Elasticity Modification of Biomaterials Used in 3D Printing with an Elastin-Silk-like Recombinant Protein.

The recombinant structural protein described in this study was designed based on sequences derived from elastin and silk. Silk-elastin hybrid copolymers are characterized by high solubility while maintaining high product flexibility. The phase transition temperature from aqueous solution to hydrogel, as well as other physicochemical and mechanical properties of such particles, can differ significantly depending on the number of sequence repeats. We present a preliminary characterization of the EJ17zipR protein obtained in high yield in a prokaryotic expression system and efficiently purified via a multistep process. Its addition significantly improves biomaterial's rheological and mechanical properties, especially elasticity. As a result, EJ17zipR appears to be a promising component for bioinks designed to print spatially complex structures that positively influence both shape retention and the internal transport of body fluids. The results of biological studies indicate that the addition of the studied protein creates a favorable microenvironment for cell adhesion, growth, and migration.

Characterization of a Chimeric Resilin-Elastin Structural Protein Dedicated to 3D Bioprinting as a Bioink Component.

In this study we propose to use for bioprinting a bioink enriched with a recombinant RE15mR protein with a molecular weight of 26 kDa, containing functional sequences derived from resilin and elastin. The resulting protein also contains RGD sequences in its structure, as well as a metalloproteinase cleavage site, allowing positive interaction with the cells seeded on the construct and remodeling the structure of this protein in situ. The described protein is produced in a prokaryotic expression system using an E. coli bacterial strain and purified by a process using a unique combination of known methods not previously used for recombinant elastin-like proteins. The positive effect of RE15mR on the mechanical, physico-chemical, and biological properties of the print is shown in the attached results. The addition of RE15mR to the bioink resulted in improved mechanical and physicochemical properties and promoted the habitation of the prints by cells of the L-929 line.

Chitosan-based formulation of hemagglutinin antigens for oculo-nasal booster vaccination of chickens against influenza viruses.

Avian influenza viruses (AIVs) and especially highly pathogenic (HP) AIVs of the H5 and H7 subtypes are of both veterinary and public health concern worldwide. In response to the demand for effective vaccines against H5N1 HPAIVs, we produced recombinant protein based on hemagglutinin (HA), a protective viral antigen. A fragment of the HA ectodomain, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. Finally, the protein was formulated in Tris-HCl buffer of pH 8.0 or PBS of pH 7.4 to obtain antigens denoted rH5-1 and rH5-2, respectively. The systemic prime and boost immunizations proved that rH5-1 adsorbed to aluminum hydroxide induces anti-H5 HA neutralizing antibodies and protective immune responses against H5N1 HPAIVs in chickens. The present studies were aimed at stimulating immune responses via the mucosal routes using the systemic prime-mucosal boost strategy. Efficacy trials were performed in commercial layer chickens. For systemic and mucosal immunizations, H5 HA antigens were adjuvanted with aluminum hydroxide and chitosan glutamate, respectively. The first dose of rH5-2 was administered subcutaneously, while its second dose was administered subcutaneously, intraocularly, oculo-nasally, or intranasally. rH5-1 was delivered to the subcutaneously primed chickens by the intranasal route. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition tests. Intraocularly and oculo-nasally delivered rH5-2 mixed with chitosan glutamate was capable of stimulating anti-H5 HA IgY antibody responses in the subcutaneously primed chickens; however, it was ineffective when administered by the intranasal route. Efficient intranasal boosting was achieved using rH5-1. The enhanced production of antigen-specific antibodies was reflected in the development of H5-subtype specific and hemagglutination inhibiting antibodies. Conclusively, the subcutaneous prime and oculo-nasal boost vaccination is proposed as the target strategy for future optimization.

A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin.

BACKGROUND: H5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA). METHODS: In the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1-H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis. RESULTS: The H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively. CONCLUSIONS: The H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.

Chemical modification of melphalan as a key to improving treatment of haematological malignancies.

