Search Results (64)

In recent years, pseudorabies virus (PRV) variants have resulted in an epidemic in swine herds and huge economic losses in China. Therefore, it is essential to develop an efficacious vaccine against the spread of PRV variants. Here, the triple-gene-deletion virus and the triple-gene-deletion plus gC virus were constructed by homologous recombination (HR). And then, their growth capacity, proliferation ability, and immune efficacy were evaluated. The results showed that the growth kinetics of the recombinant viruses were similar to those of the parental strain PRV-AH. Compared with the triple-gene-deletion virus group, the more dominant level of neutralizing antibody (NA) can be induced in the triple-gene-deletion plus gC virus group with the same 10^6.0 TCID₅₀ dose after 4 and 6 weeks post-initial immunization (PII) (p < 0.0001). In addition, the antibody titers in mice immunized with the triple-gene-deletion plus gC virus were significantly higher than those immunized with triple-gene deletion virus with the same 10^5.0 TCID₅₀ dose after 6 weeks PII (p < 0.001). More importantly, in the triple-gene-deletion plus gC virus group with 10^5.0 TCID₅₀, the level of NA was close to that in the triple-gene deletion virus group with 10^6.0 TCID₅₀ at 6 weeks PII. Meanwhile, the cytokines IL-4 and IFN-γ in sera were tested by enzyme-linked immunosorbent assay (ELISA) in each group. The highest level of IL-4 or IFN-γ was also elicited in the triple-gene deletion plus gC virus group at a dose of 10^6.0 TCID₅₀. After challenge with PRV-AH, the survival rates of the triple-gene deletion plus gC virus immunized groups were higher than those of other groups. In immunized groups with 10^5.0 TCID₅₀, the survival rate shows a significant difference between the triple-gene deletion plus gC virus group (75%, 6/8) and the triple-gene deletion virus group (12.5%, 1/8). In general, the immune efficacy of the PRV TK/gI/gE-deleted virus can be increased with additional gC insertion in mice, which has potential for developing an attenuated vaccine candidate for PRV control. Full article

Activin A is known to impede tubular repair following renal ischemia, whereas exogenous follistatin, an activin A antagonist, has been shown to ameliorate kidney damage in rats. Despite these findings, the precise role of endogenous follistatin in the kidney has yet to be elucidated. In this study, we investigated the localization of follistatin in the normal human kidney and its potential utility as a marker for acute kidney injury (AKI). In a total of 118 AKI patients and 16 healthy adults, follistatin levels in serum and urine were quantified using ELISA, and correlations with clinical parameters were analyzed. Follistatin-producing cells were positive for Na-Cl co-transporter and uromodulin, but negative for aquaporin 1 and aquaporin 2. Unlike healthy adults, urinary follistatin significantly increased in AKI patients, correlating positively with AKI severity. Urinary follistatin levels were notably higher in patients needing renal replacement therapy. Significant correlations were observed with urinary protein, α1 microglobulin, and urinary NGAL, but not with urinary KIM-1, urinary L-FABP, urinary NAG, urinary β2 microglobulin, or serum creatinine. Interestingly, no correlation between urinary and serum follistatin levels was identified, indicating a renal origin for urinary follistatin. In conclusion, follistatin, produced by distal tubules, is detectable in the urine of AKI patients, suggesting its potential as a valuable marker for monitoring acute tubular damage severity in AKI. Full article

