Electrochemical Immunosensing of ST2: A Checkpoint Target in Cancer Diseases

A magnetic beads (MB)-involved amperometric immunosensor for the determination of ST2, a member of the IL1 receptor family, is reported in this work. The method utilizes a sandwich immunoassay and disposable screen-printed carbon electrodes (SPCEs). Magnetic immunoconjugates built on the surface of carboxylic acid-microsized magnetic particles (HOOC-MBs) were used to selectively capture ST2. A biotinylated secondary antibody further conjugated with a streptavidin peroxidase conjugate (Strep-HRP) was used to accomplish the sandwiching of the target protein. The immune platform exhibits great selectivity and a low limit of detection (39.6 pg mL−1) for ST2, allowing the determination of soluble ST2 (sST2) in plasma samples from healthy individuals and patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) in only 45 min once the immunoconjugates have been prepared. The good correlation of the obtained results with those provided by an ELISA kit performed using the same immunoreagents demonstrates the potential of the developed strategy for early diagnosis and/or prognosis of the fatal PDAC disease.

Highly Portable and Sensitive Filter-Based Antibody-or DNA Aptamer-Enzyme-Linked Fluorescence Detection of Potential Bacterial Pathogens Proximal to Agricultural Fields

A novel, sensitive, rapid and highly portable system consisting of a membrane filtration unit connected to a large syringe with tubing was used to detect Escherchia coli and Salmonella enterica bacteria in a mobile laboratory setting. These simple fluorescent enzyme-linked molecular assays consisted of biotinylated DNA aptamers or antibodies which were allowed to bind the target bacteria and be deposited on 0.45 μm pore size membrane filters by suction, followed by binding of streptavidin-peroxidase conjugate, wash steps and development in Amplex® UltraRed (AUR; resazurin derivative) with hydrogen peroxide activation. Fluorescence values of developed AUR from the filter surface were assessed with a commercially-available handheld fluorometer. Preliminary data indicate detection limits in the range of 50–110 E. coli and Salmonella bacterial cells per sample with good specificity and total processing times under one hour.

ANALYSIS OF ASSOCIATION BETWEEN THE DENSITY OF INFILTRATION IN PRIMARY CARCINOMA OF THE MAMMARY GLAND BY TUMOR-ASSOCIATED MACROPHAGES AND POSTOPERATIVE PROGNOSIS

Tumor-associated macrophages (TAM) of the M2-type dominate in tumors and produce molecules, favorable for their growth, stimulating tumor growth. However, changing the M2-type for M1 can slow down or arrest this growth. For realization of the M1 / M2 modulation direction in the treatment of carcinoma / breast cancer (BC), a substantiated diagnosis and confirmation of the TAM negative prognosis is necessary. Therefore, the aim of the study was to evaluate the relation of tumor associated macrophages to the postoperative prognosis / survival of patients with 5 molecular-biological types of breast carcinoma. Materials of the study were intraoperative tissues of tumors and ipsilateral lymph nodes in radically removed mammary glands. Pathomorphological study of lymph nodes was conducted to clarify the diagnosis in relation to N0/1. The density of TAM infiltration was determined by immunohistochemical staining of CD68 and CD163 in 30 samples of five molecular biological types of breast cancer (three clinical cases of each type). Immunohistochemical (IHC) studies for the determination of TAM and M2-like macrophages were conducted using streptavidin-peroxidase method. The quantitative representation of CD68 + and CD163 + Mph is very different from patient to patient and also within one sample, which depends, in particular, on the morphological characteristics of breast cancer, studied by the biopsy. The density of infiltration by CD163 + macrophages of the BC focus negatively correlated with postoperative survival, which did not reach statistical significance, but is included in the general concept of a negative prognosis of infiltration by M2-like macrophages. Further research is needed to confirm the negative significance of the ТАМ infiltration density in the BC primary focus for postoperative prognosis. Promising is the development of differential diagnosis and approach to the treatment of breast cancer, taking into account the levels of its infiltration by subpopulations of TAM.

Detection of bovine respiratory syncytial virus, Pasteurella multocida, and Mannheimia haemolytica by immunohistochemical method in naturally-infected cattle

Introduction: The aim of this study was to determine the predisposing effect of bovine respiratory syncytial virus (BRSV) on Pasteurella spp. infection in naturally-induced pneumonia in cattle by immunohistochemical labelling.Material and Methods: Lungs of cattle slaughtered in the slaughterhouse were examined macroscopically, and 100 pneumonic samples were taken. The samples were fixed in 10% neutral formalin and embedded in paraffin by routine methods. Sections 5 μm in thickness were cut. The streptavidin-peroxidase method (ABC) was used to stain the sections for immuno-histochemical examination.Results: BRSV antigens were found in the cytoplasm of epithelial cells of bronchi, bronchioles, and alveoles and within inflammatory cell debris and inflammatory exudate in bronchial lumens. Pasteurella spp. antigens were detected in the cytoplasm of the epithelial cells of bronchi and bronchioles, and in cells in the lumens of bronchi and bronchioles. Eleven cases were positive for only one pathogen (six for BRSV and five for Pasteurella spp.), while 35 cases were positive for 2 pathogens: BRSV plus P. multocida (n = 21) or M. haemolytica (n = 14).Conclusion: The presence of high levels of BRSV in dual infections indicates that BSRV may be the main pneumonia-inducing agent and an important predisposing factor for the formation of Pasteurella spp. infections in cattle naturally afflicted with pneumonia.

