The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

The expression of the HER2 (human epidermal growth factor receptor 2) protein in cancer cells is a well-established cancer marker used for diagnostic and therapeutic purposes in modern treatment protocols, especially in breast cancer. The gold-standard immunohistochemical diagnostic methods with the specific anti-HER2 antibodies are utilized in the clinic to measure expression level of the membrane-bound receptor. However, a soluble extracellular domain (ECD) of HER2 is released to the extracellular matrix, thus the blood assays for HER2 measurements present an attractive way for HER2 level determination. There is a need for accurate and validated assays that can be used to correlate the concentration of the circulating HER2 protein with disease clinical manifestations. Here we describe two monoclonal antibodies binding HER2 with a unique sequence of the complementarity-determining regions that recognize HER2 ECD. Development and validation of the sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the soluble HER2 in a variety of biological samples is also presented. The assay provides HER2 quantitation within a concentrations range from 1.56 to 100 ng/ml with sensitivity at the level of 0.5 ng/ml that meets the expectations for measurements of HER2 in the blood and tumor tissue samples. The method presents satisfactory intra- and inter-assay precision and accuracy for immunochemical quantification of biomarkers in biological samples. The utility of the generated monoclonal anti-HER2 antibodies has been confirmed for use in the precise measurement of HER2 (both cell-bound and soluble) in several types of biological material, including serum, solid tumor tissue, and cell culture medium. Additionally, the developed immunochemical tools have a potential for HER2 detection, not only in a wide range of sample types but also independently of the sample storage/pre-processing, allowing for comprehensive HER2 analysis in tissue (IHC), cultured cells (immunofluorescence) and blood (ELISA).

Influence of the Scatter Index of Non-Metallic Inclusions in Structural Steel on the Fatigue Resistance Coefficient.

One of the main parameters characterizing steel is tensile strength. Conducting actual research is time consuming and expensive. For this reason, the technique uses simplified methods that allow one to quickly estimate the resistance of the material to fatigue. They are conducted mainly by computer methods. For the proper development of programs to determine the fatigue parameters of steel, solid data preparation is necessary. Unfortunately, some studies are performed on materials produced in laboratory conditions, which is only an approximation of the actual production conditions. Real alloys contain natural impurities which can affect their properties. Therefore, it is important to use real results obtained on an industrial scale for analysis including computer simulations. One of the important parameters that can be used to describe the properties of steel is the scatter index. It is the quotient of the average distance between the pollution and the average size of the pollution. This parameter makes it possible to take into account the fatigue strength of steel, taking into account the size of impurities and the distance between these impurities. The paper attempted to determine the scatter index and its impact on the fatigue resistance coefficient for steel melted in an industrial 140 ton electric furnace. The tests were carried out on structural steel with an average carbon content of 0.26%. The steel was hardened and tempered in all temperature tempering ranges (low, medium, and high). The fatigue resistance coefficient in the scatter index function was determined and discussed for each of the applied heat treatment parameters.

IFNß-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages.

IFN-I is the key regulatory component activating and modulating the response of innate and adaptive immune system to bacterial as well as viral pathogens. IFN-I promotes the expression of IFN-induced genes (ISG) and, consequently, the production of chemokines, e.g., CXCL10. Those chemokines control migration and localization of immune cells in tissues, and, thus, are critical to the function of the innate immune system during infection. Consequently, the regulation of IFN-I signaling is essential for the proper induction of an immune response. Our previous study has shown that E3 ubiquitin ligase Pellino3 positively regulates IFNß expression and secretion. Herein, we examined the role of Pellino3 ligase in regulating CXCL10 expression in response to IFNß stimulation. Our experiments were carried out on murine macrophage cell line (BMDM) and human monocytes cell line (THP-1) using IFNß as a IFNAR ligand. We demonstrate that Pellino3 is important for IFNß-induced phosphorylation and nuclear translocation of STAT1/STAT2/IRF9 complex which interacts with CXCL10 promoter and enhances its expression. In this study, we characterize a novel molecular mechanism allowing Pellino3-dependent modulation of the IFNß-induced response in BMDM and THP-1 cell lines.

Influence of Non-Metallic Inclusions on Bending Fatigue Strength of High-Quality Carbon Constructional Steel Heated in an Industrial Electric Arc Furnace.

Non-metallic inclusions are one of the many factors influencing the strength of materials operating under variable loads. Their influence on the strength of the material depends not only on the morphology of the impurities themselves, but it is also closely related to the microstructure of the material. This microstructure is the matrix for non-metallic inclusions. This article discusses the results of a study investigating the effect of non-metallic inclusions on the fatigue strength of structural steel during rotary bending. The study was performed at 12 heats produced in an industrial plant's 140-ton electric furnaces. Six heats were desulphurised, and six were refined with argon. This paper presents the bending fatigue strength of steel hardened and tempered at different temperatures, subject to the relative volume of inclusions. This paper also presents the dimensional structure of non-metallic inclusions divided by different two technologies. The research shows that the main fraction of non-metallic inclusions is Al2O3; the most numerous were impurities with a diameter of less than 2 µm; argon refining does not affect the proportion of non-metallic inclusions of large dimensions (with a diameter of over 15 µm); the influence of non-metallic inclusions on the strength of the steel is also related to the microstructure of the steel constituting the matrix of inclusions.