Osteoarthritis (OA) is a multifactorial disease depending on molecular, genetic, and environmental factors like mechanical strain. Next to the cartilage and the subchondral bone, OA also affects the synovium, which is critically involved in the maintenance of joint homeostasis. As there is a correlation between the extracellular sodium content in the knee joint and OA, this study investigates the impact of sodium on OA-associated processes like inflammation and bone remodeling without and with mechanical loading in synovial fibroblasts. For that purpose, murine synovial fibroblasts from the knee joint were exposed to three different extracellular sodium chloride concentrations (−20 mM, ±0 mM and +50 mM NaCl) in the absence or presence of compressive or intermittent tensile strain. In addition to the intracellular Na⁺ content and gene expression of the osmoprotective transcription factor nuclear factor of activated T cells 5 (Nfat5), the gene and protein expression of inflammatory mediators (interleukin-6 (IL6), prostaglandin endoperoxide synthase-2 (Ptgs2)/prostaglandin E₂ (PGE₂)), and factors involved in bone metabolism (receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG)) were analyzed by qPCR and ELISA. Mechanical strain already increased intracellular Na⁺ and Nfat5 gene expression at standard salt conditions to levels obtained by exposure to increased extracellular Na⁺ content. Both high salt and compressive strain resulted in elevated IL6 and PGE₂ release. Intermittent tensile strain did not increase Il6 mRNA expression or IL6 protein secretion but triggered Ptgs2 expression and PGE₂ production. Increased extracellular Na⁺ levels and compressive strain increased RANKL expression. In contrast, intermittent tension suppressed RANKL expression without this response being subject to modification by extracellular sodium availability. OPG expression was only induced by compressive strain. Changes in extracellular Na⁺ levels modified the inflammatory response and altered the expression of mediators involved in bone metabolism in cells exposed to mechanical strain. These findings indicate that Na⁺ balance and Nfat5 are important players in synovial fibroblast responses to mechanical stress. The integration of Na⁺ and Na⁺-dependent signaling will help to improve the understanding of the pathogenesis of osteoarthritis and could lead to the establishment of new therapeutic targets. Full article

Purpose: Chemical eye injury is an acute emergency that can result in vision loss. Neurotrophic keratitis (NK) is the most common long-term manifestation of chemical injury. NK due to alkali burn affects ocular surface health and is one of its most common causes. Here, we established a rabbit model of corneal alkali burns to evaluate the severity of NK-associated changes. Material methods: Alkali burns were induced in NZ rabbits by treating the cornea with (i) a 5 mm circular filter paper soaked in 0.75 N NaOH for 10 s (Mild NK) and (ii) trephination using a guarded trephine (5 mm diameter and 150-micron depth), followed by alkali burn, with a 5 mm circular filter paper soaked in 0.75 N NaOH for 10 s (a severe form of NK). Immediately after, the cornea was rinsed with 10 mL of normal saline to remove traces of NaOH. Clinical features were evaluated on Day 0, Day 1, Day 7, Day 15, and Day 21 post-alkali burn using a slit lamp, Pentacam, and anterior segment optical coherence tomography (AS-OCT). NK-like changes in epithelium, sub-basal nerve plexus, and stroma were observed using in vivo confocal microscopy (IVCM), and corneal sensation were measured using an aesthesiometer post alkali injury. After 21 days, pro-inflammatory cytokines were evaluated for inflammation through ELISA. Results: Trephination followed by alkali burn resulted in the loss of epithelial layers (manifested using fluorescein stain), extensive edema, and increased corneal thickness (550 µm compared to 380 µm thickness of control) evaluated through AS-OCT and increased opacity score in alkali-treated rabbit (80 compared to 16 controls). IVCM images showed complete loss of nerve fibers, which failed to regenerate over 30 days, and loss of corneal sensation—conditions associated with NK. Cytokines evaluation of IL6, VEGF, and MMP9 indicated an increased angiogenic and pro-inflammatory milieu compared to the milder form of NK and the control. Discussion: Using clinical parameters, we demonstrated that the alkali-treated rabbit model depicts features of NK. Using IVCM in the NaOH burn animal model, we demonstrated a complete loss of nerve fibers with poor self-healing capability associated with sub-basal nerve degeneration and compromised corneal sensation. This pre-clinical rabbit model has implications for future pre-clinical research in neurotrophic keratitis. Full article

