DNA-binding protein-A promotes kidney ischemia/reperfusion injury and participates in mitochondrial function.

DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation, and tight junction communication. In chronic nephritis, DbpA is upregulated. However, its activities in acute injury models, such as kidney ischemia/reperfusion injury (IRI), are unclear. To study this, mice harboring Ybx3+/+, Ybx3+/- or the Ybx3-/- genotype were characterized over 24 months and following experimental kidney IRI. Mitochondrial function, number and integrity were analyzed by mitochondrial stress tests, MitoTracker staining and electron microscopy. Western Blot, immunohistochemistry and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA was found to be dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells. Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, lipid uptake and metabolism, oxygen consumption rates and glycolytic activities of tubular epithelial cells. Ybx3-/- mice demonstrated protection from IRI with less immune cell infiltration, endoplasmic reticulum stress and tubular cell damage. A presumed protective mechanism was identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 was deleted. Thus, our studies reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted. Hence, preemptive DbpA targeting in situations with expected IRI, such as kidney transplantation or cardiac surgery, may preserve post-procedure kidney function.

Comparative Analysis of Acute Kidney Injury Models and Related Fibrogenic Responses: Convergence on Methylation Patterns Regulated by Cold Shock Protein.

BACKGROUND: Fibrosis is characterized by excessive extracellular matrix formation in solid organs, disrupting tissue architecture and function. The Y-box binding protein-1 (YB-1) regulates fibrosis-related genes (e.g., Col1a1, Mmp2, and Tgfß1) and contributes significantly to disease progression. This study aims to identify fibrogenic signatures and the underlying signaling pathways modulated by YB-1. METHODS: Transcriptomic changes associated with matrix gene patterns in human chronic kidney diseases and murine acute injury models were analyzed with a focus on known YB-1 targets. Ybx1-knockout mouse strains (Ybx1ΔRosaERT+TX and Ybx1ΔLysM) were subjected to various kidney injury models. Fibrosis patterns were characterized by histopathological staining, transcriptome analysis, qRT-PCR, methylation analysis, zymography, and Western blotting. RESULTS: Integrative transcriptomic analyses revealed that YB-1 is involved in several fibrogenic signatures related to the matrisome, the WNT, YAP/TAZ, and TGFß pathways, and regulates Klotho expression. Changes in the methylation status of the Klotho promoter by specific methyltransferases (DNMT) are linked to YB-1 expression, extending to other fibrogenic genes. Notably, kidney-resident cells play a significant role in YB-1-modulated fibrogenic signaling, whereas infiltrating myeloid immune cells have a minimal impact. CONCLUSIONS: YB-1 emerges as a master regulator of fibrogenesis, guiding DNMT1 to fibrosis-related genes. This highlights YB-1 as a potential target for epigenetic therapies interfering in this process.

Endostatin, soluble tumour necrosis factor receptor 1 and soluble tumour necrosis factor receptor 2 cannot predict new onset of microalbuminuria in patients with type 2 diabetes.

AIMS: Inflammation and angiogenesis play an important role in the development of early diabetic kidney disease. We investigated the association of soluble Tumour Necrosis Factor Receptor 1 (sTNF-R1), sTNF-R2 and endostatin with new onset microalbuminuria in normoalbuminuric patients with diabetes mellitus type 2. METHODS: We conducted a case control study to assess serum levels of sTNF-R1, sTNF-R2 and endostatin in 169 patients with new onset microalbuminuria and in 188 matched normoalbuminuric, diabetic controls. Baseline serum samples from participants of the ROADMAP (Randomized Olmesartan and Diabetes Microalbuminuria Prevention) and observational follow-up (ROADMAP-OFU) studies were used. RESULTS: Endostatin and sTNF-R1 but not sTNF-R2 were increased at baseline in patients with future microalbuminuria. In the multivariate analysis, each log2 increment in endostatin levels was associated with an increase of only 6% in the risk of development of microalbuminuria (adjusted HR (95% CI) 1.006 (1.001-1011). sTNF-R1 and sTNF-R2 levels were conversely associated with microalbuminuria, but the results did not reach statistical significance. The respective adjusted HRs (95% CI) were 1.305 (0.928-1.774) and 0.874 (0.711-1.074). CONCLUSIONS: sTNF-R1 and sTNF-R2 failed to predict the occurrence of microalbuminuria in normoalbuminuric patients with type 2 diabetes. Likewise, the utility of endostatin in predicting new onset proteinuria is limited.

