Significance of Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) for the Monitoring of Treatment Response to Cyclosporine in Patients with Psoriasis.

Neutrophil gelatinase-associated lipocalin (NGAL) may promote development of inflammation in psoriasis, whereas proprotein convertase subtilisin/kexin type 9 (PCSK9) may account for dyslipidemia in some psoriatic patients. The aim of the study was to analyze the influence of cyclosporine therapy on serum levels of NGAL and PCSK9 in patients with psoriasis vulgaris. METHODS: Serum samples were obtained before and after three months cyclosporine therapy. Patients were grouped into responders and non-responders to cyclosporine depending on whether they achieved at least 50% reduction of Psoriatic Activity Score Index (PASI), or not. Serum levels of PCSK9 and NGAL were assayed using commercially available ELISA tests. Lipid levels were measured with an enzymatic method. RESULTS: There were 40 patients enrolled. A significant decrease in serum NGAL level was seen in cyclosporine responders. No similar dependance was found for PCSK9. Serum PCSK9 concentration correlated with total cholesterol (TChol) and LDL at baseline and after three month treatment. CONCLUSIONS: Cyclosporine therapy contributes to the reduction of the NGAL serum but not the PCSK9 concentration. Correlation between the PCSK9 serum level and TChol as well as LDL concentration may help to understand drug induced dyslipidemia after cyclosporine.

IgYs: on her majesty's secret service.

There has been an increasing interest in using Immunoglobulin Y (IgY) antibodies as an alternative to "classical" antimicrobials. Unlike traditional antibiotics, they can be utilized on a continual basis without leading to the development of resistance. The veterinary IgY antibody market is growing because of the demand for minimal antibiotic use in animal production. IgY antibodies are not as strong as antibiotics for treating infections, but they work well as preventative agents and are natural, nontoxic, and easy to produce. They can be administered orally and are well tolerated, even by young animals. Unlike antibiotics, oral IgY supplements support the microbiome that plays a vital role in maintaining overall health, including immune system function. IgY formulations can be delivered as egg yolk powder and do not require extensive purification. Lipids in IgY supplements improve antibody stability in the digestive tract. Given this, using IgY antibodies as an alternative to antimicrobials has garnered interest. In this review, we will examine their antibacterial potential.

The prevalence and role of functional autoantibodies to angiotensin-converting-enzyme-2 in patients with systemic sclerosis.

INTRODUCTION: Systemic sclerosis (SSc) is an autoimmune disease caused by the imbalance between the activity of angiotensin II and angiotensin-(1-7). Their balance should be controlled by angiotensin-converting enzyme 2 (ACE2), which degrades angiotensin II into angiotensin-(1-7). Previously, autoantibodies to ACE2 (anti-ACE2) were identified in patients with vasculopathy due to different connective tissue diseases, including SSc, but their frequency in SSc was not further analyzed. The aim of the research was to investigate the prevalence and potential role of those anti-ACE2 antibodies in SSc patients. MATERIALS AND METHODS: There were enrolled 27 patients with SSc and 23 healthy donors. ELISA assay determined the presence of anti-ACE2 autoantibodies in serum samples. The results were compared to plasma measurements of angiotensin-(1-7) level via commercial ELISA. RESULTS: The presence of anti-ACE2 autoantibodies was confirmed in five patients with SSc and two healthy controls. Two of those SSc subjects were anti-Scl70+, another two were double anti-Scl70+ and anti-Ro/SSA+, and anti-PM/Scl antibodies were detected in one patient. Median plasma level of Ang-(1-7) in anti-ACE2 negative patients was 47.4 pg/ml and stayed below the detection level in anti-ACE2 positive subjects. The plasma level of Ang-(1-7) was undetectable in four SSc patients, and three of them were anti-ACE2 positive. CONCLUSIONS: Anti-ACE2 antibodies appear to be other functional autoantibodies with the potential to dysregulate the balance between Ang II and Ang-(1-7). They are non-specific for SSc and probably result from polyautoimmunity which affect some of SSc patients. Their occurrence in SSc settings may be associated with a severe depletion of plasma Ang-(1-7).

PssJ Is a Terminal Galactosyltransferase Involved in the Assembly of the Exopolysaccharide Subunit in Rhizobium Leguminosarum bv. Trifolii.