Background: Neutrophils are thought to play a pivotal role in the pathogenesis of many inflammatory diseases, such as hepatitis, liver cirrhosis, etc. Activated human neutrophils release human neutrophil peptides (HNP1-3) or alpha-defensins that are antimicrobial peptides in azurophil granules. Furthermore, HNP1-3 build a scaffold of neutrophil extracellular traps (NETs) and promote the process of programmed cell death called NETosis. Our study aimed to investigate the role of alpha-defensins in the pathogenesis of alcohol-related liver cirrhosis (ALC). Methods: The concentrations of alpha-defensins in the plasma of 62 patients with ALC and 24 healthy subjects were measured by ELISA. The patients with ALC were prospectively recruited based on the severity of liver dysfunction according to the Child-Pugh and Model of End-Stage Liver Disease-Natrium (MELD-Na) scores, modified Maddrey’s Discriminant Function (mDF), and the presence of ALC complications. Results: The concentrations of alpha-defensins in plasma were significantly higher in the ALC patients than in the controls. The plasma levels of HNP1-3 correlated with the MELD and mDF scores. ALC subgroups with MELD > 20 and mDF > 32 displayed significantly higher HNP1-3 concentrations. The plasma levels of HNP1-3 revealed a good predictive AUC for hepatic encephalopathy and ascites development (0.81 and 0.74, respectively) and for patient survival (0.87) in those over 40 years of age. Conclusion: These findings suggest that alpha-defensins play an important role in the assessment of ALC. Full article

We explored metastasis-associated protein 1 (MTA1) promoter methylation in the development of brain arteriovenous malformation (BAVM). The clinical data of 148 sex- and age-matched BAVMs and controls were collected, and the MTA1 DNA methylation in peripheral white blood cells (WBC) was assessed by bisulfite pyrosequencing. Among them, 18 pairs of case–control samples were used for WBC mRNA detection, 32 pairs were used for WBC MTA1 protein measurement, and 50 pairs were used for plasma inflammatory factor analysis. Lipopolysaccharide (LPS) treatment was used to induce an inflammatory injury cell model of human brain microvascular endothelial cells (BMECS). 5-Aza-2′-deoxycytidine (5-AZA), nicotinic acid (NA), and MTA1 siRNAs were used in functional experiments to examine BMECS behaviors. RT-qPCR, Western blot, and ELISA or cytometric bead arrays were used to measure the expression levels of MTA1, cytokines, and signaling pathway proteins in human blood or BMECS. The degree of MTA1 promoter methylation was reduced in BAVM compared with the control group and was inversely proportional to MTA1 expression. Plasma ApoA concentrations in BAVM patients were significantly lower than those in controls and correlated positively with MTA1 promoter methylation and negatively with MTA1 expression. The expression of cytokine was markedly higher in BAVM than in controls. Cell experiments showed that 5-AZA decreased the methylation level of MTA1 and increased the expression of MTA1 protein. LPS treatment significantly increased cytokine concentrations (p < 0.05). NA and MTA1 silencing could effectively reverse the LPS-mediated increase in IL-6 and TNF-α expression through the NF-κB pathway. Our study indicated that NA may regulate MTA1 expression by affecting promoter DNA methylation, improve vascular inflammation through the NF-κB pathway, and alleviate the pathological development of BAVM. Full article

Pseudorabies virus (PRV), an alpha herpesvirus, induces significant economic losses to the swine industry and infects multiple kinds of animals. Therefore, it is of great importance to explore anti-PRV compounds. In this study, to explore the anti-PRV compounds, a library of natural compounds was screened through a cell-based ELISA assay, and it was discovered that bufalin, a Na⁺/K⁺-ATPase inhibitor, had a robust inhibitory effect on PRV replication. A time-of-addition experiment and temperature-shift assay showed that bufalin significantly inhibited the entry stage of PRV. NaCl- or KCl-treatment showed that NaCl could enhance the inhibitory effect of bufalin on PRV replication, whereas there was no significant effect under the treatment of KCl. Meanwhile, it was also found that bufalin possessed antiviral activity against other alpha herpesviruses, including human herpes simplex virus type 1 (HSV-1) and chicken Marek’s disease virus (MDV). Finally, it was found that bufalin could decrease the viral load in multiple tissues, and reduce the morbidity and mortality in PRV-challenged BALB/c mice. Overall, our findings demonstrated that bufalin has the potential to be developed as an anti-PRV compound. Full article

