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Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI) encoding a type-IV secretion system (T4SS) for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs) and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5) during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-α cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-α cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IkB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-κB. Finally, NF-κB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression of TLR-2 and TLR-5 can qualitatively shift cagPAI-dependent to cagPAI-independent pro-inflammatory signalling pathways with possible impact on the outcome of H. pylori-associated diseases. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is the causative agent of gastric pathologies ranging from chronic gastritis to peptic ulcers and even cancer. Virulent strains carrying both the cag pathogenicity island ( cagPAI) and the vacuolating cytotoxin VacA are key players in disease development. The cagPAI encodes a type IV secretion system (T4SS) which forms a pilus for injection of the CagA protein into gastric epithelial cells. Injected CagA undergoes tyrosine phosphorylation and induces actin-cytoskeletal rearrangements involved in host cell scattering and elongation. We show here that the CagA-induced responses can be inhibited in strains expressing highly active VacA. Further investigations revealed that VacA does not interfere with known activities of phosphorylated CagA such as inactivation of Src kinase and cortactin dephosphorylation. Instead, we demonstrate that VacA exhibits inactivating activities on the epidermal growth factor receptor EGFR and HER2/Neu, and subsequently Erk1/2 MAP kinase which are important for cell scattering and elongation. Inactivation of vacA gene, downregulation of the VacA receptor RPTP-α, addition of EGF or expression of constitutive-active MEK1 kinase restored the capability of H. pylori to induce the latter phenotypes. These data demonstrate that VacA can downregulate CagA's effects on epithelial cells, a novel molecular mechanism showing how H. pylori can avoid excessive cellular damage. [ABSTRACT FROM AUTHOR]

The Helicobacter pylori CagA protein induces profound morphological changes in the host cytoskeleton and cell scattering, but the signalling involved is poorly understood. Pseudomonas aeruginosa also affects host actin cytoskeleton in a variety of ways by injecting the ExoS and ExoT toxins which encode N-terminal GTPase activating protein and C-terminal ADP-ribosyltransferase (ADPRT) activities. In this study we developed a novel coinfection assay to gain new insights into CagA effector protein functions. We found that P. aeruginosa injecting either ExoT or ExoS efficiently prevented the H. pylori-induced scattering phenotype. Both the Rho-GAP and the ADPRT domains of ExoS were needed to block the H. pylori-induced actin cytoskeletal rearrangements, whereas either domain of ExoT was sufficient for this activity. This strategy revealed common pathways subverted by different pathogens, and aided in the definition of signalling cascades that control the CagA-mediated cell scattering and elongation. We identified Crk adapter proteins, Rac1 and H-Ras, but not RhoA or Cdc42, which are the ExoS and/or ExoT targets, as crucial components of the CagA-induced phenotype. In addition, we show that ADP-ribosylation of CrkII by ExoT blocks phosphorylation of CrkII at Y-221, which is also important for the CagA-induced signalling. [ABSTRACT FROM AUTHOR]

The Helicobacter pylon immunodominant protein, CagA, is associated with severe gastritis and carcinoma. Injection of CagA into gastric epithelial cells by type IV secretion leads to actin-cytoskeletal rearrangements and cell scattering. CagA has been reported to have no role in the induction of transcription factor NF-κB and IL-8, which are crucial determinants for chronic inflammation. Here, we provide several lines of evidence showing that CagA is able to induce IL-8 in a time- and strain-dependent manner. We also show that by exchanging specific cagA genes, high IL-S-inducing H. pylon strains could be converted into low inducing strains and vice versa. Our results suggest that IL-S release induced by CagA occurs via a Ras→Raf→Mek→Erk→NF-κB signaling pathway in a Shp-2- and c-Met-independent manner. Thus, CagA is a multifunctional protein capable of effecting both actin remodeling and potentiation of chemokine release. [ABSTRACT FROM AUTHOR]

Background: There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is causally associated with the development of equine genital squamous cell carcinomas (SCCs). Early stages of disease present clinically as plaques or wart-like lesions which can gradually progress to tumoural lesions. Histologically these lesions are inconsistently described as benign hyperplasia, papilloma, penile intraepithelial neoplasia (PIN), carcinoma in situ (CIS) or SCC. Guidelines for histological classification of early SCC precursor lesions are not precisely defined, leading to potential misdiagnosis. The aim of this study was to identify histologic criteria and diagnostic markers allowing for a more accurate diagnosis of EcPV2-associated equine penile lesions. Results: A total of 61 archived equine penile lesions were histologically re-assessed and classified as benign hyperplasia, papilloma, CIS or SCC. From these, 19 representative lesions and adjacent normal skin were comparatively analysed for the presence of EcPV2 DNA and transcripts using PCR and RNA in situ hybridisation (RISH). All lesional samples were positive by EcPV2 PCR and RISH, while adjacent normal skin was negative. RISH analysis yielded signal distribution patterns that allowed distinction of early (hyperplasia, papilloma) from late stage lesions (CIS, SCC). Subsequently, the 19 lesions were further assessed for expression of p53, Ki67, MCM7 and MMP1 by immunohistochemistry (IHC). All four proteins were expressed in both normal and lesional tissue. However, p53 expression was up-regulated in basal keratinocyte layers of papillomas, CIS and SCCs, as well as in upper keratinocyte layers of CIS and SCCs. MCM7 expression was only up-regulated in upper proliferating keratinocyte layers of papillomas, CIS and SCCs. Conclusion: This study proposes combining a refined histological protocol for analysis of equine penile lesions with PCR- and/or RISH based EcPV2-screening and p53/MCM7 IHC to more accurately determine the type of lesion. This may help to guide the choice of optimum treatment strategy, especially at early stages of disease. [ABSTRACT FROM AUTHOR]