Glomerular-tubular crosstalk via cold shock Y-box binding protein-1 in the kidney.

Glomerular-tubular crosstalk within the kidney has been proposed, but the paracrine signals enabling this remain largely unknown. The cold-shock protein Y-box binding protein 1 (YBX1) is known to regulate inflammation and kidney diseases but its role in podocytes remains undetermined. Therefore, we analyzed mice with podocyte specific Ybx1 deletion (Ybx1ΔPod). Albuminuria was increased in unchallenged Ybx1ΔPod mice, which surprisingly was associated with reduced glomerular, but enhanced tubular damage. Tubular toll-like receptor 4 (TLR4) expression, node-like receptor protein 3 (NLRP3) inflammasome activation and kidney inflammatory cell infiltrates were all increased in Ybx1ΔPod mice. In vitro, extracellular YBX1 inhibited NLRP3 inflammasome activation in tubular cells. Co-immunoprecipitation, immunohistochemical analyses, microscale cell-free thermophoresis assays, and blunting of the YBX1-mediated TLR4-inhibition by a unique YBX1-derived decapeptide suggests a direct interaction of YBX1 and TLR4. Since YBX1 can be secreted upon post-translational acetylation, we hypothesized that YBX1 secreted from podocytes can inhibit TLR4 signaling in tubular cells. Indeed, mice expressing a non-secreted YBX1 variant specifically in podocytes (Ybx1PodK2A mice) phenocopied Ybx1ΔPod mice, demonstrating a tubular-protective effect of YBX1 secreted from podocytes. Lipopolysaccharide-induced tubular injury was aggravated in Ybx1ΔPod and Ybx1PodK2A mice, indicating a pathophysiological relevance of this glomerular-tubular crosstalk. Thus, our data show that YBX1 is physiologically secreted from podocytes, thereby negatively modulating sterile inflammation in the tubular compartment, apparently by binding to and inhibiting tubular TLR4 signaling. Hence, we have uncovered an YBX1-dependent molecular mechanism of glomerular-tubular crosstalk.

Feline SCCs of the Head and Neck Display Partial Epithelial-Mesenchymal Transition and Harbor Stem Cell-like Cancer Cells.

Squamous cell carcinoma of the head and neck (HNSCC) is a malignant cancer disease in humans and animals. There is ample evidence that the high plasticity of cancer cells, i.e., their ability to switch from an epithelial to a mesenchymal, endothelial, and stem cell-like phenotype, chiefly contributes to progression, metastasis, and multidrug resistance of human HNSCCs. In feline HNSCC, the field of cancer cell plasticity is still unexplored. In this study, fourteen feline HNSCCs with a known feline papillomavirus (FPV) infection status were subjected to histopathological grading and subsequent screening for expression of epithelial, mesenchymal, and stem cell markers by immunohistochemistry (IHC) and immunofluorescence staining (IF). Irrespective of the FPV infection status, all tumors except one corresponded to high-grade, invasive lesions and concurrently expressed epithelial (keratins, E-cadherin, ß-catenin) and mesenchymal (vimentin, N-cadherin, CD146) proteins. This finding is indicative for partial epithelial-mesenchymal transition (pEMT) events in the lesions, as similarly described for human HNSCCs. IF double staining revealed the presence of CD44/CD271 double-positive cells notably within the tumors' invasive fronts that likely correspond to cancer stem cells. Taken together, the obtained findings suggest that feline HNSCCs closely resemble their human counterparts with respect to tumor cell plasticity.

Cold Shock Domain Protein DbpA Orchestrates Tubular Cell Damage and Interstitial Fibrosis in Inflammatory Kidney Disease.

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.

Fracture healing in a mouse model of Hajdu-Cheney-Syndrome with high turnover osteopenia results in decreased biomechanical stability.

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.

Excessive sodium chloride ingestion promotes inflammation and kidney fibrosis in aging mice.

