Characterization of the interleukin 1 receptor-associated kinase 4 (IRAK4)-encoding gene in salmonid fish: the functional copy is rearranged in Oncorhynchus mykiss and that factor can impair TLR signaling in mammalian cells.

The interleukin 1 receptor-associated kinase 4 (IRAK4) is an essential factor for TLR-mediated activation of the host's immune functions subsequent to pathogen contact. We have characterized the respective cDNA and gene sequences from three salmonid species, salmon, rainbow trout and maraena whitefish. The gene from salmon is structured into eleven exons, as is the mammalian homologue, while exons have been fused in the genes from the two other salmonid species. Rainbow trout expresses also a pseudogene at low levels. Its basic structure resembles more closely the primordial gene than the functional copy does. The N-terminal death domain and the C-terminal protein kinase domain of the factors are better conserved throughout evolution than the linker domain. The deduced amino acid sequences of the factors from all three species group together in an evolutionary tree of IRAK4 factors. Scrutinizing expression and function of IRAK4 from rainbow trout, we found its highest expression in head kidney and spleen and lowest expression in muscle tissue. Infecting fish with Aeromonas salmonicida did not modulate its expression during 72 h of observation. Expression of a GFP-tagged trout IRAK4 revealed, expectedly, its cytoplasmic localization in human HEK-293 cells. However, this factor significantly quenched in a dose-dependent fashion not only the pathogen-induced stimulation of NF-κB factors in the HEK-293 reconstitution system of TLR2 signaling, but also the basal NF-κB levels in unstimulated control cells. Our data unexpectedly imply that IRAK4 is involved in establishing threshold levels of active NF-κB in resting cells.

GRP94 is encoded by two differentially expressed genes during development of rainbow trout (Oncorhynchus mykiss).

The glucose-regulated protein, 94 kDa (GRP94), is an endoplasmic reticulum (ER)-localized heat shock protein that plays among other functions a crucial role in folding and exports of Toll-like receptors (TLRs) and some other immune-relevant factors. We identified two copies of the GRP94-encoding gene in rainbow trout sharing 91% DNA sequence identity. The conceptually translated ORFs encode a 795-aa GRP94a and a 510-aa GRP94b protein variant, respectively, with characteristic domains and amino acid residues. However, the shorter variant lacks motifs required for its localization in the ER and might thus represent an isoform of the putative mammalian ortholog GRP94a. Heat stress only slightly affects the expression of the two GRP94-encoding trout genes, as reported for mammals. We recorded the abundances of transcripts coding for both GRP94 variants as well as for a broad panel of TLRs representing their potential targets. In embryonic and larval trout, only the mRNAs encoding TLR1, -2, -9, and -20 were found in significant concentrations, while the expression of nine other TLRs was hardly detectable. The GRP94a-encoding gene showed constantly high expression levels indicating that this isoform is vitally required throughout the life cycle of rainbow trout. The concentration of the GRP94b-encoding mRNA was only ~0.1% compared to the GRP94a mRNA level. These structural and gene expression data together suggest that the two GRP94 gene products fulfill different physiological roles.

Development of a Novel Valve-Controlled Drug-Elutable Microstent for Microinvasive Glaucoma Surgery: In Vitro and Preclinical In Vivo Studies.

Purpose: Microinvasive glaucoma surgery (MIGS) has become an important treatment approach for primary open-angle glaucoma, although the safe and long-term effective lowering of intraocular pressure with currently available implants for MIGS is not yet achieved to a satisfactory extent. The study focusses on the development and in vitro and in vivo testing of a novel microstent for MIGS. Methods: A silicone elastomer-based microstent was developed. Implants were manufactured using dip coating, fs-laser cutting, and spray coating. Within the current study no antifibrotic drug was loaded into the device. Sterilized microstents were analyzed in vitro regarding pressure-flow characteristics and biocompatibility. Six New Zealand white rabbits were implanted with a microstent draining the aqueous humor from the anterior chamber into the subconjunctival space. Drainage efficacy was evaluated using oculopressure tonometry as a transient glaucoma model. Noninvasive imaging was performed. Results: Microstents were manufactured successfully and characterized in vitro. Implantation in vivo was successful for four animals with additional device fixation. Without additional fixation, dislocation of microstents was found in two animals. Safe and effective intraocular pressure reduction was observed for the four eyes with correctly implanted microstent during the 6-month trial period. Conclusions: The described microstent represents an innovative treatment approach for MIGS. The incorporation of a selectively antifibrotic drug into the microstent drug-elutable coating will be addressed in future investigations. Translational Relevance: The current preclinical study successfully provided proof of concept for our microstent for MIGS which is suitable for safe and effective intraocular pressure reduction and offers promising perspectives for the clinical management of glaucoma.