Chemical modification of known, effective drugs is one method to improve chemotherapy. Thus, the object of this study was to generate melphalan derivatives with improved cytotoxic activity in human cancer cells (RPMI8226, HL60 and THP1). Several melphalan derivatives were synthesised, modified in their two important functional groups. Nine analogues were tested, including melphalan compounds modified: only at the amino group, by replacing the amine with an amidine group containing a morpholine ring (MOR-MEL) or with an amidino group and dipropyl chain (DIPR-MEL); only at the carboxyl group to form methyl and ethyl esters of melphalan (EM-MEL, EE-MEL); and in a similar manner at both functional groups (EM-MOR-MEL, EE-MOR-MEL, EM-DIPR-MEL, EE-DIPR-MEL). Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity (comet assay) and the ability to induce apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling, TUNEL, phosphatidylserine externalisation, chromatin condensation, activity of caspases 3/7, 8 and 9 and intracellular concentration of calcium ions) in comparison with the parent drug. Almost all derivatives, with the exception of MOR-MEL and DIPR-MEL, were found to be more toxic than melphalan in all cell lines evaluated. Treatment of cultures with the derivatives generated a significant higher level of DNA breaks compared to those treated with melphalan, especially after longer incubation times. In addition, all the melphalan derivatives demonstrated a high apoptosis-inducing ability in acute monocytic and promyelocytic leukemia cells. This study showed that the mechanism of action of the tested compounds differed depending on the cell line, and allowed the selection of the most active compounds for further, more detailed investigations.

Bacterial expression and characterization of an anti-CD22 single-chain antibody fragment.

Single-chain variable fragment (scFv) antibodies are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. They possess unique and superior features compared to whole antibodies for immunotherapy of various carcinomas, including hematologic B-cell malignancies. In the presented study we obtained efficient production of the recombinant anti-CD22 scFv in Escherichia coli expression system. The active recombinant protein was successfully recovered from inclusion bodies. Assays were performed to assess the in vitro targeting properties and specificity of the obtained anti-CD22 scFv antibody in the CD22 positive and negative lymphoma cell lines.

Prime-Boost Vaccination With a Novel Hemagglutinin Protein Produced in Bacteria Induces Neutralizing Antibody Responses Against H5-Subtype Influenza Viruses in Commercial Chickens.

The highly pathogenic (HP) avian influenza virus (AIV), H5N1 and reassortant H5-subtype HPAIVs, H5N2, H5N6, and H5N8, cause high mortality in domestic birds, resulting in economic losses in the poultry industry. H5N1 and H5N6 also pose significant public health risks and H5N1 viruses are a permanent pandemic threat. To control HPAIVs, eukaryotic expression systems have traditionally been exploited to produce vaccines based on hemagglutinin (HA), a protective viral antigen. In contrast, we used a bacterial expression system to produce vaccine targeting the HA protein. A fragment of the HA ectodomain from H5N1, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. The resulting antigen, rH5-E. coli, was validated in terms of conformational integrity and oligomerization status. Previously, the protective efficacy of rH5-E. coli adjuvanted with aluminum hydroxide, has been positively verified by challenging the specific pathogen-free layer chickens with homologous and heterologous H5N1 HPAIVs. Protection was provided primarily by the H5 subtype-specific antibodies, as detected in the FluAC H5 test. The present studies were conducted to assess the performance of alum-adjuvanted rH5-E. coli in commercial birds. Broiler chickens were vaccinated twice with 25 µg of rH5-E. coli at 2- and 4-week intervals, while the layer chickens were vaccinated with 5- to 25-µg antigen doses at 4- and 6-week intervals. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition (HI) tests. Prime-boost immunizations with rH5-E. coli elicited H5 HA-specific antibodies in all the chickens tested. Two antigen doses administered at 4- or 6-week intervals were sufficient to induce neutralizing antibodies against H5-subtype HAs; however, they were ineffective when applied with a 2-week delay. In the layers, 80% to 100% of individuals developed antibodies that were active in the FluAC H5 and/or HI tests. A dose-sparing effect was seen when using the longer prime-boost interval. In the broiler chickens, 62.5% positivity was achieved in the FluAC H5 and/or HI tests. The trials confirmed the vaccine potential of rH5-E. coli and provided indications for anti-influenza vaccination with respect to the chicken type and immunization scheme.