In this study, broccoli grown with deep sea water minerals (DSWMs) confirmed anticancer effects in HT-29 colorectal cancer cells and anti-inflammatory effects in C57BL/6 mouse splenocytes. Natural dream cultured broccoli (NB) grown with DSWMs had elevated sodium (Na) and calcium (Ca) levels and enhanced the expression of p53 and p21, both of which are associated with cell cycle arrest in HT-29 colorectal cancer cells. It also decreased the expression of Bax, Bad, Bim, Bak, caspase-9, and caspase-3. In lipopolysaccharide (LPS)-treated C57BL/6 mouse splenocytes, NB produced little nitric oxide (NO). ELISA research indicated that NB decreased IL-1β, IL-6, TNF-α, IFN-γ, and IL-12 expression while increasing NK cell activity. As a result, broccoli cultivated with deep water minerals has better anticancer and anti-inflammatory properties than conventional and organic farming. Full article

Background: Identification of progressive liver disease necessitates the finding of novel non-invasive methods to identify and monitor patients in need of early intervention. Investigating patients with early-liver injury may help identify unique biomarkers. Early-liver injury is characterized by remodeling of the hepatocyte basement membrane (BM) of the extracellular matrix. Thus, we quantified biomarkers targeting two distinct neo-epitopes of the major BM collagen, type IV collagen (PRO-C4 and C4M), in patients spanning the non-alcoholic fatty liver disease (NAFLD) spectrum. Methods: We evaluated PRO-C4 and C4M in a cross-sectional study with 97 patients with NAFLD confirmed on histology. Serological levels of PRO-C4 and C4M were quantified using validated competitive enzyme-linked immunosorbent assays (ELISA). Using the fatty liver inhibition of progression (FLIP) algorithm, we stratified patients into two groups: non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). Biomarker levels were investigated in the two groups in patients stratified by the NAFLD activity score (NAS). In both groups, biomarker measurements were analyzed in relation to histological scorings of steatosis, inflammation, ballooning, and fibrosis. Results: Patients had a body mass index (BMI) of 30.9 ± 5.6 kg/m², age of 53 ± 13 years and a NAS range of 1–8. Upon stratification by FLIP, the NASH patients had higher platelets, ALT, and AST levels than the NAFL group. Both PRO-C4 (p = 0.0125) and C4M (p = 0.003) increased with increasing NAS solely within the NAFL group; however, a large variability was present in the NASH group. Furthermore, both markers were significantly associated with lobular inflammation (p = 0.020 and p = 0.048) and steatosis (p = 0.004 and p = 0.015) in patients with NAFL. Conclusions: This study found that type IV collagen turnover increased with the increase in NAS in patients with NAFL; however, this was not the case in patients with NASH. These findings support the assessments of the BM turnover using biomarkers in patients with early-disease development. These biomarkers may be used to track specific processes involved in the early pathobiology of NAFL. Full article

The innate immune system is the first line of defense of the body composed of anatomical barriers, such as skin and mucosa, as well as effector cells, antimicrobial peptides, soluble mediators, and cell receptors able to detect and destroy viruses and bacteria and to sense trauma and wounds to initiate repair. The human β-defensins belong to a family of antimicrobial small cationic peptides produced by epithelial cells, and show immunomodulatory and pro-healing activities. Laser biostimulation is a therapy widely used to contrast microbial infection and to accelerate wound healing through biological mechanisms that include the creation of oxidative stress. In this paper, we explored laser biostimulation’s ability to modulate the production of two β-defensins, hBD-1 and hBD-2, in human keratinocytes and whether this modulation was, at least in part, oxidative-stress-dependent. Human spontaneously immortalized keratinocytes (HaCaT) were stimulated using laser irradiation at a 980 nm wavelength, setting the power output to 1 W (649.35 mW/cm²) in the continuous mode. Cells were irradiated for 0 (negative control), 5, 10, 25 and 50 s, corresponding to an energy stimulation of 0, 5, 10, 25 and 50 J. Positive control cells were treated with lipopolysaccharide (LPS, 200 ng/mL). After 6 and 24 h of treatment, the cell conditioned medium was collected and analyzed via ELISA assay for the production of hBD-1 and hBD-2. In another set of experiments, HaCaT were pre-incubated for 45 min with antioxidant drugs—vitamin C (Vit. C, 100 µM), sodium azide (NaN₃, 1 mM); ω-nitro-L-arginine methyl ester (L-NAME, 10 mM) and sodium pyruvate (NaPyr, 100 µM)—and then biostimulated for 0 or 50 s. After 6 h, the conditioned medium was collected and used for the ELISA analysis. The hBD-1 and hBD-2 production by HaCaT was significantly increased by single laser biostimulation after 6 h in an energy-dependent fashion compared to basal levels, and both reached production levels induced by LPS. After 24 h, only hBD-2 production induced by laser biostimulation was further increased, while the basal and stimulated hBD-1 levels were comparable. Pre-incubation with antioxidative drugs was able to completely abrogate the laser-induced production of both hBD-1 and hBD-2 after 6 h, with the exception of hBD-1 production in samples stimulated after NaN₃ pre-incubation. A single laser biostimulation induced the oxidative-stress-dependent production of both hBD-1 and hBD-2 in human keratinocytes. In particular, the pro-healing hBD-2 level was almost three times higher than the baseline level and lasted for 24 h. These findings increase our knowledge about the positive effects of laser biostimulation on wound healing. Full article

