Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same

Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.

Image based annotation of Chemogenomic Libraries for Phenotypic Screening

Phenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still poses a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies.

Intraarterial anti-leptin therapy via ICA protects ipsilateral CA1 neurons subjected to ischemia and reperfusion

Background Brain reperfusion following an ischemic event is essential for tissue viability, however, it also involves processes that promote neuronal cell death. We have recently shown that local expression of the hormone leptin in cardiovascular organs drives deleterious remodeling. As cerebral ischemia-reperfusion (IR) lesions derive expression of both the leptin hormone and its receptor, we hypothesized that blocking leptin activity in the injured brain area will reduce the deleterious effects of IR injury. Methods C57BL6 male mice underwent bilateral common carotid artery and external carotid artery ligation. The right hemisphere was reperfused after 12 minutes, followed by intraarterial injection of either a low-dose leptin antagonist or saline solution via the ipsilateral ICA. The left common carotid artery remained ligated. Fifteen IR/leptin antagonist-injected and fourteen IR/saline-injected mice completed the experiment. Five days after surgery brains were collected and samples of the hippocampal CA1 region were analyzed for cell viability (H&E) and apoptosis (TUNEL and caspase3), for neuroinflammation (Iba1), and for signaling pathways of pSTAT3 and pSmad2. Results The right hemisphere hippocampal CA1 region subjected to IR and saline injection exhibited increased apoptosis and necrosis of pyramidal cells. Also, increased density of activated microglia/macrophages was evident around the CA1 region. Comparatively, leptin antagonist treatment at reperfusion reduced apoptosis and necrosis of pyramidal cells, as indicated by increased number of viable cells (p < 0.01), and reduced TUNEL (p < 0.001) and caspase3-positive cells (p<0.05). Furthermore, this treatment reduced the density of activated microglia/macrophages (p < 0.001) in the CA1 region. Signaling pathway analysis revealed that while pSTAT3 and pSmad2-positive cells were found surrounding the stratum pyramidal in saline-treated animals, pSTAT3 signal was undetected and pSmad2 was greatly reduced in this territory following leptin antagonist treatment (p < 0.01). Conclusions Inhibition of leptin activity in hemispheric IR injury preserved the viability of ipsilateral hippocampal CA1 neurons, likely by preventing apoptosis and local inflammation. These results indicate that intraarterial anti-leptin therapy may have clinical potential in reducing hemispheric brain IR injury.

Infection of Galleria mellonella (Lepidoptera) Larvae With the Entomopathogenic Fungus Conidiobolus coronatus (Entomophthorales) Induces Apoptosis of Hemocytes and Affects the Concentration of Eicosanoids in the Hemolymph

Apoptosis and autophagy, the mechanisms of programmed cell death, play critical roles in physiological and pathological processes in both vertebrates and invertebrates. Apoptosis is also known to play an important role in the immune response, particularly in the context of entomopathogenic infection. Of the factors influencing the apoptotic process during infection, two of the lesser known groups are caspases and eicosanoids. The aim of this study was to determine whether infection by the entomopathogenic soil fungus Conidiobolus coronatus is associated with apoptosis and changes in caspase activity in the hemocytes of Galleria mellonella larvae, and to confirm whether fungal infection may affect eicosanoid levels in the host. Larvae were exposed for 24 h to fully grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24 group) or 24 h later (F48 group). Apoptosis/necrosis tests were performed in hemocytes using fluorescence microscopy and flow cytometry, while ELISA tests were used to measure eicosanoid levels. Apoptosis and necrosis occurred to the same degree in F24, but necrosis predominated in F48. Fungal infection resulted in caspase activation, increased PGE1, PGE2, PGA1, PGF2α, and 8-iso-PGF2α levels and decreased TXB2 levels, but had no effect on TXA2 or 11-dehydro-TXB2 concentrations. In addition, infected larvae demonstrated significantly increased PLA2 activity, known to be involved in eicosanoid biosynthesis. Our findings indicate that fungal infection simultaneously induces apoptosis in insects and stimulates general caspase activity, and this may be correlated with changes in the concentrations of eicosanoids.