Production of highly and broad-range specific monoclonal antibodies against hemagglutinin of H5-subtype avian influenza viruses and their differentiation by mass spectrometry.

BACKGROUND: The highly pathogenic avian influenza viruses of the H5 subtype, such as the H5N1 viral strains or the novel H5N8 and H5N2 reassortants, are of both veterinary and public health concern worldwide. To combat these viruses, monoclonal antibodies (mAbs) against H5 hemagglutinin (HA) play a significant role. These mAbs are effective diagnostic and therapeutic agents and powerful tools in vaccine development and basic scientific research. The aim of this study was to obtain diagnostically valuable mAbs with broad strain specificity against H5-subtype AIVs. RESULTS: We applied the hybridoma method to produce anti-HA mAbs. The cloning and screening procedures resulted in the selection of 7 mouse hybridoma cell lines and their respective antibody clones. Preliminary immunoreactivity studies showed that these newly established mAbs, all of the IgG1 isotype, had high specificity and broad-range activities against the H5 HAs. However, these studies did not allow for a clear distinction among the selected antibodies and mAb-secreting hybridoma clones. To differentiate the analyzed mAbs and determine the exact number of hybridoma clones, peptide mapping of the Fc and Fab fragments was performed using a Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF/TOF) mass spectrometer. Detailed analyses of the acquired MS and MS/MS spectra confirmed that the Fc fragments constituted highly conserved species- and isotype-immunoglobulin components, whereas the Fab fragments exhibited considerable variation in the sequences that determine antibody specificity. This approach enabled unambiguous characterization of the selected mAbs according to their peptide composition. As a result, 6 different clones were distinguished. CONCLUSIONS: Our work provided a unique panel of anti-H5 HA mAbs, which meets the demand for novel, high-specificity analytical tools for use in serologic surveillance. Applications of these mAbs in areas other than diagnostics are also possible. Moreover, we demonstrated for the first time that peptide mapping of antibody fragments with mass spectrometry is an efficient method for the differentiation of antibody clones and relevant antibody-producing cell lines. The method may be successfully used to characterize mAbs at the protein level.

Correction: A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies.

[This corrects the article DOI: 10.1371/journal.pone.0172008.].

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies.

The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.

Efficient Source of Cells in Proximal Oviduct for Testing Non-Viral Expression Constructs in the Chicken Bioreactor Model and for Other in Vitro Studies.

This work shows the usefulness of chicken oviduct epithelial cells (COEC) in evaluating the efficacy of non-viral expression vectors carrying human therapeutic genes. Secondly, an efficient source of progenitor COEC for in vitro studies is described. Within the distal part of the oviduct, weak to moderate expression of a trans membrane glycoprotein (CD44) was observed. Single cells presenting only weak expression of CD44 were found in magnum sections. in vitro cultured oviduct cells originating from the distal oviduct were suitable for subculturing and showed a stable proliferation potential up to the 2nd passage. However, the pavimentous epithelial-like morphology of COEC was progressively lost over time and mainly a fibroblast-like monolayer was established in consecutive passages. Moreover, various commercial transfection agents including FuGENE6 and XtremeGENE9 DNA were used to optimize delivery of human interferon alfa-2a, (IFNα2a) a therapeutic protein gene under an ovalbumin promoter. The transfection efficiency of adherent COEC was estimated for up to 40% at a ratio of 6:1 of transfectant to pOVA5EIFN + GFP plasmid. Expression of IFNα2a was confirmed by western blotting in transformed COEC. In conclusion, the population of epithelial progenitor cells sourced from the distal oviduct can significantly contribute to in vitro culture of COEC, representing an efficient model to develop the production of avian bioreactors and other in vitro studies related to oviduct tissue.

Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification.

The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.

Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response.

Efficient sorting of TNF-alpha to rodent mast cell granules is dependent on N-linked glycosylation.