Rhizobium leguminosarum bv. trifolii produces exopolysaccharide (EPS) composed of glucose, glucuronic acid, and galactose residues at a molar ratio 5:2:1. A majority of genes involved in the synthesis, modification, and export of exopolysaccharide are located in the chromosomal Pss-I region. In the present study, a ΔpssJ deletion mutant was constructed and shown to produce EPS lacking terminal galactose in the side chain of the octasaccharide subunit. The lack of galactose did not block EPS subunit translocation and polymerization. The in trans delivery of the pssJ gene restored the production of galactose-containing exopolysaccharide. The mutant was compromised in several physiological traits, e.g., motility and biofilm production. An impact of the pssJ mutation and changed EPS structure on the symbiotic performance was observed as improper signaling at the stage of molecular recognition, leading to formation of a significant number of non-infected empty nodules. Terminal galactosyltransferase PssJ was shown to display a structure typical for the GT-A class of glycosyltransferases and interact with other GTs and Wzx/Wzy system proteins. The latter, together with PssJ presence in soluble and membrane protein fractions indicated that the protein plays its role at the inner membrane interface and as a component of a larger complex.

Structure-based design, synthesis, and evaluation of the biological activity of novel phosphoroorganic small molecule IAP antagonists.

One of the strategies employed by novel anticancer therapies is to put the process of apoptosis back on track by blocking the interaction between inhibitor of apoptosis proteins (IAPs) and caspases. The activity of caspases is modulated by the caspases themselves in a caspase/procaspase proteolytic cascade and by their interaction with IAPs. Caspases can be released from the inhibitory influence of IAPs by proapoptotic proteins such as secondary mitochondrial activator of caspases (Smac) that share an IAP binding motif (IBM). The main purpose of the present study was the design and synthesis of phosphorus-based peptidyl antagonists of IAPs that mimic the endogenous Smac protein, which blocks the interaction between IAPs and caspases. Based on the structure of the IAP antagonist and recently reported thiadiazole derivatives, we designed and evaluated the biochemical properties of a series of phosphonic peptides bearing the N-Me-Ala-Val/Chg-Pro-OH motif (Chg: cyclohexylglycine). The ability of the obtained compounds to interact with the binding groove of the X-linked inhibitor of apoptosis protein baculovirus inhibitor of apoptosis protein repeat (XIAP BIR3) domain was examined by a fluorescence polarization assay, while their potential to induce autoubiquitination followed by proteasomal degradation of cellular IAP1 was examined using the MDA-MB-231 breast cancer cell line. The highest potency against BIR3 was observed among peptides containing C-terminal phosphonic phenylalanine analogs, which displayed nanomolar Ki values. Their antiproliferative potential as well as their proapoptotic action, manifested by an increase in caspase-3 activity, was examined using various cell lines.

Temporal inhibition of mouse mammary gland cancer metastasis by CORM-A1 and DETA/NO combination therapy.

Carbon monoxide and nitric oxide are two of the most important vasoprotective mediators. Their downregulation observed during vascular dysfunction, which is associated with cancer progression, leads to uncontrolled platelet activation. Therefore, the aim of our studies was to improve vasoprotection and to decrease platelet activation during progression of mouse mammary gland cancer by concurrent use of CO and NO donors (CORM-A1 and DETA/NO, respectively). Methods: Mice injected intravenously with 4T1-luc2-tdTomato or orthotopically with 4T1 mouse mammary gland cancer cells were treated with CORM-A1 and DETA/NO. Ex vivo aggregation and activation of platelets were assessed in the blood of healthy donors and breast cancer patients. Moreover, we analyzed the compounds' direct effect on 4T1 mouse and MDA-MB-231 human breast cancer cells proliferation, adhesion and migration in vitro. Results: We have observed antimetastatic effect of combination therapy, which was only transient in orthotopic model. During early stages of tumor progression concurrent use of CORM-A1 and DETA/NO demonstrated vasoprotective ability (decreased endothelin-1, sICAM and sE-selectin plasma level) and downregulated platelets activation (decreased bound of fibrinogen and vWf to platelets) as well as inhibited EMT process. Combined treatment with CO and NO donors diminished adhesion and migration of breast cancer cells in vitro and inhibited aggregation as well as TGF-ß release from breast cancer patients' platelets ex vivo. However, antimetastatic effect was not observed at a later stage of tumor progression which was accompanied by increased platelets activation and endothelial dysfunction related to a decrease of VASP level. Conclusion: The therapy was shown to have antimetastatic action and resulted in normalization of endothelial metabolism, diminution of platelet activation and inhibition of EMT process. The effect was more prominent during early stages of tumor dissemination. Such treatment could be applied to inhibit metastasis during the first stages of this process.

