and red blood cell lysis assays. Studies in tumor-bearing (nu/nu) athymic mice showed a negligible distribution of UCNC-APP-NPs to reticuloendothelial tissues. Further, distribution of UCNC-APP-NPs to various tissues was in the order (highest to lowest): Lungs &gt; Tumor &gt; Kidneys &gt; Liver &gt; Spleen &gt; Brain &gt; Blood &gt; Heart. In all, the work highlighted some important factors that may influence the effectiveness, reproducibility and biocompatibility of drug delivery systems that operate on the process of photon-upconversion.

In-vitro assessment of antimicrobial properties and lymphocytotoxicity assay of benzoisochromanequinones polyketide from Streptomyces sp JRG-04

The chromanequinone (BIQ) compound produced by the mangrove estuary derived strain, Streptomyces sp. JRG-04 was effective even at low MIC level concentration against Methicillin resistant S. aureus and other clinical pathogens. In this study, we have investigated the antimicrobial potential of chromanequinone compound by using various microscopy and imaging techniques. The flow cytometry (FACS) analysis suggested the BIQ aromatic polyketide compound produced by the Streptomyces sp. JRG-04 has toxic effect on MRSA cell membrane by increased up take of propidium iodide dye. The bacterial imaging analysis by high content screening experiment (HCS) revealed the increased number of dead MRSA cells than the live MRSA populations with chromanequinone treatment. Furthermore, atomic force microscopic study proved the MRSA cell surface ultra-structure changes when the cells exposed to chromanequinone compound at 3 h and 6 h. Further, in-vitro lymphocytotoxicity effect of chromanequinone compound at different concentrations with the combination of complement was performed on human lymphocytes by cell lysis assay. Interestingly, we have found that the higher concentration of BIQ chromanequinone (10 mg/mL) compound without complement induced apoptosis of human lymphocytes. The present investigation reveals that the toxic potential of chromanequinone on human lymphocytes might be associated with the complement dependent. This study strongly suggests that the chromanequinone compound produced by the Streptomyces strain with bioactive property can be developed as a therapeutic leads for various pharmaceutical applications.

Molecular and Biocompatibility Characterization of Red Blood Cell Membrane Targeted and Cell-Penetrating-Peptide-Modified Polymeric Nanoparticles.

Red blood cells (RBCs) express a variety of immunomodulatory markers that enable the body to recognize them as self. We have shown that RBC membrane glycophorin A (GPA) receptor can mediate membrane attachment of protein therapeutics. A critical knowledge gap is whether attaching drug-encapsulated nanoparticles (NPs) to GPA and modification with cell-penetrating peptide (CPP) will impact binding, oxygenation, and the induction of cellular stress. The objective of this study was to formulate copolymer-based NPs containing model fluorescent-tagged bovine serum albumin (BSA) with GPA-specific targeting ligands such as ERY1 (ENPs), single-chain variable antibody (scFv TER-119, SNPs), and low-molecular-weight protamine-based CPP (LNPs) and to determine their biocompatibility using a variety of complementary high-throughput in vitro assays. Experiments were conducted by coincubating NPs with RBCs at body temperature, and biocompatibility was evaluated by Raman spectroscopy, hemolysis, complement lysis, and oxidative stress assays. Data suggested that LNPs effectively targeted RBCs, conferring 2-fold greater uptake in RBCs compared to ENPs and SNPs. Raman spectroscopy results indicated no adverse effect of NP attachment or internalization on the oxygenation status of RBCs. Cellular stress markers such as glutathione, malondialdehyde, and catalase were within normal limits, and complement-mediated lysis due to NPs was negligible in RBCs. Under the conditions tested, our data demonstrates that molecular targeting of the RBC membrane is a feasible translational strategy for improving drug pharmacokinetics and that the proposed high-throughput assays can prescreen diverse NPs for preclinical and clinical biocompatibility.

In vitro and in silico studies on fibrinolytic activity of nattokinase: A clot buster from Bacillus sp.

Nattokinase (NK) is a serine protease enzyme of the subtilisin family. It exhibits a strong fibrinolytic activity. The fibrinolytic enzymes from Bacillus sp. have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process including plasmin activation. In the present study, VIT garden soil was collected and subjected to isolation process in order to screen for the NK production. Screening for NK enzyme was performed by radial caseinolytic assay. The production of NK enzyme was done in two different production medium for comparative studies. The NK enzyme was purified by gel permeation chromatography. The activity of the purified NK was checked by clot lysis and casein digestion assay. To investigate the structural basis of NK and fibrinogen interaction and also to identify the best binding mode, molecular dynamics and docking studies were performed. Based on the morphological and biochemical characterization, the isolate was identified as Bacillus sp. The overall purification fold of NK was about 3 with the specific activity of 664U/mg and 9.9% yield. Homogeneity of the purified enzyme was analyzed and confirmed by the single band obtained in SDS-PAGE. Molecular weight of the purified protease was estimated as 25 kDa. Purified NK enzyme exhibited 97% of effective clot lysis activity. The NK was docked in to the knob region of the fibrinogen at its binding site using Dock server. A total of 26 residues of fibrinogen and 29 residues of NK constitute the interface region. However, 9 residues of fibrinogen (THR238, MET264, LYS266, ARG275, THR277, ALA279, ASN308, MET310, and LYS321) and 8 residues of NK (GLY61, SER63, THR99, PHE189, LEU209, TYR217, ASN218, and MET222) are involved in intact binding. A significant amount of NK enzyme was obtained from Bacillus sp. The docking analysis revealed that the NK and fibrinogen adopt an extended binding pattern and interacts with the crucial residues to exhibit their activity.