Mast cells play an important role at the early stages of immunological response to bacterial infections and parasite infestations. One of the major mast cell proinflammatory mediators is TNF-alpha. Mast cells are considered the only cells capable of storing TNF-alpha in cytoplasmic granules and rapidly releasing it upon activation. To determine what pathway is utilized to direct TNF-alpha to cytoplasmic granules and what motifs are responsible for the sorting process, we constructed a fusion protein covering the full sequence of TNF-alpha, N-terminally fused to enhanced green fluorescent protein (EGFP). In rodent mast cells, such protein was sorted to secretory granules, and this process was inhibited by both brefeldin A and monensin. Considering the relationship between lysosomes and secretory granules and following TNF-alpha sequence analysis, it was determined whether TNF-alpha is sorted through the mannose-6-phosphate receptor (MPR)-dependent pathway. We observed that ammonium chloride and tunicamycin blocked TNF-alpha-EGFP fusion protein delivery to secretory granules. In situ mutagenesis experiments confirmed the necessity of N-linked glycosylation for efficient sorting of TNF-alpha into rodent mast cell granules. In this work we established that TNF-alpha travels from the ER to mast cell granules via a brefeldin A- and monensin-sensitive route, utilizing the MPR-dependent pathway, although this dependency does not seem to be absolute.

"Fluorescent Cell Chip" for immunotoxicity testing: development of the c-fos expression reporter cell lines.

The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals.

Detection of immunotoxicity using T-cell based cytokine reporter cell lines ("Cell Chip").

Safety assessment of chemicals and drugs is an important regulatory issue. The evaluation of potential adverse effects of compounds on the immune system depends today on animal experiments. An increasing demand, however, exists for in vitro alternatives. Cytokine measurement is a promising tool to evaluate chemical exposure effects on the immune system. Fortunately, this type of measurement can be performed in conjunction with in vitro exposure models. We have taken these considerations as the starting point to develop an in vitro method to efficiently screen compounds for potential immunotoxicity. The T-cell lymphoma cell line EL-4 was transfected with the regulatory sequences of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma or actin fused to the gene for enhanced green fluorescent protein (EGFP) in either a stabile or a destabilised form. Consequently, changes in fluorescence intensity represent changes in cytokine expression with one cell line per cytokine. We used this prototype "Cell Chip" to test, by means of flow cytometry, the immunomodulatory potential of 13 substances and were able to detect changes in cytokine expression in 12 cases (successful for cyclosporine, rapamycin, pentamidine, thalidomide, bis(tri-n-butyltin)oxide, house dust mite allergen (Der p I), 1-chloro-2,4-dinitrobenzene, benzocaine, tolylene 2,4-diisocyanate, potassium tetrachloroplatinate, sodium dodecyl sulphate and mercuric chloride; unsuccessful for penicillin G). In conclusion, this approach seems promising for in vitro screening for potential immunotoxicity, especially when additional cell lines besides T-cells are included.

Development of the "Cell Chip": a new in vitro alternative technique for immunotoxicity testing.

Predictive testing of immunotoxicity associated with chemical compounds is complicated and cannot be accomplished with a single test. As most of the existing tests for immunotoxicity employ experimental animals, there is an increasing need for alternative tests in vitro. We have developed a new system for in vitro immunotoxicity testing, which employs changes in cytokine expression observed in vitro as an endpoint indicating potential for perturbation of the immune system in vivo. This system named "fluorescent cell chip" (FCC) is based on a number of genetically modified cell lines that regulate the expression of a transgene coding for fluorescent protein enhanced green fluorescent protein (EGFP) in a similar way as they regulate expression of IL-1beta, IL-2, IL-4, IFN-gamma, IL-10, TNF-alpha, and beta-actin. Morphological and functional features of selected cell lines expressing EGFP under the control of cytokine promotors were compared with maternal cell lines and this comparison showed that critical functional features of the maternal cell lines were preserved in EGFP expressing cells. Two chemicals with known immunotoxic activities, cyclosporine A and potassium tetrachloro-platinate(II), mediated compound-specific pattern of inhibition and activation of reporter gene expression. Thus, the "fluorescent cell chip" has demonstrated potential for application as a predictive screening test for immunomodulatory activities of chemicals. The major advantage of this approach is the possibility to apply this test in high throughput screening of high number of compounds for their well defined biological activity.