Development and Evaluation of an Immunoglobulin Y-Based ELISA for Measuring Prostate Specific Antigen in Human Serum.

BACKGROUND: Measurement of serum prostate specific antigen (PSA) concentrations remains one of the leading methods for diagnosing prostate cancer. We developed and evaluated an immunoglobulin Y (IgY)-based ELISA to measure total PSA (tPSA) concentrations in human serum that could be used as an alternative to commercially available in vitro diagnostic assays that rely on mouse monoclonal IgG. METHODS: A sandwich ELISA based on an anti-PSA IgY antibody was developed. We evaluated the ability of the anti-PSA IgY antibody to detect free and complexed PSA at the same molar ratio. The assay was optimized, and its analytical performance was verified by calculating limit of background (LoB), limit of detection (LoD), and limit of quantification (LoQ). We performed correlation and regression analyses between tPSA concentrations measured by our ELISA and those from commercial assays: Cobas 6000 (Roche Diagnostics, Warszawa, Poland) and PSA total ELISA (IBL International, Hamburg, Germany). RESULTS: LoB, LoD, and LoQ, were 0.061, 0.083, and 0.100 ng/mL, respectively, and linearity range was 0.100-3.375 ng/mL. tPSA concentrations from our IgY-based ELISA strongly correlated with those from the commercial assays. CONCLUSIONS: Our IgY-based ELISA is an efficient equivalent to the above commercial assays. The use of IgY as the detecting agent could reduce the risk of false positive results, as well as decrease the overall cost of analysis.

Discovering simple phenylboronic acid and benzoxaborole derivatives for experimental oncology - phase cycle-specific inducers of apoptosis in A2780 ovarian cancer cells.

Objective The aim of the study was to evaluate the antiproliferative potential of simple phenylboronic acid and benzoxaborole derivatives as well as to provide preliminary insight into their mode of action in cancer cells in vitro. Methods The antiproliferative activity was assessed in five diverse cancer cell lines via the SRB method (sulforhodamine B) or MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method after 72 h of treatment. Further studies of the mechanism of action consisted of the influence of the compounds on cell cycle progression and apoptosis induction, which was assessed by flow cytometry, caspase-3 enzymatic activity, fluorescence microscopy and western blot analysis. Results A clear structure-activity relationship was observed for both groups of compounds with several representatives evaluated as highly active antiproliferative agents with low micromolar [Formula: see text] values. 2-Fluoro-6-formylphenylboronic acid (18) and 3-morpholino-5-fluorobenzoxaborole (27) exhibited strong cell cycle arrest induction in G2/M associated with caspase-3 activation in an A2780 ovarian cancer cell line. These events were accompanied by a mitotic catastrophe cell morphology and an increased percentage of aneuploid and tetraploid cells. Further experiments indicated that the compounds were phase cycle-specific agents since cells co-treated with hydroxyurea were less sensitive. The observed cell cycle arrest resulted from significant p21 accumulation and was associated neither with cyclin B1 nor ß-tubulin degradation. Conclusion Phenylboronic acid and benzoxaborole derivatives were found to be highly promising antiproliferative and proapoptotic compounds with a cell cycle-specific mode of action. The presented data support their candidacy for further studies as a novel class of potential anticancer agents.

Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application.

Adenosine deaminase (ADA) is currently used as a diagnostic marker for tuberculous pleuritis. Although ADA has been suggested as a potential marker for several types of cancer, the importance of each of ADA isoforms as well as their levels and enzymatic activities in tumors need to be further investigated. Herein we developed avian immunoglobulin Y highly specific to human ADA via hens immunization with calf adenosine deaminase. The obtained antibodies were used for the development of a sensitive double-egg yolk immunoglobulin (IgY) sandwich ELISA assay with an ADA detection limit of 0.5 ng/ml and a linearity range of up to 10 ng/ml. Specific, affinity-purified IgYs were able to recognize human recombinant ADA and ADA present in human cancer cell lines. In addition, antigen-specific IgY antibodies were able to inhibit catalytic activity of calf ADA with an IC50 value of 47.48 nM. We showed that generated IgY antibodies may be useful for ADA detection, thus acting as a diagnostic agent in immunoenzymatic assays.

Method for generation of peptide-specific IgY antibodies directed to Staphylococcus aureus extracellular fibrinogen binding protein epitope.

