Investigation of canine parvovirus occurrence in cats with clinical signs of feline panleukopenia in Slovakia - pilot study.

Introduction: Feline panleukopenia is a contagious viral disease caused by the feline panleukopenia virus (FPV). A closely related pathogen is canine parvovirus (CPV), and amino acid substitutions in this virus allow it to acquire a feline host range. In feline hosts, the disease induced by CPV manifests with similar symptoms to those caused by FPV or milder ones, leading to its underdiagnosis. The aim of this study was to determine the presence of CPV type 2 (CPV-2) in cats with clinical symptoms of panleukopenia and to assess the use of commercial CPV antigen tests for the clinical diagnosis of FPV. Material and Methods: Samples from 59 cats from central Slovakia were included in the study. Rectal swabs were collected and clinically tested for parvovirus infection using a commercial antigen test. Antigen-positive samples were confirmed by PCR targeting the viral VP2 gene. The sequences of the PCR products were established with the Sanger method. Results: Of 59 samples, 23 were revealed to be positive for parvovirus infection by both antigen and PCR test (38.9%). Analysis with the National Center for Biotechnology Information BLASTn application showed 99.78-100% pairwise identity with FPV. The mortality rate of parvovirus-infected cats included in this study was 8.69% (2/23). Conclusion: Although feline disease with CPV-2 was not confirmed, the CPV antigen test was able to detect FPV infection.

The role of primed and non-primed MSC-derived conditioned media in neuroregeneration.

Introduction: With growing significance in nervous system repair, mesenchymal stem cell-derived conditioned media (MSCCM) have been used in cell-free therapies in regenerative medicine. However, the immunomodulatory and neuroregenerative effects of MSCCM and the influence of priming on these effects are still poorly understood. Methods: In this study, by various methods focused on cell viability, proliferation, neuron-like differentiation, neurite outgrowth, cell migration and regrowth, we demonstrated that MSCCM derived from adipose tissue (AT-MSCCM) and amniotic membrane (AM-MSCCM) had different effects on SH-SY5Y cells. Results and discussion: AT-MSCCM was found to have a higher proliferative capacity and the ability to impact neurite outgrowth during differentiation, while AM-MSCCM showed more pronounced immunomodulatory activity, migration, and re-growth of SH-SY5Y cells in the scratch model. Furthermore, priming of MSC with pro-inflammatory cytokine (IFN-γ) resulted in different proteomic profiles of conditioned media from both sources, which had the highest effect on SH-SY5Y proliferation and neurite outgrowth in terms of the length of neurites (pAT-MSCCM) compared to the control group (DMEM). Altogether, our results highlight the potential of primed and non-primed MSCCM as a therapeutic tool for neurodegenerative diseases, although some differences must be considered.

Evaluation of Biocompatibility of PLA/PHB/TPS Polymer Scaffolds with Different Additives of ATBC and OLA Plasticizers.

One of the blends that is usable for 3D printing while not being toxic to cell cultures is the lactic acid (PLA)/polyhydroxybutyrate (PHB)/thermoplastic starch (TPS) blend. The addition of plasticizers can change the rate of biodegradation and the biological behavior of the material. In order to evaluate the potential of the PLA/PHB/TPS material in combination with additives (plasticizers: acetyl tributyl citrate (ATBC) and oligomeric lactic acid (OLA)), for use in the field of biomedical tissue engineering, we performed a comprehensive in vitro characterization of selected mixture materials. Three types of materials were tested: I: PLA/PHB/TPS + 25% OLA, II: PLA/PHB/TPS + 30% ATBC, and III: PLA/PHB/TPS + 30% OLA. The assessment of the biocompatibility of the materials included cytotoxicity tests, such as monitoring the viability, proliferation and morphology of cells and their deposition on the surface of the materials. The cell line 7F2 osteoblasts (Mus musculus) was used in the experiments. Based on the test results, the significant influence of plasticizers on the material was confirmed, with their specific proportions in the mixtures. PLA/PHB/TPS + 25% OLA was evaluated as the optimal material for biocompatibility with 7F2 osteoblasts. The tested biomaterials have the potential for further investigation with a possible change in the proportion of plasticizers, which can have a fundamental impact on their biological properties.

Impact of Canine Amniotic Mesenchymal Stem Cell Conditioned Media on the Wound Healing Process: In Vitro and In Vivo Study.