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial–mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein. [ABSTRACT FROM AUTHOR]

Equine sarcoids are caused by infection with bovine papillomavirus (BPV) types 1, 2, and possibly 13. However, a number of sarcoids lack BPV DNA, and new potential etiological agents for sarcoid diseases need to be considered. High-performance digital droplet polymerase chain reaction (ddPCR) was used for the quantitative detection of ovine papillomavirus (OaPV) types 1–4 DNA from 63 sarcoid DNA samples collected in Austria. All samples were comparatively evaluated for OaPV DNA loads by qPCR. Conventional PCR and amplicon sequencing were used to validate the data. Of the 63 sarcoid DNA isolates, ddPCR was able to detect 22 samples harboring OaPV DNA (34.92%), whereas only five of the OaPV-positive samples were revealed by qPCR (22.72%). The differences in detection by ddPCR and qPCR were statistically significant (p < 0.05). The detected OaPV types were OaPV1, 3, and 4. Both methods failed to detect OaPV2 DNA, which could be due to the limited number of examined samples. Importantly, ddPCR detected multiple types of OaPV DNA in seven cases, whereas the qPCR failed to detect multiple infections. This study is the first to provide evidence of the presence of OaPV types 1, 3, and 4 DNA in a subset of equine sarcoids. The comparative detection approach underscores the superior sensitivity of ddPCR compared to that of qPCR. [ABSTRACT FROM AUTHOR]

Background: Skin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium. Methods: Conditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium. Results: Conditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers. Conclusions: Our results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation. [ABSTRACT FROM AUTHOR]

Abstract Background and aims In dialysis patients, vascular morbidities are highly prevalent and linked to leukocyte extravasation, especially of polarized monocytes. Experimental data demonstrate that phenotypic changes in monocytes require Y-box binding protein-1 (YB-1) upregulation. Methods We determined YB-1 expression in circulating and vessel-invading monocytes from healthy controls and dialysis patients to correlate results with intima plaque formation and systemic inflammation. Results Compared to healthy subjects, dialysis patients have fewer classical and more intermediate and non-classical monocytes. Post-translationally modified YB-1 (lysine 301/304 acetylation) is detected at high levels in the nucleus of adherent and invading CD14+CD68+ monocytes from umbilical cord and atherosclerosis-prone vessels. The content of non-acetylated YB-1 is significantly decreased (p < 0.001), whereas acetylated YB-1 is correspondingly increased (p < 0.001) throughout all monocyte subpopulations, such that the overall content remains unchanged. Conclusions In dialysis patients the YB-1 acetylation status is higher with prevailing diabetes and intima plaque formation. Pro-inflammatory mediators TNFα, IL-6, uPAR, CCL2, M-CSF, progranulin, ANP, and midkine, as well as anti-inflammatory IL-10 are significantly increased in dialysis patients, emphasizing a systemic inflammatory milieu. Strong positive correlations of monocytic YB-1 content are seen with ANP, IP-10, IL-6, and IL-10 serum levels. This is the first study demonstrating an association of cold shock protein YB-1 expression with inflammation in hemodialysis patients. Highlights • In hemodialysis (HD) patients the level of acetylated YB-1 is increased (p < 0.001) in all monocytic subpopulations. • In HD patients with vascular damage (intima plaques, increased intima media thickness) alterations of monocytic YB-1 content are apparent. • Serum cytokine levels show a close correlation between monocytic YB-1 content and systemic inflammatory status. • The correlation of monocytic YB-1 content with disease and systemic inflammation suggests a causal link. [ABSTRACT FROM AUTHOR]