Ligase Pellino3 Regulates Macrophage Action and Survival in Response to VSV Infection in RIG-I-Dependent Path.

Sensing of viral particles and elements that initiate mechanisms of immune response is an intrinsic ability of mammalian cells. Regulatory cytokines and antiviral mediators are released after triggering of complex signaling cascades in response to interaction of pathogen particles with pattern recognition receptors (PRRs) leading to the production of interferons (IFN) and proinflammatory cytokines. Viral RNA in the cytoplasm constitute a potent danger molecule that recognition is performed by RIG-I-like receptors, the most common group of receptors in mammalian cells, capable to recognize a foreign RNA. It is known that the E3 ubiquitin ligase Pellino3 plays an important role in antibacterial and antiviral response, but its involvement in the RLR pathways remains poorly understood. In this study, we investigate the molecular mechanisms of the innate immune response in BMDMs (immortalized macrophages from mouse bone marrow) during VSV infection. Here, we present evidence that the activation of the RIG-I/Pellino3/ERK1/2 pathway in BMDMs is crucial for the protection against VSV. We demonstrate that during infection, viral particles replicate in Pellino3 knockout BMDMs more effectively than in wild-type cells. Increased viral replication resulting in cell lysis and death is aid by impaired synthesis of IFN-I and inflammatory cytokines as a consequence of disturbances in the ERK1/2 pathway regulation.

[The development of methods for obtaining monoclonal antibody-producing cells]. / Rozwój metod otrzymywania komórek wytwarzajacych przeciwciala monoklonalne.

Monoclonal antibodies (mAbs) are biomolecules of great scientific and practical significance. In contrast to polyclonal antibodies from immune sera, they are homogeneous and monospecific, since they are produced by hybridoma cells representing a clone arising from a single cell. The successful technology was described for the first time in 1975; the inventors were later awarded the Nobel Prize. Currently, mAbs are broadly used as a research tool, in diagnostics and medicine in particular for the treatment of cancer or in transplantology. About 47 therapeutics based on monoclonal antibodies are now available in the US and Europe, and the number is still growing. Production of monoclonal antibodies is a multistage, time-consuming and costly process. Growing demand for these molecules creates space for research focused on improvements in hybridoma technology. Lower costs, human labor, and time are important goals of these attempts. In this article, a brief review of current methods and their advances is given.

Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker specific for glandular epithelium and nervous tissue. Further studies should be performed to determine the structure of the tissue epitope recognized.

Synthesis of antifungal vaccines by conjugation of ß-1,2 trimannosides with T-cell peptides and covalent anchoring of neoglycopeptide to tetanus toxoid.

Selective strategies for the construction of novel three component glycoconjugate vaccines presenting Candida albicans cell wall glycan (ß-1,2 mannoside) and polypeptide fragments on a tetanus toxoid carrier are described. The first of two conjugation strategies employed peptides bearing an N-terminal thiopropionyl residue for conjugation to a trisaccharide equipped with an acrylate linker and a C-terminal S-acetyl thioglycolyl moiety for subsequent linking of neoglycopeptide to bromoacetylated tetanus toxoid. Michael addition of acrylate trisaccharides to peptide thiol under mildly basic conditions gave a mixture of N- and C- terminal glyco-peptide thioethers. An adaptation of this strategy coordinated S-acyl protection with anticipated thioester exchange equilibria. This furnished a single chemically defined fully synthetic neoglycopeptide conjugate that could be anchored to a tetanus toxoid carrier and avoids the introduction of exogenous antigenic groups. The second strategy retained the N-terminal thiopropionyl residue but replaced the C-terminal S-acetate functionality with an azido group that allowed efficient, selective formation of neoglycopeptide thioethers and subsequent conjugation of these with propargylated tetanus toxoid, but introduced potentially antigenic triazole linkages.

Synthesis and immunochemical characterization of S-linked glycoconjugate vaccines against Candida albicans.

Replacement of the glycosidic oxygen atom by a sulphur atom is a promising technique for creating glycoconjugates with increased resistance to hydrolysis by endogenous glycosidases. The synthesis and antigenic properties of two distinct (1-->2)-beta-mannan trisaccharides with inter residue-S-linked mannopyranose residues are described. Syntheses were based on an oxidation-reduction strategy to construct the O-linked beta-mannopyranoside bonds and a SN2 inversion to provide 1-thio-beta-mannopyranoside residues. Subsequently the allyl trisaccharide glycosides were subjected to photo addition with cysteine amine and coupled to tetanus toxoid and bovine serum albumin with good efficiency via an adipic acid tether. Rabbit immunization studies revealed that the antibodies elicited by the two glycoconjugates were able to recognize the corresponding O-linked trisaccharide epitope conjugated to BSA and the native cell wall antigen of Candida albicans.