Characterization and Cytotoxic Activity of Microwave-Assisted Extracted Crude Fucoidans from Different Brown Seaweeds.

Microwave-assisted extraction (MAE) is recognized as a green method for extraction of natural products. The current research aimed to explore the MAE for fucoidans extraction from different brown seaweeds, including Fucus vesiculosus, F. spiralis, and Laminaria saccharina. Following several solvent-extraction pre-treatment steps and MAE optimization, the algal biomasses were extracted in a ratio of 1:25 in 0.1 M HCl containing 2 M CaCl2 for 1.0 min. The results showed that L. saccharina's extract was different from the others, regarding the highest sugar content reached 0.47 mg glucose equivalent/mg extract being confirmed by monosaccharide composition analysis and the lowest fucoidan content and sulfation degree at 0.09 mg/mg extract and 0.13, respectively. Moreover, these findings were confirmed by tentative structural elucidation based on Fourier-transform infrared spectrometry which also showed a different spectrum. However, the MAE enhanced melanoidins formation in products, which was confirmed by the intense band at 1420 cm-1. Interestingly, the results of monomeric composition showed that fucoidan extract by MAE from F. vesiculosus belonged to sulfated galactofucans which are known for their potential bioactivities. Furthermore, the cytotoxic activity of the four fucoidans in concentrations ranging from 4.9 µg/mL to 2500 µg/mL was investigated and correlated with the chemical characterization showing that F. vesiculosus_MAE fucoidan was the most potent and safest. The current research revealed the chemical heterogeneity of fucoidans regarding taxonomical class and used greener extraction method of fucoidans toward the achievement of the UN sustainability goals.

Human-Based Immune Responsive In Vitro Infection Models for Validation of Novel TLR4 Antagonists Identified by Computational Discovery.

Infectious diseases are still a major problem worldwide. This includes microbial infections, with a constant increase in resistance to the current anti-infectives employed. Toll-like receptors (TLRs) perform a fundamental role in pathogen recognition and activation of the innate immune response. Promising new approaches to combat infections and inflammatory diseases involve modulation of the host immune system via TLR4. TLR4 and its co-receptors MD2 and CD14 are required for immune response to fungal and bacterial infection by recognition of microbial cell wall components, making it a prime target for drug development. To evaluate the efficacy of anti-infective compounds early on, we have developed a series of human-based immune responsive infection models, including immune responsive 3D-skin infection models for modeling fungal infections. By using computational methods: pharmacophore modeling and molecular docking, we identified a set of 46 potential modulators of TLR4, which were screened in several tests systems of increasing complexity, including immune responsive 3D-skin infection models. We could show a strong suppression of cytokine and chemokine response induced by lipopolysacharide (LPS) and Candida albicans for individual compounds. The development of human-based immune responsive assays provides a more accurate and reliable basis for development of new anti-inflammatory or immune-modulating drugs.

A helicase-primase drug candidate with sufficient target tissue exposure affects latent neural herpes simplex virus infections.

More than 50% of the world population is chronically infected with herpesviruses. Herpes simplex virus (HSV) infections are the cause of herpes labialis (cold sores), genital herpes, and sight-impairing keratitis. Less frequently, life-threatening disseminated disease (encephalitis and generalized viremia) can also occur, mainly in immunocompromised patients and newborns. After primary infection, HSV persists for life in a latent state in trigeminal or sacral ganglia and, triggered by diverse stimuli, disease recurs in more than 30% of patients up to several times a year. Current therapy with nucleoside analogs targeting the viral polymerase is somewhat effective but limited by poor exposure in the nervous system, and latent infections are not affected by therapy. Here, we report on an inhibitor of HSV helicase-primase with potent in vitro anti-herpes activity, a different mechanism of action, a low frequency of HSV resistance, and a favorable pharmacokinetic and safety profile. Improved target tissue exposure results in superior efficacy in preventing and treating HSV infection and disease in animal models as compared to standard of care. Therapy of primary HSV infections with drug candidate IM-250 {(S)-2-(2',5'-difluoro-[1,1'-biphenyl]-4-yl)-N-methyl-N-(4-methyl-5-(S-methylsulfon-imidoyl)thiazol-2-yl)acetamide} not only reduces the duration of disease symptoms or time to healing but also prevents recurrent disease in guinea pigs. Treatment of recurrent infections reduces the frequency of recurrences and viral shedding, and, unlike nucleosidic drugs, IM-250 remains effective for a time after cessation of treatment. Hence, IM-250 has advantages over standard-of-care therapies and represents a promising therapeutic for chronic HSV infection, including nucleoside-resistant HSV.