In aging kidneys, a decline of function resulting from extracellular matrix (ECM) deposition and organ fibrosis is regarded as "physiological." Whether a direct link between high salt intake and fibrosis in aging kidney exists autonomously from arterial hypertension is unclear. This study explores kidney intrinsic changes (inflammation, ECM derangement) induced by a high-salt diet (HSD) in a murine model lacking arterial hypertension. The contribution of cold shock Y-box binding protein (YB-1) as a key orchestrator of organ fibrosis to the observed differences is determined by comparison with a knockout strain (Ybx1DRosaERT + TX). Comparisons of tissue from mice fed with normal-salt diet (NSD, standard chow) or high-salt diet (HSD, 4% NaCl in chow; 1% NaCl in water) for up to 16 mo revealed that with HSD tubular cell numbers decrease and tubulointerstitial scarring [periodic acid-Schiff (PAS), Masson's trichrome, Sirius red staining] prevails. In Ybx1DRosaERT + TX animals tubular cell damage, a loss of cell contacts with profound tubulointerstitial alterations, and tubular cell senescence was seen. A distinct tubulointerstitial distribution of fibrinogen, collagen type VI, and tenascin-C was detected under HSD, transcriptome analyses determined patterns of matrisome regulation. Temporal increase of immune cell infiltration was seen under HSD of wild type, but not Ybx1DRosaERT + TX animals. In vitro Ybx1DRosaERT + TX bone marrow-derived macrophages exhibited a defect in polarization (IL-4/IL-13) and abrogated response to sodium chloride. Taken together, HSD promotes progressive kidney fibrosis with premature cell aging, ECM deposition, and immune cell recruitment that is exacerbated in Ybx1DRosaERT + TX animals. NEW & NOTEWORTHY Short-term experimental studies link excessive sodium ingestion with extracellular matrix accumulation and inflammatory cell recruitment, yet long-term data are scarce. Our findings with a high-salt diet over 16 mo in aging mice pinpoints to a decisive tipping point after 12 mo with tubular stress response, skewed matrisome transcriptome, and immune cell infiltration. Cell senescence was aggravated in knockout animals for cold shock Y-box binding protein (YB-1), suggesting a novel protective protein function. [ABSTRACT FROM AUTHOR]

Notch signaling regulates cell fate in multiple tissues including the skeleton. Hajdu–Cheney-Syndrome (HCS), caused by gain-of-function mutations in the Notch2 gene, is a rare inherited disease featuring early-onset osteoporosis and increased risk for fractures and non-union. As the impact of Notch2 overactivation on fracture healing is unknown, we studied bone regeneration in mice harboring a human HCS mutation. HCS mice, displaying high turnover osteopenia in the non-fractured skeleton, exhibited only minor morphologic alterations in the progression of bone regeneration, evidenced by static radiological and histological outcome measurements. Histomorphometry showed increased osteoclast parameters in the callus of HCS mice, which was accompanied by an increased expression of osteoclast and osteoblast markers. These observations were accompanied by inferior biomechanical stability of healed femora in HCS mice. Together, our data demonstrate that structural indices of bone regeneration are normal in HCS mice, which, however, exhibit signs of increased callus turnover and display impaired biomechanical stability of healed fractures. [ABSTRACT FROM AUTHOR]

DNA-binding protein A (DbpA) belongs to the Y-box family of cold shock domain proteins that exert transcriptional and translational activities in the cell via their ability to bind and regulate mRNA. To investigate the role of DbpA in kidney disease, we utilized the murine unilateral ureter obstruction (UUO) model, which recapitulates many features of obstructive nephropathy seen in humans. We observed that DbpA protein expression is induced within the renal interstitium following disease induction. Compared with wild-type animals, obstructed kidneys from Ybx3-deficient mice are protected from tissue injury, with a significant reduction in the number of infiltrating immune cells as well as in extracellular matrix deposition. RNAseq data from UUO kidneys show that Ybx3 is expressed by activated fibroblasts, which reside within the renal interstitium. Our data support a role for DbpA in orchestrating renal fibrosis and suggest that strategies targeting DbpA may be a therapeutic option to slow disease progression. [ABSTRACT FROM AUTHOR]

Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on Clostridioides difficile toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised C. difficile toxins. Our findings emphasise that canine intestinal organoids can be used to test different components and suggest that they can be further refined to also mirror complex interactions between the intestinal epithelium and other cells. [ABSTRACT FROM AUTHOR]

Papillomaviruses (PVs) are a family of small DNA tumor viruses that can induce benign lesions or cancer in vertebrates. The observation that animal PV capsid-proteins spontaneously self-assemble to empty, highly immunogenic virus-like particles (VLPs) has led to the establishment of vaccines that efficiently protect humans from specific PV infections and associated diseases. We provide an overview of PV-induced tumors in horses and other equids, discuss possible routes of PV transmission in equid species, and present recent developments aiming at introducing the PV VLP-based vaccine technology into equine medicine. [ABSTRACT FROM AUTHOR]