The IgY antibodies offer an attractive alternative to mammalian IgGs in research, diagnosis and medicine. The isolation of immunoglobulin Y from the egg yolks is efficient and economical, causing minimal suffering to animals. Here we present the methodology for the production of IgY antibodies specific to Staphylococcus aureus fibrinogen binding protein (Efb) and its peptidyl epitope (spanning residues 127-140). The Efb is an extracellular, adhesion protein which binds both human fibrinogen and complement C3 protein thus contributing to the high infectious potential of this pathogen. The selected epitope of Efb protein is responsible for the interaction with C3. The immunochemical characterization of both anti-Efb and epitope-specific IgY antibodies revealed their similar avidity, titer, and reactivity profile, although some differences in the hen's immune response to administered antigens is discussed.

Adjuvant-dependent immunogenicity of Staphylococcus aureus Efb and Map proteins in chickens.

The avian IgY antibodies generated in hens and isolated from egg yolk have gained in popularity as they present an alternative source of antibodies for diagnostic as well as therapeutic applications. One of the advantages of IgY technology are the large amounts of produced antibodies from a single animal combined with their high reactivity representing an attractive alternative for mammalian antibodies. Despite many known protocols for the immunization of chickens, the administration of new antigens often requires additional modification such as antigen dose or use of an adjuvant in order to elicit a significant immune response. We investigated the immunogenicity of three Staphylococcus aureus antigens including two extracellular proteins Map and Efb and one selected Efb105-124 epitope conjugated to KLH that were administered to the animals. Additionally, the immunization protocol included two adjuvant systems: Freund's complete adjuvant and Emulsigen-D. The results demonstrated a high immunostimulatory potency of Freund's complete adjuvant, especially in case of Efb compared to the immune response elicited by Emulsigen-D. However, after immunization with the KLH-Efb105-124 conjugate, the obtained antibodies showed similar reactivity regardless of adjuvant system used with the only exception being their avidity.

Generation and application of polyclonal IgY antibodies specific for full-length and nicked prostate-specific antigen.

BACKGROUND: The prostate-specific antigen (PSA) is considered an important serum marker for prostate cancer detection, monitoring and staging. The purpose of this study was to generate IgY class antibodies that recognize native PSA and selected epitopes. METHODOLOGY: Hens immunized with either full-length human PSA or its peptidyl fragment-conjugates produced specific antibodies that were isolated from egg yolks. We developed a monoclonal/IgY sandwich ELISA with a PSA detection limit of 50 pg/ml and a linear range of 0.05-1.0 ng/ml. CONCLUSION: Because the signal observed for the PSA-specific IgY antibodies by ELISA and the reactivity profile of the epitope-derived IgYs were comparable to those of mouse monoclonal IgG antibodies, avian antibodies may be a cost-effective alternative to mammalian antibodies for prostate cancer diagnostics.

Synthesis of novel phosphonic-type activity-based probes for neutrophil serine proteases and their application in spleen lysates of different organisms.

Neutrophils are a type of granulocyte important in the "first line of defense" of the innate immune system. Upon activation, they facilitate the destruction of invading microorganisms by the production of superoxide radicals, as well as the release of the enzymatic contents of their lysozymes. These enzymes include specific serine proteases: cathepsin G, neutrophil elastase, proteinase 3, as well as the recently discovered neutrophil serine protease 4 (NSP4). Under normal conditions, the proteolytic activity of neutrophil proteases is tightly regulated by endogenous serpins; however, this mechanism can be subverted during tissue stress, thereby resulting in the uncontrolled activity of serine proteases, which induce chronic inflammation and subsequent pathology. Herein, we describe the development of low-molecular-weight activity-based probes that specifically target the active sites of neutrophil proteases.

Highly sensitive detection of cancer antigen 15-3 using novel avian IgY antibodies.

Early detection of cancer development is crucial for successful therapy and for monitoring patient outcome. Various immunodiagnostic methods are able to detect pathological changes in the human body ahead of symptomatic manifestation of the disease. Most immunological examinations are based on the detection of specific tumor markers in body fluids. Of the various cancer-specific proteins used for breast cancer diagnostics, one of the most commonly applied is the cancer antigen 15-3 (CA 15-3). An elevation in its serum level (>25-40 U/ml) usually correlates with tumor malignancy. The CA 15-3 antigen is also used for monitoring patients after surgical treatment and for measuring therapeutic efficacy. Herein, we present the generation of polyclonal IgY antibodies isolated from egg yolks of immunized hens and their application for CA 15-3 detection. The developed sandwich ELISA assay showed a detection limit of 0.028 U/ml, thus demonstrating its potential for clinical applications.