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 (Ybx1ΔRosaERT+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 Ybx1ΔRosaERT+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 Ybx1ΔRosaERT+TX animals. In vitro Ybx1ΔRosaERT+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 Ybx1ΔRosaERT+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.

Immunotherapy of Equine Sarcoids-From Early Approaches to Innovative Vaccines.

Horses and other equid species are frequently affected by bovine papillomavirus type 1 and/or 2 (BPV1, BPV2)-induced skin tumors termed sarcoids. Although sarcoids do not metastasize, they constitute a serious health problem due to their BPV1/2-mediated resistance to treatment and propensity to recrudesce in a more severe, multiple form following accidental or iatrogenic trauma. This review provides an overview on BPV1/2 infection and associated immune escape in the equid host and presents early and recent immunotherapeutic approaches in sarcoid management.

Detection of Equine Papillomaviruses and Gamma-Herpesviruses in Equine Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) seriously compromises the health and welfare of affected horses. Although robust evidence points to equine papillomavirus type 2 (EcPV2) causing genital lesions, the etiopathogenesis of equine SCC is still poorly understood. We screened a series of SCCs from the head-and-neck (HN), (peri-)ocular and genital region, and site-matched controls for the presence of EcPV2-5 and herpesvirus DNA using type-specific EcPV PCR, and consensus nested herpesvirus PCR followed by sequencing. EcPV2 DNA was detected in 45.5% of HN lesions, 8.3% of (peri-)ocular SCCs, and 100% of genital tumors, whilst control samples from tumor-free horses except one tested EcPV-negative. Two HNSCCs harbored EcPV5, and an ocular lesion EcPV4 DNA. Herpesvirus DNA was detected in 63.6%, 66.6%, 47.2%, and 14.2% of horses with HN, ocular, penile, and vulvar SCCs, respectively, and mainly identified as equine herpesvirus 2 (EHV2), 5 (EHV5) or asinine herpesvirus 5 (AsHV5) DNA. In the tumor-free control group, 9.6% of oral secretions, 46.6% of ocular swabs, 47% of penile samples, and 14.2% of vaginal swabs scored positive for these herpesvirus types. This work further highlights the role of EcPV2 as an oncovirus and is the first to provide information on the prevalence of (gamma-)herpesviruses in equine SCCs.

Papillomavirus-like Particles in Equine Medicine.

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.

Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach.

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.

Normoxic HIF-1α Stabilization Caused by Local Inflammatory Factors and Its Consequences in Human Coronary Artery Endothelial Cells.

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-1ß, and IGF-I in both cell lines, as well. Treatment with a cocktail consisting of TNF-α, INF-γ, and IL-1ß 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.

Treatment of digital dermatitis using salicylic acid in European bison (Bison bonasus) reveals promising results.

Digital dermatitis (DD) associated with the presence of multiple Treponema spp. was recently described for the first time in European bison (Bison bonasus). DD is characterized by skin inflammation in the distal foot area in various ungulates. The objective of this proof of concept study was to test a treatment protocol adopted from cattle for its applicability in this wildlife species using five animals. Keratolytic salicylic acid paste was administered topically under bandages for seven days to enable removal of the affected skin. All interventions were performed under general anesthesia. To evaluate the treatment efficacy, photographs and biopsies were taken pre- and post-treatment. The biopsies were examined histologically, by PCR for the presence of different bacterial species, by Treponema-specific fluorescent in situ hybridization (FISH), and by transmission electron microscopy. Based on photographs, complete clinical healing of the 15 feet with macroscopical DD lesions was achieved. Histological examination showed mild to moderate dermatitis in 17/20 feet before, and in 12/20 feet after treatment. 17/20 feet were Treponema spp. PCR positive before, and none was positive after treatment. Dichelobacter nodosus, Fusobacterium necrophorum, and Porphyromonas levii could not be detected in any of the samples. By FISH and electron microscopy, Treponema spp. could be visualized in the stratum corneum before, but not after treatment. These results suggest that this treatment method can be applied as standard practice prior to transporting DD affected European bison to prevent the spread of this contagious disease.