Equine sarcoids are common, locally aggressive skin tumors induced by bovine papillomavirus types 1, 2, and possibly 13 (BPV1, BPV2, BPV13). Current in vitro models do not mimic de novo infection. We established primary fibroblasts from horse skin and succeeded in infecting these cells with native BPV1 and BPV2 virions. Subsequent cell characterization was carried out by cell culture, immunological, and molecular biological techniques. Infection of fibroblasts with serial 10-fold virion dilutions (2 × 106-20 virions) uniformly led to DNA loads settling at around 150 copies/cell after four passages. Infected cells displayed typical features of equine sarcoid cells, including hyperproliferation, and loss of contact inhibition. Neither multiple passaging nor storage negatively affected cell hyperproliferation, viral DNA replication, and gene transcription, suggestive for infection-mediated cell immortalization. Intriguingly, extracellular vesicles released by BPV1-infected fibroblasts contained viral DNA that was most abundant in the fractions enriched for apoptotic bodies and exosomes. This viral DNA is likely taken up by non-infected fibroblasts. We conclude that equine primary fibroblasts stably infected with BPV1 and BPV2 virions constitute a valuable near-natural model for the study of yet unexplored mechanisms underlying the pathobiology of BPV1/2-induced sarcoids. [ABSTRACT FROM AUTHOR]

Highlights: A HIF-1α biosensor was generated to facilitate studies on the role of different agents in normoxic HIF-1α stabilization. Micromilieu factors (MFs) induce normoxic HIF-1α stabilization with two different kinetics in HCAECs. There is an unknown indirect regulation of normoxic HIF-1α stabilization by NF-κB. Normoxic endothelial HIF-1α stabilization plays an important role in the inflammatory response of ECs to MFs, which could play a crucial role in the progression of atherosclerosis. Knowledge about normoxic hypoxia-inducible factor (HIF)-1α stabilization is limited. We investigated normoxic HIF-1α stabilization and its consequences using live cell imaging, immunoblotting, Bio-Plex multiplex immunoassay, immunofluorescence staining, and barrier integrity assays. We demonstrate for the first time that IL-8 and M-CSF caused HIF-1α stabilization and translocation into the nucleus under normoxic conditions in both human coronary endothelial cells (HCAECs) and HIF-1α-mKate2-expressing HEK-293 cells. In line with the current literature, our data show significant normoxic HIF-1α stabilization caused by TNF-α, INF-γ, IL-1β, and IGF-I in both cell lines, as well. Treatment with a cocktail consisting of TNF-α, INF-γ, and IL-1β caused significantly stronger HIF-1α stabilization in comparison to single treatments. Interestingly, this cumulative effect was not observed during simultaneous treatment with IL-8, M-CSF, and IGF-I. Furthermore, we identified two different kinetics of HIF-1α stabilization under normoxic conditions. Our data demonstrate elevated protein levels of HIF-1α-related genes known to be involved in the development of atherosclerosis. Moreover, we demonstrate an endothelial barrier dysfunction in HCAECs upon our treatments and during normoxic HIF-1α stabilization comparable to that under hypoxia. This study expands the knowledge of normoxic HIF-1α stabilization and activation and its consequences on the endothelial secretome and barrier function. Our data imply an active role of HIF-1α in vivo in the vasculature in the absence of hypoxia. [ABSTRACT FROM AUTHOR]

Equine sarcoids develop upon bovine papillomavirus type 1 or 2 (BPV1, BPV2) infection in conjunction with trauma and represent the most common tumour disease in horses and other equids, including donkeys. In face of a sarcoid outbreak involving 12 of 111 donkeys and mules at the ‘Rifugio degli Asinelli’, a subsidiary charity organization of The Donkey Sanctuary, non-invasively collected sample material including crusts, dandruff, swabs and hair roots was collected from sarcoid-affected and 26 healthy donkeys, as well as dandruff from a grooming kit and tabanids caught from or in the vicinity of sarcoid patients. In addition five previously collected sarcoids stored in formalin were provided. DNA isolated from collected material was tested for the presence of the BPV1/2 E5 oncogene using PCR. Positive samples were further analysed by E2/E4 and LCR PCR and amplicon sequencing to determine a possible common source of infection via comparative alignment of intralesional BPV1/2 gene variants. IC/PCR was used to assess sample aliquots for the presence of BPV1/2 virions, and IHC to analyse five tumours for BPV1 E5 and L1 protein expression. All sarcoid-affected donkeys, two of 55 tabanids and dandruff from a curry comb tested positive for BPV1/2 E5, yet negative by IC/PCR. Healthy animals were BPV1/2-free. IHC revealed different levels of intralesional E5 and L1 expression. A series of BPV1 E5, E2, and LCR variants and BPV2 E5 were detected from donkeys, indicating that they had accidently developed sarcoids at about the same time rather than having acquired disease from each other. [ABSTRACT FROM AUTHOR]