Thermal, Mechanical and Biocompatibility Analyses of Photochemically Polymerized PEGDA250 for Photopolymerization-Based Manufacturing Processes.

Novel fabrication techniques based on photopolymerization enable the preparation of complex multi-material constructs for biomedical applications. This requires an understanding of the influence of the used reaction components on the properties of the generated copolymers. The identification of fundamental characteristics of these copolymers is necessary to evaluate their potential for biomaterial applications. Additionally, knowledge of the properties of the starting materials enables subsequent tailoring of the biomaterials to meet individual implantation needs. In our study, we have analyzed the biological, chemical, mechanical and thermal properties of photopolymerized poly(ethyleneglycol) diacrylate (PEGDA) and specific copolymers with different photoinitiator (PI) concentrations before and after applying a post treatment washing process. As comonomers, 1,3-butanediol diacrylate, pentaerythritol triacrylate and pentaerythritol tetraacrylate were used. The in vitro studies confirm the biocompatibility of all investigated copolymers. Uniaxial tensile tests show significantly lower tensile strength (82% decrease) and elongation at break (76% decrease) values for washed samples. Altered tensile strength is also observed for different PI concentrations: on average, 6.2 MPa for 1.25% PI and 3.1 MPa for 0.5% PI. The addition of comonomers lowers elongation at break on average by 45%. Moreover, our observations show glass transition temperatures (Tg) ranging from 27 °C to 56 °C, which significantly increase with higher comonomer content. These results confirm the ability to generate biocompatible PEGDA copolymers with specific thermal and mechanical properties. These can be considered as resins for various additive manufacturing-based applications to obtain personalized medical devices, such as drug delivery systems (DDS). Therefore, our study has advanced the understanding of PEGDA multi-materials and will contribute to the future development of tools ensuring safe and effective individual therapy for patients.

Swelling characteristics and biocompatibility of ionic liquid based hydrogels for biomedical applications.

Polymers are commonly used in medical device manufacturing, e.g. for drug delivery systems, bone substitutes and stent coatings. Especially hydrogels exhibit very promising properties in this field. Hence, the development of new hydrogel systems for customized application is of great interest, especially regarding the swelling behavior and mechanical properties as well as the biocompatibility. The aim of this work was the preparation and investigation of various polyelectrolyte and poly-ionic liquid based hydrogels accessible by radical polymerization. The obtained polymers were covalently crosslinked with N,N'-methylenebisacrylamide (MBAA) or different lengths of poly(ethyleneglycol)diacrylate (PEGDA). The effect of different crosslinker-to-monomer ratios has been examined. In addition to the compression curves and the maximum degree of swelling, the biocompatibility with L929 mouse fibroblasts of these materials was determined in direct cell seeding experiments and the outcome for the different hydrogels was compared.

Synthesis of novel carbohydrate based pyridinium ionic liquids and cytotoxicity of ionic liquids for mammalian cells.

The large pool of naturally occurring carbohydrates with their diversity in chirality and structure led to the idea of a systematic investigation of carbohydrate based ILs. To this end, we investigated the influence of different ether groups, mainly methyl or ethyl ether, on the secondary OH groups as well as different configurations on physical properties such as melting point, thermostability and especially the influence on cell toxicity. For this investigation we chose α- and ß-methyl-, ß-allyl- and ß-phenyl d-glucopyranose as well as four 1-deoxy-pentoses. In order to be able to classify the results, more ionic liquids with different structural motives were examined for cytotoxicity. Here, we present data that confirm the biocompatibility of such ILs consisting of naturally occurring molecules or their derivatives. The synthesized carbohydrate based ILs were tested for their suitability as additives in coatings for medical applications such as drug-eluting balloons.