Bovine Papillomavirus Type 1 or 2 Virion-Infected Primary Fibroblasts Constitute a Near-Natural Equine Sarcoid Model.

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.

"Non-healing" claw horn lesions in dairy cows: Clinical, histopathological and molecular biological characterization of four cases.

The increasing prevalence of bovine digital dermatitis (BDD) contributes to a higher occurrence of secondary infections of exposed corium with Treponema spp. in bovine claws. "Non-healing" claw horn lesions (NHL) clinically resemble BDD lesions. They are severe, cause chronic lameness, and may persist for several months. They poorly respond to standard treatments of BDD and represent a serious welfare issue. In this study, four cases of NHL were classified clinically either as BDD-associated axial horn fissures (BDD-HFA; n = 3) or BDD-associated sole ulcer (BDD-SU; n = 1). In all four cases, pronounced multifocal keratinolysis of the stratum corneum, ulceration, and severe chronic lymphoplasmacytic perivascular to interstitial dermatitis were observed. All lesional samples tested positive for Treponema spp., Fusobacterium (F.) necrophorum, and Porphyromonas (P.) levii by PCRs. BDD-HFA lesions contained Treponema pedis as revealed by genetic identities of 93, 99, and 100%. Treponemes in the BDD-SU lesion were 94% homologous to Treponema phylotype PT3. Fluorescent in situ hybridization (FISH) revealed extensive epidermal infiltration by treponemes that made up > 90% of the total bacterial population in all four lesions. FISH also tested positive for P. levii and negative for F. necrophorum in all four cases, whilst only one BDD-HFA contained Dichelobacter nodosus. Our data point to BDD-associated treponemes and P. levii constituting potential etiological agents in the development of "non-healing" claw horn lesions in cattle.

Proof of an optimized salicylic acid paste-based treatment concept of ulcerative M2-stage digital dermatitis lesions in 21 dairy cows.

The efficacy of salicylic acid paste (SA) in the treatment of ulcerative bovine digital dermatitis (BDD) was assessed by combining clinical and histopathological analyses with molecular biological techniques. The latter were conducted in a blinded manner to reach maximum objectivity. Prior to treatment, M2-stage BDD lesions (n = 26, diagnosed in 21 dairy cows) exhibited ulceration, with severe perivascular, chronic, lymphoplasmacytic dermatitis and extensive keratinolysis being noted in most cases. Pretreatment biopsy samples (n = 12) followed by povidone-iodine ointment under bandage for one week before administration of SA paste were tested positive for Treponema spp. by blinded PCR and fluorescent in situ hybridization (FISH). Subsequent treatment consisted of application of SA and bandaging at weekly intervals until lesions had completely resolved. The treatment duration ranged between 2 and 4 weeks. Complete healing was achieved in 100% of cases, with 2/21 animals requiring a second round of treatment upon disease reoccurrence. Importantly, only 3/26 biopsies taken from previously affected sites still tested positive by Treponema PCR, and in another biopsy, the outermost layers of the stratum corneum scored weakly positive by Treponema-specific FISH. None of these Treponema DNA-positive biopsies showed signs of ulceration. One case exhibited focal keratinolysis. Positive PCR or FISH in these cases may have arisen from DNA traces of dead bacteria or environmental contamination during biopsy harvesting. To our knowledge, this is the first study on blinded molecular biological monitoring of the therapeutic efficacy of SA with respect to treponemal infection, and on complete BDD M2-stage remission in all animals achieved by SA treatment according to an optimized protocol. Although the etiology of BDD is considered as multifactorial, our data further support the concept that treponemes have a decisive role in BDD pathogenesis.

Investigation of multiple Felis catus papillomavirus types (-1/-2/-3/-4/-5/-6) DNAs in feline oral squamous cell carcinoma: a multicentric study.

Recent evidence suggests a possible association of Felis catus papillomavirus type 2 (FcaPV-2) DNA with feline oral squamous cell carcinoma (FOSCC). In this study, type-specific PCR targeting two genes (L1/E6 or E1/E6) of FcaPV-1/-2/-3/-4/-5/-6 was performed to detect viral DNA in a large amount of FOSCC samples collected in Italy and Austria. FcaPV-1/-2/-3/-4/-5 were detected in 7/113 (6.2%), 7/93 (7.5%), 6/113 (5.3%), 1/113 (0.9%) and 2/113 (1.8%) specimens, respectively, with different prevalences in Italian vs. Austrian samples, whilst FcaPV-6 went undetected. Our results confirms that FcaPV-2 is the most prevalent in FOSCC, followed by FcaPV-1/-3 and suggest that FcaPVs have variable circulation rates in European countries.