The aim of this study was to provide a beneficial treatment effect of mesenchymal stem cell products derived from the canine amniotic membrane (AM-MSC) on the complicated wound healing process in dogs. AM-MSCs were characterized in terms of morphology, phenotypic profile, and multilineage differentiation potential. The in vitro study of the effect of canine amniotic mesenchymal stem cell conditioned media (AMMSC-CM) on a primary skin fibroblast cell culture scratch assay showed a decrease in the measured scratch area of about 66.39% against the negative control (Dulbecco's Modified Eagle's Medium-32.55%) and the positive control (Dulbecco's Modified Eagle's Medium supplemented with FGF2, N2, B27, and EGF-82.077%) after 72 h treatment. In the experimental study, seven dogs with complicated nonhealing wounds were treated with a combination of antibiotics, NSAIDs, and local AMMSC-CM application. After 15 days of therapy, we observed a 98.47% reduction in the wound surface area as opposed to 57.135% in the control group treated by conventional therapy based on debridement of necrotic tissue, antibiotic therapy, pain management, and change of wound dressing.

A Comparative Study of Canine Mesenchymal Stem Cells Isolated from Different Sources.

In this study, we provide comprehensive analyses of mesenchymal stem cells (MSCs) isolated from three types of canine tissues: bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and amniotic tissue (AM-MSCs). We compare their morphology, phenotype, multilineage potential and proliferation activity. The BM-MSCs and AM-MSCs showed fibroblast-like shapes against the spindle shape of the AT-MSCs. All populations showed strong osteogenic and chondrogenic potential. However, we observed phenotypic differences. The BM-MSCs and AT-MSCs revealed high expression of CD29, CD44, CD90 and CD105 positivity compared to the AM-MSCs, which showed reduced expression of all the analysed CD markers. Similarly, the isolation yield and proliferation varied depending on the source. The highest isolation yield and proliferation were detected in the population of AT-MSCs, while the AM-MSCs showed a high yield of cells, but the lowest proliferation activity, in contrast to the BM-MSCs which had the lowest isolation yield. Thus, the present data provide assumptions for obtaining a homogeneous MSC derived from all three canine tissues for possible applications in veterinary regenerative medicine, while the origin of isolated MSCs must always be taken into account.

Signaling events evoked by domain III of envelop glycoprotein of tick-borne encephalitis virus and West Nile virus in human brain microvascular endothelial cells.

Tick-borne encephalitis virus and West Nile virus can cross the blood-brain barrier via hematogenous route. The attachment of a virion to the cells of a neurovascular unit, which is mediated by domain III of glycoprotein E, initiates a series of events that may aid viral entry. Thus, we sought to uncover the post-attachment biological events elicited in brain microvascular endothelial cells by domain III. RNA sequencing of cells treated with DIII of TBEV and WNV showed significant alteration in the expression of 309 and 1076 genes, respectively. Pathway analysis revealed activation of the TAM receptor pathway. Several genes that regulate tight-junction integrity were also activated, including pro-inflammatory cytokines and chemokines, cell-adhesion molecules, claudins, and matrix metalloprotease (mainly ADAM17). Results also indicate activation of a pro-apoptotic pathway. TLR2 was upregulated in both cases, but MyD88 was not. In the case of TBEV DIII, a MyD88 independent pathway was activated. Furthermore, both cases showed dramatic dysregulation of IFN and IFN-induced genes. Results strongly suggest that the virus contact to the cell surface emanates a series of events namely viral attachment and diffusion, breakdown of tight junctions, induction of virus uptake, apoptosis, reorganization of the extracellular-matrix, and activation of the innate immune system.

Engineering the Single Domain Antibodies Targeting Receptor Binding Motifs Within the Domain III of West Nile Virus Envelope Glycoprotein.

West Nile virus (WNV) is a mosquito-borne neurotrophic flavivirus causing mild febrile illness to severe encephalitis and acute flaccid paralysis with long-term or permanent neurological disorders. Due to the absence of targeted therapy or vaccines, there is a growing need to develop effective anti-WNV therapy. In this study, single-domain antibodies (sdAbs) were developed against the domain III (DIII) of WNV's envelope glycoprotein to interrupt the interaction between DIII and the human brain microvascular endothelial cells (hBMEC). The peripheral blood mononuclear cells of the llama immunized with recombinant DIIIL297-S403 (rDIII) were used to generate a variable heavy chain only (VHH)-Escherichia coli library, and phage display was performed using the M13K07ΔpIII Hyperphages system. Phages displaying sdAbs against rDIII were panned with the synthetic analogs of the DIII receptor binding motifs, DIII-1G299-K307 and DIII-2V371-R388, and the VHH gene from the eluted phages was subcloned into E. coli SHuffle. Soluble sdAbs purified from 96 E. coli SHuffle clones were screened to identify 20 candidates strongly binding to the synthetic analogs of DIII-1G299-K307 and DIII-2V371-R388 on a dot blot assay. Among them, sdAbA1, sdAbA6, sdAbA9, and sdAbA10 blocked the interaction between rDIII and human brain microvascular endothelial cells (hBMECs) on Western blot and cell ELISA. However, optimum stability during the overexpression was noticed only for sdAbA10 and it also neutralized the WNV-like particles (WNV-VLP) in the Luciferase assay with an half maximal effective concentration (EC50) of 1.48 nm. Furthermore, the hemocompatibility and cytotoxicity of sdAbA10 were assessed by a hemolytic assay and XTT-based hBMEC proliferation assay resulting in 0.1% of hemolytic activity and 82% hBMEC viability, respectively. Therefore, the sdAbA10 targeting DIII-2V371-R388 of the WNV envelope glycoprotein is observed to be suitable for in vivo trials as a specific therapy for WNV-induced neuropathogenesis.