Computationally Designed Bispecific MD2/CD14 Binding Peptides Show TLR4 Agonist Activity.

Toll-like receptor 4 plays an important role in the regulation of the innate and adaptive immune response. The majority of TLR4 activators currently in clinical use are derivatives of its prototypic ligand LPS. The discovery of innovative TLR4 activators has the potential of providing new therapeutic immunomodulators and adjuvants. We used computational design methods to predict and optimize a total of 53 cyclic and linear peptides targeting myeloid differentiation 2 (MD2) and cluster of differentiation 14 (CD14), both coreceptors of human TLR4. Activity of the designed peptides was first assessed using NF-κB reporter cell lines expressing either TLR4/MD2 or TLR4/CD14 receptors, then binding to CD14 and MD2 confirmed and quantified using MicroScale Thermophoresis. Finally, we incubated select peptides in human whole blood and observed their ability to induce cytokine production, either alone or in synergy with LPS. Our data demonstrate the advantage of computational design for the discovery of new TLR4 peptide activators with little structural resemblance to known ligands and indicate an efficient strategy with which to identify TLR4 targeting peptides that could be used as easy-to-produce alternatives to LPS-derived molecules in a variety of settings.

Central Role for Dermal Fibroblasts in Skin Model Protection against Candida albicans.

The fungal pathogen Candida albicans colonizes basically all human epithelial surfaces, including the skin. Under certain conditions, such as immunosuppression, invasion of the epithelia occurs. Not much is known about defense mechanisms against C. albicans in subepithelial layers such as the dermis. Using immune cell-supplemented 3D skin models we defined a new role for fibroblasts in the dermis and identified a minimal set of cell types for skin protection against C. albicans invasion. Dual RNA sequencing of individual host cell populations and C. albicans revealed that dermal invasion is directly impeded by dermal fibroblasts. They are able to integrate signals from the pathogen and CD4+ T cells and shift toward an antimicrobial phenotype with broad specificity that is dependent on Toll-like receptor 2 and interleukin 1ß. These results highlight a central function of dermal fibroblasts for skin protection, opening new possibilities for treatment of infectious diseases.

Computational Discovery and Experimental Confirmation of TLR9 Receptor Antagonist Leads.

Toll-like receptors (TLR) are receptors of innate immunity that recognize pathogen associated molecular patterns. They play a critical role in many pathological states, in acute and chronic inflammatory processes. TLR9 is a promising target for drug discovery, since it has been implicated in several pathologies, including defense against viral infections and psoriasis. Immune-modulators are promising molecules for therapeutic intervention in these indications. TLR9 is located in the endosome and activated by dsDNA with CpG motives encountered in microbial DNA. Here we report on a combined approach to discover new TLR9 antagonists by computational chemistry and cell based assays. We used our in-house iterative stochastic elimination (ISE) algorithm to create models that distinguish between TLR9 antagonists ("actives") and other molecules ("inactives"), based on molecular physicochemical properties. Subsequent screening and scoring of a data set of 1.8 million commercially available molecules led to the purchasing of top scored molecules, which were tested in a new cell based system based on human pattern recognition receptors (PRRs) stably expressed in NIH3T3 fibroblasts. As described previously, this cell line shows a very low endogenous PRR-activity and contains a reporter gene which is selectively activated by the integrated human PRR enabling rapid screening of potential ligands. IC50 values of each of these top scored molecules were determined. Out of 60 molecules tested, 56 showed antagonistic activity. We discovered 21 new highly potential antagonists with IC50 values lower than 10 µM, with 5 of them having IC50 values under 1 µM.

Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.

An In Vitro HSV-1 Reactivation Model Containing Quiescently Infected PC12 Cells.