Tumor Cell Plasticity in Equine Papillomavirus-Positive Versus-Negative Squamous Cell Carcinoma of the Head and Neck.

Squamous cell carcinoma of the head and neck (HNSCC) is a common malignant tumor in humans and animals. In humans, papillomavirus (PV)-induced HNSCCs have a better prognosis than papillomavirus-unrelated HNSCCs. The ability of tumor cells to switch from epithelial to mesenchymal, endothelial, or therapy-resistant stem-cell-like phenotypes promotes disease progression and metastasis. In equine HNSCC, PV-association and tumor cell phenotype switching are poorly understood. We screened 49 equine HNSCCs for equine PV (EcPV) type 2, 3 and 5 infection. Subsequently, PV-positive versus -negative lesions were analyzed for expression of selected epithelial (keratins, ß-catenin), mesenchymal (vimentin), endothelial (COX-2), and stem-cell markers (CD271, CD44) by immunohistochemistry (IHC) and immunofluorescence (IF; keratins/vimentin, CD44/CD271 double-staining) to address tumor cell plasticity in relation to PV infection. Only EcPV2 PCR scored positive for 11/49 equine HNSCCs. IHC and IF from 11 EcPV2-positive and 11 EcPV2-negative tumors revealed epithelial-to-mesenchymal transition events, with vimentin-positive cells ranging between <10 and >50%. CD44- and CD271-staining disclosed the intralesional presence of infiltrative tumor cell fronts and double-positive tumor cell subsets independently of the PV infection status. Our findings are indicative of (partial) epithelial-mesenchymal transition events giving rise to hybrid epithelial/mesenchymal and stem-cell-like tumor cell phenotypes in equine HNSCCs and suggest CD44 and CD271 as potential malignancy markers that merit to be further explored in the horse.

Midkine release during hemodialysis is predictive of hypervolemia and associates with excess (cardiovascular) mortality in patients with end-stage renal disease: a prospective study.

BACKGROUND: In end-stage renal disease, a high cardiovascular risk profile and endothelial damage prevails. The heparin-binding growth factor midkine stimulates neo-angiogenesis in ischemic diseases, coordinates neutrophil influx, and raises blood pressure through stimulated angiotensin synthesis. METHODS: We determined changes of midkine serum levels during hemodialysis sessions under the assumption that endothelial cell-derived midkine is released. Periprocedural differences (∆midkine) were calculated and correlated with cardiovacular biomarkers and fluid status (clinical assessment, V. cava collapse, comet tail phenomenon), cardiovascular morbidities, mortality rates. Blood was collected before and after dialysis from hemodialysis patients (n = 171; diabetes: n = 70; hypervolemia: n = 83; both: n = 32). RESULTS: Baseline midkine levels were ~ fourfold elevated compared to healthy controls (n = 100). Further, on average a tenfold rise was detected during dialysis, the extent of which was partially related to non-fractionated heparin application (r2 = 0.17). Inter-individual differences were highly reproducible. Hypervolemic patients responded with a less than average rise in midkine levels during dialysis (p < 0.02), this difference became more obvious with co-existing diabetes (p < 0.001 for long dialysis-free interval) and was confirmed in an independently enrolled dialysis cohort (n = 88). In Kaplan Meier survival curves, low delta midkine levels correlated with cardiovascular/overall mortality rates, similar to elevated uPAR levels, whereas other markers (NTproANP, galectin, tenascin-C) were less predictive. Following intervention with successful fluid removal in hypervolemic dialysis patients to optimize fluid homeostasis, midkine values increased (p < 0.002), which was not observed in patients that failed to decrease weight. CONCLUSION: Thus, for dialysis patients inadequate periprocedural midkine upregulation is linked with hypervolemia and associates with cardiovascular events.