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).

Karl Fisher determination of residual moisture in veterinary vaccines -- practical implementation in market monitoring.

Residual moisture content plays a significant role in assessing the stability of veterinary vaccines. Analysis of water amount is often a critical parameter, which determines the quality of product, its appearance as well as the expiration date. The aim of the study was to validate a coulometric Karl Fisher method for practical use in the national monitoring of veterinary vaccines market. Immunological veterinary medicinal product (ivmp) for three different animal species - cats, dogs and rabbits - were used. Automated coulometric analysis in chamber without diaphragm was used, as well as a solution for titration, which was a mixture of diethanolamine, imidazole, methanol and sulfur dioxide. The weight of a single sample was 15-100 mg. The most important concern was optimization of the way of transferring a vaccine sample into titration cell, so that atmospheric moisture would not affect baseline drift and repeatability of the results. Humidity level in lyophilized biopharmaceuticals was validated in accordance with the guidelines. The method was linear in the range of one to five percent of water content with R(2) = 0.9998. Repeatability for different sample types was found to be not higher than CV% = 5.9. The method was used for vaccines market monitoring in 2010 and 2011. Thirteen vaccines from the market were tested and all were found to be compliant with official EU guidelines.

Draft Genome Sequence of Avian Chlamydia abortus Genotype G1 Strain 15-70d24, Isolated from Eurasian Teal in Poland.

Here, we report the draft genome sequence of avian Chlamydia abortus genotype G1 strain 15-70d24, isolated from Eurasian teal in Poland. The total genome assembly length is 1,149,382 bp.

Parthenolide inhibits proliferation of fibroblast-like synoviocytes in vitro.

Parthenolide is a bioactive constituent of an aromatic herb Feverfew (Tanacetum parthenium). It has been found that both parthenolide and extract of feverfew have anti-inflammatory and antinociceptive properties. Moreover, they demonstrate antiproliferative activities on different human tumour cells. The massive hyperplasia of synovial fibroblasts is the one of the most striking features of rheumatoid arthritis. It is not known whether this is due to the proliferation of synovial fibroblasts or to defective apoptosis. We investigated the effect of parthenolide on the proliferation of rabbit synoviocytes cell line HIG-82, rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and human skin fibroblasts (HSF) in vitro. Cell proliferation was assessed by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 5'-bromo-2'-deoxy-uridine methods. Parthenolide inhibited proliferation of HIG-82 and human RA-FLS. The proliferation of HSF was inhibited less effectively. The antiproliferative potential of parthenolide was demonstrated.

Free-living and captive turtles and tortoises as carriers of new Chlamydia spp.

A variety of Chlamydia species belonging to the Chlamydiaceae family have been reported in reptilian hosts but scarce data about their occurrence in turtles and tortoises are available. In this study, research was conducted to acquire information on invasive alien species (IAS) of turtles and indigenous turtles and tortoises, living both free and in captivity, as possible reservoirs of Chlamydiaceae. Analysis of specimens (pharyngeal and cloacal swabs and tissues) from 204 turtles and tortoises revealed an overall Chlamydiaceae prevalence of 18.3% and 28.6% among free-living and captive animals respectively, with variable levels of shedding. Further testing conducted with a species-specific real-time PCR and microarray test was unsuccessful. Subsequently sequencing was applied to genotype the Chlamydiaceae-positive samples. Almost the full lengths of the 16S rRNA and ompA genes as well as the 16S-23S intergenic spacer (IGS) and 23S rRNA domain I were obtained for 14, 20 and 8 specimens respectively. Phylogenetic analysis of 16S rRNA amplicons revealed two distinct branches. Group 1 (10 specimens), specific to freshwater turtles and reported here for the first time, was most closely related to Chlamydia (C.) pneumoniae strains and the newly described Candidatus C. sanzinia. Group 2 (four specimens), detected in Testudo spp. samples, showed highest homology to C. pecorum strains but formed a separate sub-branch. Finally, molecular analysis conducted on positive samples together with their geographical distribution in places distant from each other strongly suggest that Group 1 specimens correspond to a new species in the Chlamydiaceae family. In-depth studies of Chlamydia spp. from turtles and tortoises are needed to further characterise these atypical strains and address arising questions about their pathogenicity and zoonotic potential.

Poultry in Poland as Chlamydiaceae Carrier.

INTRODUCTION: The study was conducted to investigate the prevalence and genetic diversity of Chlamydia spp. in poultry in Poland and estimate possible transmission to humans. MATERIAL AND METHODS: Molecular diagnostic methods followed by sequencing and strain isolation were used on cloacal/faecal swabs collected from 182 apparently healthy poultry flocks including chickens, turkeys, ducks, and geese. Serum samples obtained from people exposed (study group) and non-exposed (control group) to birds were tested by complement fixation test to acquire data on Chlamydia spp. antibody level. RESULTS: Overall, 15.9% of the tested flocks were Chlamydiaceae-positive and three Chlamydia spp. were identified. Predominant chlamydial agent found was C. gallinacea occurring in 65.5% of all positive poultry flocks and in 73.0% of positive chicken flocks. The sequences from four chicken flocks were assigned to C. abortus, whereas C. psittaci was confirmed in one duck and one goose flock. The analysis of ompA variable domains revealed at least nine genetic variants of C. gallinacea. Chlamydial antibodies were detected in 19.2% of human serum samples in the study group in comparison with 10.8% in the controls. CONCLUSION: The obtained results confirm that chlamydiae are common among chicken flocks in Poland with C. gallinacea as a dominant species. Moreover, the presence of C. abortus in chickens is reported here for the first time. Further investigation should focus on possible zoonotic transmission of C. gallinacea and C. abortus as well as potential pathogenic effects on birds' health and poultry production.

Dissemination and genetic diversity of chlamydial agents in Polish wildfowl: Isolation and molecular characterisation of avian Chlamydia abortus strains.

Wild birds are considered as a reservoir for avian chlamydiosis posing a potential infectious threat to domestic poultry and humans. Analysis of 894 cloacal or fecal swabs from free-living birds in Poland revealed an overall Chlamydiaceae prevalence of 14.8% (n = 132) with the highest prevalence noted in Anatidae (19.7%) and Corvidae (13.4%). Further testing conducted with species-specific real-time PCR showed that 65 samples (49.2%) were positive for C. psittaci whereas only one was positive for C. avium. To classify the non-identified chlamydial agents and to genotype the C. psittaci and C. avium-positive samples, specimens were subjected to ompA-PCR and sequencing (n = 83). The ompA-based NJ dendrogram revealed that only 23 out of 83 sequences were assigned to C. psittaci, in particular to four clades representing the previously described C. psittaci genotypes B, C, Mat116 and 1V. Whereas the 59 remaining sequences were assigned to two new clades named G1 and G2, each one including sequences recently obtained from chlamydiae detected in Swedish wetland birds. G1 (18 samples from Anatidae and Rallidae) grouped closely together with genotype 1V and in relative proximity to several C. abortus isolates, and G2 (41 samples from Anatidae and Corvidae) grouped closely to C. psittaci strains of the classical ABE cluster, Matt116 and M56. Finally, deep molecular analysis of four representative isolates of genotypes 1V, G1 and G2 based on 16S rRNA, IGS and partial 23S rRNA sequences as well as MLST clearly classify these isolates within the C. abortus species. Consequently, we propose an expansion of the C. abortus species to include not only the classical isolates of mammalian origin, but also avian isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.