An Imaging and Computational Algorithm for Efficient Identification and Quantification of Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) are associated with multiple disease pathologies including sepsis, asthma, rheumatoid arthritis, cancer, systemic lupus erythematosus, acute respiratory distress syndrome, and COVID-19. NETs, being a disintegrated death form, suffered inconsistency in their identification, nomenclature, and quantifications that hindered therapeutic approaches using NETs as a target. Multiple strategies including microscopy, ELISA, immunoblotting, flow cytometry, and image-stream-based methods have exhibited drawbacks such as being subjective, non-specific, error-prone, and not being high throughput, and thus demand the development of innovative and efficient approaches for their analyses. Here, we established an imaging and computational algorithm using high content screening (HCS)—cellomics platform that aid in easy, rapid, and specific detection as well as analyses of NETs. This method employed membrane-permeable and impermeable DNA dyes in situ to identify NET-forming cells. Automated algorithm-driven single-cell analysis of change in nuclear morphology, increase in nuclear area, and change in intensities provided precise detection of NET-forming cells and eliminated user bias with other cell death modalities. Further combination with Annexin V staining in situ detected specific death pathway, e.g., apoptosis, and thus, discriminated between NETs, apoptosis, and necrosis. Our approach does not utilize fixation and permeabilization steps that disturb NETs, and thus, allows the time-dependent monitoring of NETs. Together, this specific imaging-based high throughput method for NETs analyses may provide a good platform for the discovery of potential inhibitors of NET formation and/or agents to modulate neutrophil death, e.g., NETosis-apoptosis switch, as an alternative strategy to enhance the resolution of inflammation.

Comprehensive expression profiles of mRNAs, lncRNAs and miRNAs in Kashin-Beck disease identified by RNA-sequencing

Kashin-Beck disease (KBD) is a chronic, endemic and deforming osteochondropathy, whose basic pathological alterations include apoptosis and necrosis of chondrocytes in articular cartilage and growth plates and imbalanced extracellular matrix metabolism.

Evaluation of Plasma Nucleosome Concentrations In Healthy Dogs And Dogs With A Variety of Common Cancers.

Background: Cell free DNA, in the form of nucleosomes, is released into circulation during apoptosis and necrosis in a variety of diseases. They are small fragments of chromosomes that are composed of DNA wrapped around a histone core made of four duplicate histone proteins forming an octamer. The nucleosome compartment is a relatively uninvestigated area of circulating tumor biomarkers in dogs. The objectives of this study were to quantify and better characterize nucleosomes in 528 dogs with various common malignancies and 134 healthy dogs. Results: The sensitivity of elevated circulating nucleosome concentrations for the detection of cancer in all dogs was 49.8% with a specificity of 97% with an area under the curve of 68.74%. The top 4 malignancies detected by the test included lymphoma, hemangiosarcoma, histiocytic sarcoma and malignant melanoma. The malignancies least likely to be detected were soft tissue sarcomas and mast cell tumors. Conclusions: A variety of tumor types may cause elevated nucleosome concentations in dogs. Tumors of hematopoietic origin are most likely to cause elevations and local tumors such as soft tissue sarcomas are least likely to cause elevations in plasma nucleosome concentrations.

The Role of p53 Protein in the Realization of the Exogenous Heat Shock Protein 70 Anti-Apoptotic Effect during Axotomy

The search for effective neuroprotective agents for the treatment of neurotrauma has always been of great interest to researchers around the world. Extracellular heat shock protein 70 (eHsp70) is considered a promising agent to study, as it has been demonstrated to exert a significant neuroprotective activity against various neurodegenerative diseases. We showed that eHsp70 can penetrate neurons and glial cells when added to the incubation medium, and can accumulate in the nuclei of neurons and satellite glial cells after axotomy. eHsp70 reduces apoptosis and necrosis of the glial cells, but not the neurons. At the same time, co-localization of eHsp70 with p53 protein, one of the key regulators of apoptosis, was noted. eHsp70 reduces the level of the p53 protein apoptosis promoter both in glial cells and in the nuclei and cytoplasm of neurons, which indicates its neuroprotective effect. The ability of eHsp70 to reverse the proapoptotic effect of the p53 activator WR1065 may indicate its ability to regulate p53 activity or its proteosome-dependent degradation.