A new ELISA for autoantibodies to steroid 21-hydroxylase

Background: A new ELISA for autoantibodies to steroid 21-hydroxylase (21-OH Ab) is described. Methods: In the assay test sample autoantibodies form a bridge between 21-OH coated onto the plate well and liquid phase 21-OH-biotin. Bound 21-OH-biotin is detected by the addition of streptavidin peroxidase and colorogenic peroxidase substrate. Results: Of 100 samples from patients with autoimmune Addison’s disease, 86 (86%) were positive for 21-OH Ab ELISA whereas 84 (84%) were positive in an immunoprecipitation assay based on 125I-labeled 21-OH. Six (0.6%) of 928 healthy adult blood donors and 1 (2.0%) of 49 adult patients with type 1 diabetes mellitus (T1DM) were positive by ELISA. No samples from adult patients with Graves’ disease (GD; n=50), celiac disease (n=29), systemic lupus erythematosis (n=9) or rheumatoid arthritis (n=20) were positive by ELISA. However, 2/51 (3.9%) children with GD, 3/69 (4.3%) children with Hashimoto’s thyroiditis (HT) and 3/119 (2.5%) children with T1DM alone or associated with autoimmune thyroid disorders were ELISA positive. Conclusions: The new assay should be useful for screening patients known to be at increased risk of developing clinical autoimmune Addison’s disease, in particular children with HT, GD and/or T1DM.

Parasite load in intact and ulcerative skin of dogs with leishmaniais

The skin is the site of inoculation of Leishmania spp. in susceptible hosts, and consequently dermatopathies, especially ulcerative dermatitis, are the main clinical signs observed. The aim of this study was to assess parasitism of the skin (intact and ulcerated) among dogs that were naturally infected by Leishmania spp., through immunohistochemical analysis. Skin fragments (intact and ulcerated) were collected from 13 dogs with positive parasitological (bone marrow aspiration and exfoliative skin) and serological examinations (ELISA S7® Biogene) forLeishmania spp. These samples were processed using the immunohistochemical technique, involving the streptavidin-peroxidase complex. Ulcerative lesions were mainly observed on the elbows (53.84%; 7/13), nostrils (15.38%; 2/13), ears (23.07%; 3/13) and wings of the ilium (7.69%; 1/13). A severe parasite load was detected in 46.15% and 76.92% of the intact and ulcerated skin samples tested, respectively. The parasite load on ulcerated skin was statistically higher than on intact skin (p = 0.0221). These results indicate that the intact and ulcerated skin may host a high parasite load of amastigote forms of Leishmania spp., which can favor the transmission of the parasite.

107 DETECTION OF BIOTINYLATED-OVULATION-INDUCING FACTOR (OIF) IN CEREBROSPINAL FLUID AND ITS ABILITY TO INDUCE OVULATION

Ovulation-inducing factor (OIF) is a protein in the seminal plasma of llamas that induces a preovulatory LH surge by acting directly or indirectly on the hypothalamic GnRH neurons (Silva et al. 2011 Reprod. Biol. Endocr. 9, 74). We hypothesize that OIF crosses the blood–brain barrier and reaches the hypothalamus via secretion into the cerebro-spinal fluid (CSF) through the choroid plexus. Two experiments were designed to determine whether biotinylation of OIF (as a tracer) alters its bioactivity in a llama model (Experiment 1) and whether it crosses the blood–brain barrier in a rabbit model (Experiment 2). In Experiment 1, llamas with a follicle ≥8 mm in diameter that had grown for 3 consecutive days were assigned randomly to 5 groups (n = 2/group) and given an IV dose of 1) 800 µg of OIF, 2) 800 µg of OIF biotinylated at the amino end; 3) 1600 µg of OIF biotinylated at the amino end, 4) 800 µg of OIF biotinylated at the carboxyl end, or 5) phosphate buffered saline (control). The ovaries were examined daily by transrectal ultrasonography on Day 3 and 8 after treatment (Day 0 = treatment) to detect ovulation and corpus luteum formation. In Experiment 2, adult female rabbits were assigned randomly to 3 groups and given an IV dose of (1) 250 µg of OIF, (2) 250 µg of OIF biotinylated at the amino end, or (3) 250 µg of OIF biotinylated at the carboxyl end. A 50-µL sample of CSF was collected from the cisterna magna under general anesthesia before (0 min) and 10, 20, 30, and 45 min after treatment. The presence of biotinylated OIF in CSF samples was determined by dot blot, using streptavidin-peroxidase and diaminobenzidine. In Experiment 1, the diameter of the follicle at the time of the treatment did not differ among groups (9.7 ± 0.2, 9.4 ± 0.0, 10.5 ± 1.0, 10.1 ± 0.2, 10.3 ± 0.4). Ovulation was detected in all llamas except one llama treated with 800 µg OIF biotinylated at the carboxyl end and both llamas given PBS. The diameter of the corpus luteum did not differ among OIF-treated groups. In Experiment 2, OIF biotinylated at both amino and carboxyl ends was detected in CSF samples at 10, 20, 30, and 45 min after IV administration. No signal was recorded before IV administration (0 min) or in samples from rabbits that were given nonbiotinylated OIF. We conclude that the biotinylation process did not affect OIF bioactivity, and OIF crosses the blood–brain barrier and reaches the CSF in rabbits. Research supported by FONDECYT 1120518, the Natural Sciences and Engineering Research Council of Canada, and the Alpaca Research Foundation.