Temporal changes in clot lysis and clot stability following tranexamic acid in cardiac surgery.

: Cardiac surgery induces a multifactorial coagulopathy. Regular use of tranexamic acid (TXA) is becoming standard of care. Clinical challenges include selecting optimal dosing regimen and balancing the benefit versus risk of additional dosing with antifibrinolytics. The objective was to evaluate the effect of TXA by assessing kinetic properties of plasma clot formation, clot stability, and clot fibrinolysis. The study was a prospective case follow-up of 28 patients undergoing cardiac surgery (mean age 63.9 years, 29% women). Blood samples were analysed at seven time points during the first 48 h after surgery. All patients were treated with TXA, 2 g at start surgery, 1 g during extra corporeal circulation, and 1 g after reversal of heparin. An automated clot lysis assay using tissue factor and tissue plasminogen activator (tPA) was performed to evaluate clot formation, stability, and fibrinolysis. TXA protects against facilitated fibrinolysis and induces up to 13-fold increase in clot stability. All patients showed complete resistance to tPA-induced fibrinolysis during the first 6 h after cardiac surgery declining to 33% of patients at 48 h. Impaired renal function was associated with prolonged resistance to tPA-induced fibrinolysis. Despite inhibition of fibrinolysis with TXA, the overall clot stability declines and the kinetic properties of clot formation were impaired after cardiac surgery. TXA induces a multifold increase in clot resistance to fibrinolysis but does not affect clot formation or clot stability. Monitoring the level of resistance to fibrinolysis may prevent overdosing in particular in patients with impaired renal function.

Constitutive IFN signaling to modulate the immunogenicity of glioma cells.

e13528 Background: Interferons (IFN) are cytokines that might be constitutively expressed also in the absence of acute infection and thereby regulate various physiological processes such as cellular proliferation and differentiation, cell viability or immune cell function. Alterations in IFN signaling have been observed in numerous malignancies, but their role in glioblastoma, particularly in glioma-initiating cells, has not been investigated in detail. Methods: We characterized constitutive type I IFN signaling and its functional impact in glioma cells using expression analyses, RNA interference-mediated silencing of various genes involved in the IFN signaling pathway and immune cell lysis assays. Results: We found constitutive expression of pSTAT1 and Myxovirus A (MxA), a classical IFN-response marker, in the absence of exogenous IFN-β in vitro and higher MxA expression in gliomas than in normal tissue in vivo, indicating that IFN signaling is constitutively active in these tumors. Silencing of the type I IFN receptor, IFNAR1/2, or its ligands, IFN-α or IFN-β, decreased MxA levels, providing evidence for the existence of an autocrine type I IFN signaling loop. On a functional level, we observed reduced PD-L1, MHC class I and class II expression and increased susceptibility to immune cell-mediated lysis upon disruption of IFN signaling, suggesting that constitutive IFN signaling in gliomas contributes to the immune evasion of glioma cells. Conclusions: Our findings point to an important role of constitutive IFN signaling in glioma cells in the regulation of anti-tumor immune responses.

Potent Thrombolytic Effect of N-Acetylcysteine on Arterial Thrombi.

Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N-Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization. Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection. We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis. We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.

Abstract 348: Lys 42, 43, 44 and Arg 12 of Thrombin Activable Fibrinolysis Inhibitor Comprise Thrombomodulin Binding Exosite Essential for Exerting Its Antifibrinolytic Activity

The thrombin-thrombomodulin (TM) complex activates thrombin-activable fibrinolysis inhibitor (TAFI) more efficiently than thrombin or plasmin alone. The exosite on TAFI required for its TM-dependent activation by thrombin has not been identified. Based on previous work by us and others, we generated TAFI variants with one or more of residues Lys 42, Lys 43, Lys 44 and Arg 12 within the activation peptide mutated to alanine. Mutation of one, two, or three Lys residues or the Arg residue alone decreased the catalytic efficiency of TAFI activation by thrombin-TM by 2.4-, 3.2-, 4.7-, and 15.0-fold, respectively, and increased the TAFI concentrations required for half-maximal prolongation of clot lysis times (K 1/2 ) by 3-, 4,- 15-, and 24-fold, respectively. Mutation of all four residues eliminated TM binding, decreased the catalytic efficiency of TAFI activation by 45.0-fold, increased the K 1/2 by 130-fold, and abolished antifibrinolytic activity in a clot lysis assay. When thrombin or plasmin was used as the activator, mutation of all four residues reduced the rate of activation by 1.1- and 4.0-fold compared with wild-type TAFI, respectively, suggesting that the mutation only impacted activation kinetics by thrombin-TM. Surface plasmon resonance data show that mutation of the four residues results in complete loss of binding, either in the presence or absence of thrombin. Together, our findings suggest that these four residues are critical for the interaction of TAFI with the thrombin-TM complex that modulates its antifibrinolytic activity.