Advances in the understanding of the infection and reactivation process of herpes simplex type 1 (HSV-1) are generally gained by monolayer cultures or extensive and cost-intensive animal models. So far, no reliable in vitro skin model exists either to investigate the molecular mechanisms involved in controlling latency and virus reactivation or to test pharmaceuticals. Here we demonstrate the first in vitro HSV-1 reactivation model generated by using the human keratinocyte cell line HaCaT grown on a collagen substrate containing primary human fibroblasts. We integrated the unique feature of a quiescently infected neuronal cell line, the rat pheochromocytoma line PC12, within the dermal layer of the three-dimensional skin equivalent. Transmission electron microscopy, a cell-based TCID50 assay, and polymerase chain reaction analysis were used to verify cell latency. Thereby viral DNA could be detected, whereas extracellular as well as intracellular virus activity could not be found. Further, the infected PC12 cells show no spontaneous reactivation within the in vitro skin equivalent. In order to simulate a physiologically comparable HSV-1 infection, we achieved a specific and pointed reactivation of quiescently HSV-1 infected PC12 cells by UVB irradiation at 1000 mJ/cm(2).

A screening assay based on host-pathogen interaction models identifies a set of novel antifungal benzimidazole derivatives.

Fungal infections are a serious health problem in clinics, especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses, only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapy, significant side effects, and high costs for several antifungals, there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities, we screened a compound library including more than 35,000 individual compounds derived from organic synthesis as well as combinatorial compound collections representing mixtures of compounds for antimycotic activity. In total, more than 100,000 compounds were screened using a new type of activity-selectivity assay, analyzing both the antifungal activity and the compatibility with human cells at the same time. One promising hit, an (S)-2-aminoalkyl benzimidazole derivative, was developed among a series of lead compounds showing potent antifungal activity. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole showed the highest antifungal activity and the best compatibility with human cells in several cell culture models and against a number of clinical isolates of several species of pathogenic Candida yeasts. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol pathway, in contrast to other benzimidazole derivatives, which target microtubules.

High-throughput-screening-based identification and structure-activity relationship characterization defined (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole as a highly antimycotic agent nontoxic to cell lines.

Novel nontoxic (S)-2-aminoalkylbenzimidazole derivatives were found to be effective against Candida spp. at low micromolar concentrations using high-throughput screening with infected HeLa cells. A collection of analogues defined the chemical groups relevant for activity. The most active compound was characterized by transcriptional analysis of the response of C. albicans Sc5314. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole had a strong impact on membrane biosynthesis. Testing different clinically relevant pathogenic fungi showed the selectivity of the antimycotic activity against Candida species.

A new cell-based innate immune receptor assay for the examination of receptor activity, ligand specificity, signalling pathways and the detection of pyrogens.

The pattern recognition receptors (PRRs) of the innate immune system are the first defence line of the immune system. Toll-like receptors (TLRs) are the most well known and the best examined of the PR receptors. In the last years TLRs had been studied in different ways resulting in a lot of new insights in the function and signalling pathways of these receptors. However, it was not possible to investigate individual combinations of the TLRs and their specific ligands, because of the complex network in immune signalling resulting in interference with each other. This work shows a new cell-based assay, established for the analysis of single PRRs or heterodimers. For this purpose NIH3T3 (mouse fibroblasts) were stably transfected with the NF-kappaB-inducible reporter gene secreted alkaline phosphatase (SEAP) together with the corresponding combinations of human TLRs and their co-receptors (e.g. TLR1/2, TLR2/6 and TLR4/CD14). The specificity of the respective cell lines was shown by induction with variations of specific and unspecific ligands (immune-stimulating components of microorganisms or synthetic ligands). Analysis via the NF-kappaB-dependent reporter gene SEAP allows a direct way to detect the human TLR-activity. Our results showed that this assay is highly sensitive and specific for the respective ligands. For the synthetic ligands Pam(2)CysSK(4) the assay demonstrates a detection limit of 1 pg/ml for TLR2/6. In summary, this test system allows the investigation of individual human PRR-receptors in a highly specific way, without interference with other immune components opening new avenues for novel insights in the innate immune system and its applications.

Reduction of the aortic inflammatory response in spontaneous atherosclerosis by blockade of macrophage migration inhibitory factor (MIF).