Pure titanium is limited to be used in biomedical applications due to its lower mechanical strength compared to its alloy counterpart. To enhance its properties and improve medical implants feasibility, advancements in titanium processing technologies are necessary. One such technique is equal-channel angular pressing (ECAP) for its severe plastic deformation (SPD). This study aims to surface modify commercially pure titanium using micro-arc oxidation (MAO) or plasma electrolytic oxidation (PEO) technologies, and mineral solutions containing Ca and P. The composition, metallography, and shape of the changed surface were characterized using X-ray diffraction (XRD), digital optical microscopy (OM), and scanning electron microscope (SEM), respectively. A microhardness test is conducted to assess each sample’s mechanical strength. The weight % of Ca and P in the coating was determined using energy dispersive spectroscopy (EDS), and the corrosion resistance was evaluated through potentiodynamic measurement. The behavior of human dental pulp cell and periodontal cell behavior was also studied through a biomedical experiment over a period of 1-, 3-, and 7-days using culture medium, and the cell death and viability can be inferred with the help of enzyme-linked immunosorbent assay (ELISA) since it can detect proteins or biomarkers secreted by cells undergoing apoptosis or necrosis. This study shows that the mechanical grain refinement method and surface modification might improve the mechanical and biomechanical properties of commercially pure (CP) titanium. According to the results of the corrosion loss measurements, 2PassMAO had the lowest corrosion rate, which is determined to be 0.495 mmpy. The electrode potentials for the 1-pass and 2-pass coated samples are 1.44 V and 1.47 V, respectively. This suggests that the coating is highly effective in reducing the corrosion rate of the metallic CP Ti sample. Changes in the grain size and the presence of a high number of grain boundaries have a significant impact on the corrosion resistance of CP Ti. For ECAPED and surface-modified titanium samples in a 3.6% NaCl electrolyte solution, electrochemical impedance spectroscopy (EIS) properties are similar to Nyquist and Bode plot fitting. In light of ISO 10993-5 guidelines for assessing in vitro cytotoxicity, this study contributes valuable insights into pulp and periodontal cell behavior, focusing specifically on material cytotoxicity, a critical factor determined by a 30% decrease in cell viability. Full article

Dry eye inflammation is a key step in a vicious circle and needs to be better understood in order to break it. The goals of this work were to, first, characterize alarmins and cytokines released by ocular surface cells in the hyperosmolar context and, second, study the role of NFAT5 in this process. Finally, we studied the potential action of these alarmins in ocular surface epithelial cells and macrophages via RAGE pathways. HCE and WKD cell lines were cultured in a NaCl-hyperosmolar medium and the expression of alarmins (S100A4, S100A8, S100A9, and HMGB1), cytokines (IL6, IL8, TNFα, and MCP1), and NFAT5 were assessed using RT-qPCR, ELISA and multiplex, Western blot, immunofluorescence, and luciferase assays. In selected experiments, an inhibitor of RAGE (RAP) or NFAT5 siRNAs were added before the hyperosmolar stimulations. HCE and WKD cells or macrophages were treated with recombinant proteins of alarmins (with or without RAP) and analyzed for cytokine expression and chemotaxis, respectively. Hyperosmolarity induced epithelial cell inflammation depending on cell type. NFAT5, but not RAGE or alarmins, participated in triggering epithelial inflammation. Furthermore, the release of alarmins induced macrophage migration through RAGE. These in vitro results suggest that NFAT5 and RAGE have a role in dry eye inflammation. Full article

Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of At3g50120 (DUF247-1), a member of the DUF247 multigene family containing 28 genes in Arabidopsis, results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction. Furthermore, the polymer sizes of XyGs and GXs are reduced with concomitant increases in short-chain polymers, while those of HGs and mHGs are slightly increased. Complementation using 35S:DUF247-1 partially recovers the XyG and HG content, but not those of GX and HM, suggesting that DUF247-1 is more closely associated with XyGs and HGs. DUF247-1 is expressed throughout Arabidopsis, particularly in vascular and developing tissues, and its disruption affects the expression of other gene members, indicating a regulatory control role within the gene family. Our results demonstrate that DUF247-1 is required for normal cell wall composition and structure and Arabidopsis growth. Full article

Neuroangiostrongyliasis (NAS) is an emerging tropical disease in humans and some animals which is caused by infection with the parasitic nematode Angiostrongylus cantonensis. It is the leading cause of eosinophilic meningitis worldwide. Diagnoses in humans and susceptible animals are generally presumptive and easily confused with other central nervous system disorders. The 31 kDa antigen is currently the only NAS immunodiagnostic assay that has achieved 100% sensitivity. However, little is known about the humoral immune response against the 31 kDa antigen in NAS infections, which would be critical for widespread adoption of this assay. We used the Hawai’i 31 kDa isolate in an indirect ELISA assay to confirm the presence of immunoglobulin IgG, IgM, IgA, and IgE isotypes in six-week post-infection plasma from lab-reared rats infected with 50 live, third-stage, A. cantonensis larvae isolated from a wild Parmarion martensi semi-slug. Our results confirmed the presence of all four isotypes against the Hawaii 31 kDa isolate, with sensitivity ranging from 22–100%. The IgG isotype showed 100% sensitivity in detecting A. cantonensis infection, which validates the use of IgG indirect ELISA with 31 kDa antigen as an effective immunodiagnostic assay for rats six weeks post-infection. Given each isotype may be present at different times during NAS infections, our data provides preliminary information on the humoral immune response to A. cantonensis infection in lab-reared rats and serves as a baseline for future studies. Full article

Cypermethrin (CPM) is the most toxic synthetic pyrethroid that has established neurotoxicity through oxidative stress and neurochemical agitation in experimental rats. The toxic effects are supposed to be mediated by modifying the sodium channels, reducing Na-K ATPase, acetylcholine esterase (AchE), and monoamine oxidase (MAO). The use of curcumin nanoparticles (NC) that have potent antioxidant, anti-inflammatory and antiapoptotic properties with improved bioavailability attenuates neurotoxicity in rat brains. To test this hypothesis, animals were divided into five groups, each having six animals. Group-I control received vehicle only, while Group-II was treated with 50 mg/kg CPM. Group-III and Group-IV received both CPM and NC 2.5 mg/kg and 5 mg/kg, respectively. Group-V received 5 mg of NC alone. The CPM and NC were given by oral route. Afterwards, brain antioxidant status was measured by assessing lipid peroxidation (LPO), 4-HNE, glutathione reduced (GSH), antioxidant enzyme catalase, and superoxide dismutase (SOD) along with neurotoxicity markers Na-K ATPase, AchE, and MAO. Inflammation and apoptosis indices were estimated by ELISA, qRT-PCR, and immunohistochemistry, while morphologic changes were examined by histopathology. Observations from the study confirmed CPM-induced neurotoxicity by altering Na-K ATPase, AchE, and MAO, and by decreasing the activity of antioxidant enzymes and GSH. Oxidative stress marker LPO and the level of inflammatory interleukins IL-6, IL-1β, and TNF-α were notably high, and elevated expressions of Bax, NF-kB, and caspase-3 and -9 were reported in CPM group. However, NC treatment against CPM offers protection by improving antioxidant status and lowering LPO, inflammation, and apoptosis. The neurotoxicity marker’s enzyme successfully attenuated after NC treatment. Therefore, this study supports the administration of NC effectively ameliorated CPM-induced neurotoxicity in experimental rats. Full article