Ascertainment of pharmacological activities of Allamanda neriifolia Hook and Aegialitis rotundifolia Roxb used in Bangladesh: An in vitro study

The present study was cherished to investigate in vitro thrombolytic, membrane stabilizing and antibacterial activities of Allamanda neriifolia and Aegialitis rotundifolia. Different types methanolic extracts of these two medicinal plants were tested for determining membrane stabilizing activity at a hypotonic solution and heat induce condition by comparing with reference standard acetyl salicylic acid (0.10 mg/mL), where thrombolytic activity assessment was done by employing Streptokinase as standard drug. Finally, antibacterial activity was performed against Staphylococcus aureus as a Gram-positive (+ve) and Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa as Gram negative (−ve) bacteria by using disc diffusion method. In case of membrane stabilizing studies, crude methanolic extracts of A. neriifolia at 10 mg/ml concentration, more importantly, showed 45.80% & 23.52% whereas 10 mg/ml concentration of A. rotundifolia more significantly (p < 0.01) produced 38.40% and 27.04% inhibition of hemolysis for both experimental conditions. Dose-dependently increased activity was found in the thrombolytic study where 10 mg/ml concentration of both A. neriifolia and A. rotundifolia more significantly (p < 0.01) showed 41.91% and 32.76% lysis of clot respectively by in vitro clot lysis assay method. Crude methanolic extracts of A. rotundifolia did not show any suitable antibacterial property against the test bacteria. However, the gram positive (+ve) bacteria also seemed resistant against A. neriifolia extract but this crude methanolic extracts was found to generate moderate antibacterial action against gram-negative (−ve) bacteria. The obtained results confirmed the presence of thrombolytic, membrane stabilizing activity for both plant extract along with moderate antibacterial activity for A. neriifolia.

Enzyme-Cleavable Polymeric Micelles for the Intracellular Delivery of Proapoptotic Peptides.

Peptides derived from the third Bcl-2 homology domain (BH3) renormalize apoptotic signaling by antagonizing prosurvival Bcl-2 family members. These potential peptide drugs exhibit therapeutic activities but are limited by barriers including short circulation half-lives and poor penetration into cells. A diblock polymeric micelle carrier for the BIM BH3 peptide was recently described that demonstrated antitumor activity in a B-cell lymphoma xenograft model [Berguig et al., Mol. Ther. 2015, 23, 907-917]. However, the disulfide linkage used to conjugate the BIM peptide was shown to have nonoptimal blood stability. Here we describe a peptide macromonomer composed of BIM capped with a four amino acid cathepsin B substrate (FKFL) that possesses high blood stability and is cleaved to release the drug inside of target cells. Employing RAFT polymerization, the peptide macromonomer was directly integrated into a multifunctional diblock copolymer tailored for peptide delivery. The first polymer block was made as a macro-chain transfer agent (CTA) and composed of a pH-responsive endosomolytic formulation of N,N-diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA). The second polymer block was a copolymer of the peptide and polyethylene glycol methacrylate (PEGMA). PEGMA monomers of two sizes were investigated (300 Da and 950 Da). Protein gel analysis, high performance liquid chromatography, and coupled mass spectrometry (MS) showed that incubation with cathepsin B specifically cleaved the FKFL linker and released active BIM peptide with PEGMA300 but not with PEGMA950. MALDI-TOF MS showed that incubation of the peptide monomers alone in human serum resulted in partial cleavage at the FKFL linker after 12 h. However, formulation of the peptides into polymers protected against serum-mediated peptide degradation. Dynamic light scattering (DLS) demonstrated pH-dependent micelle disassembly (25 nm polymer micelles at pH 7.4 versus 6 nm unimers at pH 6.6), and a red blood cell lysis assay showed a corresponding increase in membrane destabilizing activity (<1% lysis at pH 7.4 versus 95% lysis at pH 6.6). The full carrier-drug system successfully induced apoptosis in SKOV3 ovarian cancer cells in a dose-dependent manner, in comparison to a control polymer containing a scrambled BIM peptide sequence. Mechanistic analysis verified target-dependent activation of caspase 3/7 activity (8.1-fold increase), and positive annexin V staining (72% increase). The increased blood stability of this enzyme-cleavable peptide polymer design, together with the direct polymerization approach that eliminated postsynthetic conjugation steps, suggests that this new carrier design could provide important benefits for intracellular peptide drug delivery.