Up-Regulation of Fibroblast Growth Factor 23 Gene Expression in UMR106 Osteoblast-like Cells with Reduced Viability

Fibroblast growth factor 23 (FGF23) controls vitamin D and phosphate homeostasis in the kidney and has additional paracrine effects elsewhere. As a biomarker, its plasma concentration is associated with progression of inflammatory, renal, and cardiovascular diseases. Major stimuli of FGF23 synthesis include active vitamin D and inflammation. Antineoplastic chemotherapy treats cancer by inducing cellular damage ultimately favoring cell death (apoptosis and necrosis) and causing inflammation. Our study explored whether chemotherapeutics and other apoptosis inducers impact on Fgf23 expression. Experiments were performed in osteoblast-like UMR106 cells, Fgf23 gene expression and protein synthesis were determined by qRT-PCR and ELISA, respectively. Viability was assessed by MTT assay and NFκB activity by Western Blotting. Antineoplastic drugs cisplatin and doxorubicin as well as apoptosis inducers procaspase-activating compound 1 (PAC-1), a caspase 3 activator, and serum depletion up-regulated Fgf23 transcripts while reducing cell proliferation and viability. The effect of cisplatin on Fgf23 transcription was paralleled by Il-6 up-regulation and NFκB activation and attenuated by Il-6 and NFκB signaling inhibitors. To conclude, cell viability-decreasing chemotherapeutics as well as apoptosis stimulants PAC-1 and serum depletion up-regulate Fgf23 gene expression. At least in part, Il-6 and NFκB may contribute to this effect.

DISORDER OF SPERMATOGENESIS UNDER CONDITIONS OF EXPERIMENTAL CHRONIC KIDNEY DISEASE

Purpose - to estimate the disorder of spermatogenesis under conditions of experimentalchronic kidney disease (EChD).Material and methods. The study was carried out in two series of experiments on malemice with EChD, the model of which was created by immunizing animals with a kidneyhomogenate. The first series of experiments was devoted to the study of: sperm count(sperm concentration (mln / ml)) and the number of abnormal sperm forms; the ratio ofcells of different generations of spermatogenic epithelium (%) in the testes; pathways ofcell death of cells of the testes and epididymis (spermatocytes (primary) and spermatozoa).The fertile qualities of males were assessed in the second series of experiments, afterreplanting them to intact females. Pre- and post-implantation embryonic mortality andthe number of living fetuses per female mouse have been investigated. The research resultswere compared with the performance of animals in the control groups for each series.Results. No significant changes in the number of spermatozoa were found under EChDconditions (p> 0.05). An increase in the number of abnormal spermatozoa (22%) andthose with primary abnormalities (p <0.05) was found. Among the generations of testescells, a decrease in the number of spermatids and living spermatocytes (primary) (15%)was established, with an increase in the number of cells with apoptosis and necrosisamong them (p <0.05). The number of living cells of the epididymis (spermatozoa) alsodecreased (17.8%), with the growth of cells with apoptosis and necrosis among them (p<0.05). There was an increase in the pre- and post-implantation mortality of embryos (p<0.05); decrease in the number of living fetuses (p <0.05).Conclusions. Under conditions of four-time treatment with renal homogenate (EChD)there is a disorder of spermatogenesis in male mice. Experimental model of kidneydamage, proposed by us, can be useful for studying other aspects and consequences ofkidney pathology, and both for establishment of the features and detection of possiblepathogenetic links in the development of spermatogenesis disorder under conditions ofchronic kidney disease and search of the effective ways to correct it in future.