Atherosclerosis is an inflammatory response of the arterial wall to "injury", which is prominently driven by cytokines. The inflammatory mediator macrophage migration inhibitory factor (MIF) is a unique cytokine that was recently associated with atherogenesis. Here, we have investigated whether MIF has a role in spontaneous atherosclerosis by studying apolipoprotein E-deficient (ApoE(-/-)) mice treated with neutralizing anti-MIF monoclonal antibody and comparison with isotype IgG-treated controls. After 14 weeks, the aortas and heart valves were analyzed for inflammatory status, macrophage content and plaque areas. MIF expression in the aortic wall was elevated upon spontaneous atherogenesis, with foam cells representing a major source. Of note, MIF blockade led to a marked reduction in intimal Mac-1-positive macrophages. Similarly, treatment with anti-MIF antibody led to a reduction of a variety of inflammatory mediators typically associated with atherosclerosis including the circulating levels of fibrinogen, MIF and IL-6. Importantly, the local aortic expression of ICAM-1, MMP-2, TNF, IL-12, and CD40L was reduced by MIF blockade, as were the levels of the phospho-c-Jun and C/EBPbeta transcription factors. The observed strong reduction of inflammatory parameters by anti-MIF treatment was associated with a small, yet non-significant, reduction in aortic plaque area. Thus, although MIF's role is not directly linked to plaque volume expansion, in this mouse model of spontaneous atherogenesis, MIF plays an important role in intimal inflammation.

Binding of JAB1/CSN5 to MIF is mediated by the MPN domain but is independent of the JAMM motif.

Macrophage migration inhibitory factor (MIF) binds to c-Jun activation domain binding protein-1 (JAB1)/subunit 5 of COP9 signalosome (CSN5) and modulates cell signaling and the cell cycle through JAB1. The binding domain of JAB1 responsible for binding to MIF is unknown. We hypothesized that the conserved Mpr1p Pad1p N-terminal (MPN) domain of JAB1 may mediate binding to MIF. In fact, yeast two hybrid (YTH) and in vitro translation/coimmunoprecipitation (CoIP) analysis showed that a core MPN domain, which did not cover the functional JAB1/MPN/Mov34 metalloenzyme (JAMM) deneddylase sequence, binds to MIF comparable to full-length JAB1. YTH and pull-down analysis in conjunction with nanobead affinity matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry demonstrated that MIF(50-65) and MPN are sufficient to mediate MIF-JAB1 interaction, respectively. Finally, endogenous CoIP of MIF-CSN6 complexes from mammalian cells demonstrated that MPN is responsible for MIF-JAB1 binding in vivo, and, as CSN6 does not contain a functional JAMM motif, confirmed that the interaction does not require JAMM.

The cytokine macrophage migration inhibitory factor reduces pro-oxidative stress-induced apoptosis.

The cytokine macrophage migration inhibitory factor (MIF) exhibits pro- and anti-inflammatory activities and regulates cell proliferation and survival. We investigated the effects of MIF on apoptosis. As MIF exhibits oxidoreductase activity and participates in regulating oxidative cell stress, we studied whether MIF could affect oxidative stress-induced apoptosis. We demonstrated that MIF exhibits antiapoptotic activity in various settings. MIF suppressed camptothecin-induced apoptosis in HeLa and Kym cells and HL-60 promyeloblasts. Both exogenous MIF and endogenous MIF, induced following overexpression through tetracycline (tet) gene induction, led to significant suppression of apoptosis. Apoptosis reduction by MIF was also observed in T cells. A role for MIF in redox stress-induced apoptosis was addressed by comparing the effects of rMIF with those of the oxidoreductase mutant C60SMIF. Endogenous overexpression of C60SMIF was similar to that of MIF, but C60SMIF did not suppress apoptosis. Exogenous rC60SMIF inhibited apoptosis. A role for MIF in oxidative stress-induced apoptosis was directly studied in HL-60 leukocytes and tet-regulated HeLa cells following thiol starvation or diamide treatment. MIF protected these cells from redox stress-induced apoptosis and enhanced cellular glutathione levels. As overexpressed C60SMIF did not protect tet-regulated HeLa cells from thiol starvation-induced apoptosis, it seems that the redox motif of MIF is important for this function. Finally, overexpression of MIF inhibited phosphorylation of endogenous c-Jun induced by thiol starvation, indicating that MIF-based suppression of apoptosis is mediated through modulation of c-Jun N-terminal kinase activity. Our findings show that MIF has potent antiapoptotic activities and suggest that MIF is a modulator of pro-oxidative stress-induced apoptosis.