Structurally diverse genes encode Tlr2 in rainbow trout: The conserved receptor cannot be stimulated by classical ligands to activate NF-κB in vitro.

The mammalian toll-like receptor 2 (TLR2) is a dominant receptor for the recognition of Gram-positive bacteria. Its structure and functional properties were unknown in salmonid fish. In RT-PCR and RACE experiments, we obtained the full-length cDNA sequence encoding Tlr2 from rainbow trout (Oncorhynchus mykiss) as well as a copy of an unspliced nonsense message from a highly segmented gene. The primary structure of the encoded receptor complies with the domain structure and ligand-binding sites known from mammals and other fish species and sorts well into the evolutionary tree of teleostean Tlr2s. We retrieved a gene version encoding the receptor on a single exon (tlr2a) and also a partial sequence of a second gene variant being segmented into multiple exons (tlr2b). Surprisingly, the abundances of both transcript variants accounted only for ∼10% of all Tlr2-encoding transcripts in various tissues and cell types of healthy fish. This suggests the expression of several distinct tlr2 gene variants in rainbow trout. We expressed tlr2a in HEK-293 cells, but were unable to demonstrate its functionality through NF-κB activation. Neither synthetic lipopeptides known to stimulate mammalian TLR2 nor different bacterial challenges induced OmTLR2-mediated NF-κB activation, not in HEK-293 or in salmon CHSE-214 cells. Positive demonstration of TLR2-MYD88 interaction excluded that its functional impairment caused the failure of NF-κB activation. We discuss impaired heterodimerization with a necessary Tlr partner as one from among several alternatives to explain the dysfunction of Tlr2a in the interspecies reconstitution system of TLR signaling.

Transcriptome sequencing of maraena whitefish (Coregonus maraena).

Maraena whitefish (Coregonus maraena, Bloch, 1779) is a high-quality food fish belonging to the family Salmonidae with considerable economic relevance in the Baltic area. Aquaculture of this species is fundamental for its successful conservation and thus sustainable fisheries. Robust fishes obtained from breeding lines build the basis for effective aquaculture. Doubtless, the utilization of transcriptome sequencing and identification of genetic markers contribute to this aim. 454 FLX Titanium Sequencing provided 1.31 million sequence reads representing a first insight into the C. maraena transcriptome. The 454 Newbler Assembly arranged 29,094 contigs with an average length of 798bp. We found a whole series of transcripts highly probably resulting from ancient genome duplication and annotated 2887 different transcripts with an average length of 812bp. Functional annotation obtained a transcript composition predominantly comprising enzyme-coding genes.

Aeromonas salmonicida Infection Only Moderately Regulates Expression of Factors Contributing to Toll-Like Receptor Signaling but Massively Activates the Cellular and Humoral Branches of Innate Immunity in Rainbow Trout (Oncorhynchus mykiss).

Toll-like receptors (TLRs) are known to detect a defined spectrum of microbial structures. However, the knowledge about the specificity of teleost Tlr factors for distinct pathogens is limited so far. We measured baseline expression profiles of 18 tlr genes and associated signaling factors in four immune-relevant tissues of rainbow trout Oncorhynchus mykiss. Intraperitoneal injection of a lethal dose of Aeromonas salmonicida subsp. salmonicida induced highly increased levels of cytokine mRNAs during a 72-hour postinfection (hpi) period. In contrast, only the fish-specific tlr22a2 and the downstream factor irak1 featured clearly increased transcript levels, while the mRNA concentrations of many other tlr genes decreased. Flow cytometry quantified cell trafficking after infection indicating a dramatic influx of myeloid cells into the peritoneum and a belated low level immigration of lymphoid cells. T and B lymphocytes were differentiated with RT-qPCR revealing that B lymphocytes emigrated from and T lymphocytes immigrated into head kidney. In conclusion, no specific TLR can be singled out as a dominant receptor for A. salmonicida. The recruitment of cellular factors of innate immunity rather than induced expression of pathogen receptors is hence of key importance for mounting a first immune defense against invading A. salmonicida.