Non-Exosomal and Exosome-Derived miRNAs as Promising Biomarkers in Canine Mammary Cancer.

Canine mammary cancer (CMC), similar to human breast cancer (HBC) in many aspects, is the most common neoplasm associated with significant mortality in female dogs. Due to the limited therapy options, biomarkers are highly desirable for early clinical diagnosis or cancer progression monitoring. Since the discovery of microRNAs (miRNAs or miRs) as post-transcriptional gene regulators, they have become attractive biomarkers in oncological research. Except for intracellular miRNAs and cell-free miRNAs, exosome-derived miRNAs (exomiRs) have drawn much attention in recent years as biomarkers for cancer detection. Analysis of exosomes represents a non-invasive, pain-free, time- and money-saving alternative to conventional tissue biopsy. The purpose of this review is to provide a summary of miRNAs that come from non-exosomal sources (canine mammary tumor, mammary tumor cell lines or canine blood serum) and from exosomes as promising biomarkers of CMC based on the current literature. As is discussed, some of the miRNAs postulated as diagnostic or prognostic biomarkers in CMC were also altered in HBC (such as miR-21, miR-29b, miR-141, miR-429, miR-200c, miR-497, miR-210, miR-96, miR-18a, miR19b, miR-20b, miR-93, miR-101, miR-105a, miR-130a, miR-200c, miR-340, miR-486), which may be considered as potential disease-specific biomarkers in both CMC and HBC.

Simple and rapid pipeline for the production of cyclic and linear small-sized peptides in E. coli.

Small and medium-sized peptides are gaining popularity in biomedical applications, including therapeutic target development. As an alternative to chemical synthesis, we describe a complete pipeline for the production of linear as well as structurally constrained cyclic peptides in an E. coli expression system in this study. A plasmid vector containing a novel N terminal HOE tag (28 amino acids in length) that fuses with the peptide was created. The HOE tag contains sites for both chemical (CNBr) and enzymatic (enterokinase) cleavage, making it easy to isolate the peptide after production. A total of 21 peptides (17 cyclic and 4 linear) were synthesized, and the HOE tag was successfully removed using either CNBr (9 peptides) or enterokinase (12 peptides). The presence of a disulfide bond was confirmed in six representative cyclic peptides. In this study we have provided detailed instructions on primers design strategy, overexpression and purification of HOE tagged peptides, chemical and enzymatic cleavage, and confirmation of the cyclic form of peptides. We are confident that this pipeline will assist researchers in producing multiple recombinant peptides in a cost-effective and time-efficient manner.

Comprehensive Mapping of the Cell Response to Borrelia bavariensis in the Brain Microvascular Endothelial Cells in vitro Using RNA-Seq.

Borrelia bavariensis can invade the central nervous system (CNS) by crossing the blood-brain barrier (BBB). It is predicted that B. bavariensis evokes numerous signaling cascades in the human brain microvascular endothelial cells (hBMECs) and exploits them to traverse across the BBB. The complete picture of signaling events in hBMECs induced by B. bavariensis remains uncovered. Using RNA sequencing, we mapped 11,398 genes and identified 295 differentially expressed genes (DEGs, 251 upregulated genes and 44 downregulated genes) in B. bavariensis challenged hBMECs. The results obtained from RNA-seq were validated with qPCR. Gene ontology analysis revealed the participation of DEGs in a number of biological processes like cell communication, organization of the extracellular matrix, vesicle-mediated transport, cell response triggered by pattern recognition receptors, antigen processing via MHC class I, cellular stress, metabolism, signal transduction, etc. The expression of several non-protein coding genes was also evoked. In this manuscript, we discuss in detail the correlation between several signaling cascades elicited and the translocation of BBB by B. bavariensis. The data revealed here may contribute to a better understanding of the mechanisms employed by B. bavariensis to cross the BBB.