The objective of our in vitro study was to quantify the biochemical profile where the total polyphenol, flavonoid and phenolic acid content was determined. The antioxidant potential of microgreen extract from Trigonella foenum-graecum L., was measured molybdenum reducing power assay. Specifically, the study assessed parameters such as metabolic activity (AlamarBlueTM assay), membrane integrity (CFDA-AM assay), mitochondrial potential (JC-1 assay), as well as reactive oxygen species generation (NBT assay). In addition, the steroid hormone release in TM3 murine Leydig cells after 12 h and 24 h exposures were quantified by enzyme-linked immunosorbent assay. The gained results indicate the highest value in total flavonoid content (182.59+/-2.13 mg QE) determination, supported by a significant (108.25+/-1.27 mg TE) antioxidant activity. The effects on metabolic activity, cell membrane integrity, and mitochondrial membrane potential were found to be both time- and dose-dependent. Notably, a significant suppression in reactive oxygen species generation was confirmed at 150, 200 and 250 microg/ml after 24 h exposure. In addition, progesterone and testosterone release was stimulated up to 250 microg/ml dose of Trigonella, followed by a decline in both steroid production at 300 and 1000 microg/ml. Our results indicate, that Trigonella at lower experimental doses (up to 250 microg/ml) may positively affect majority of monitored cell parameters in TM3 Leydig cells. Overleaf, increasing experimental doses may negatively affect the intracellular parameters already after 12 h of in vitro exposure. Key words Microgreens, Trigonella foenum-graecum L., Fenugreek, Leydig cells, Male reproduction.
- MeSH
- Antioxidants pharmacology MeSH
- Cell Line MeSH
- Phytochemicals pharmacology MeSH
- Leydig Cells * drug effects metabolism MeSH
- Membrane Potential, Mitochondrial drug effects MeSH
- Mice MeSH
- Reactive Oxygen Species metabolism MeSH
- Plant Extracts * pharmacology MeSH
- Testosterone metabolism MeSH
- Trigonella * chemistry MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Berberine (BBR), a small molecule protoberberine isoquinoline alkaloid, is easy to cross the blood-brain barrier and is a potential drug for neurodegenerative diseases. Here, we explored the role and molecular mechanism of BBR in Alzheimer's disease (AD) progression. Weighted gene co-expression network analysis (WGCNA) was conducted to determine AD pathology-associated gene modules and differentially expressed genes (DEGs) were also identified. GO and KEGG analyses were performed for gene function and signaling pathway annotation. Cell counting kit-8 (CCK8) assay was applied to analyze cell viability. Immunofluorescence (IF) staining assay was conducted to measure the levels of polarization markers. The production of inflammatory cytokines was analyzed by enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) were detected using a ROS detection kit and a MMP Detection Kit (JC-1), respectively. AD pathology-associated DEGs were applied for GO function annotation and KEGG enrichment analysis, and the results uncovered that AD pathology was related to immune and inflammation. Lipopolysaccharide (LPS) exposure induced the M1 phenotype of microglia, and BBR suppressed LPS-induced M1 polarization and induced microglia toward M2 polarization. Through co-culture of microglia and neuronal cells, we found that BBR exerted a neuro-protective role by attenuating the injury of LPS-induced HMC3 on SH-SY5Y cells. Mechanically, BBR switched the M1/M2 phenotypes of microglia by activating PI3K-AKT signaling. In summary, BBR protected neuronal cells from activated microglia-mediated neuro-inflammation by switching the M1/M2 polarization in LPS-induced microglia via activating PI3K-AKT signaling. Key words Alzheimer's Disease, Berberine, Microglia polarization, Neuroinflammation, PI3K-AKT signaling.
- MeSH
- Alzheimer Disease * metabolism drug therapy pathology MeSH
- Berberine * pharmacology therapeutic use MeSH
- Phosphatidylinositol 3-Kinases * metabolism MeSH
- Humans MeSH
- Microglia * drug effects metabolism MeSH
- Mice MeSH
- Neuroprotective Agents * pharmacology MeSH
- Cell Polarity drug effects MeSH
- Proto-Oncogene Proteins c-akt * metabolism MeSH
- Signal Transduction * drug effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Tick-borne encephalitis virus (TBEV) is an emerging pathogen that initially causes flu-like symptoms and can progress to central nervous system (CNS) infections. Tick-borne encephalitis (TBE) is an endemic disease in southern coastal counties with regular human cases, while the causative agent, TBEV, is prevalent in ticks in most of the coastal regions of Norway. This study was aimed to understand TBEV infection status across Norway including both TBE endemic and non-endemic areas. For this, we analyzed a total of 1940 residual serum samples from 19 counties of Norway (as of 2016). The samples were initially screened by ELISA, followed by virus neutralization tests for TBEV confirmation. We found a similar TBEV seroprevalence of 1.7% in TBE endemic and 1.6% in non-endemic areas. Since TBE cases are only reported from endemic regions, our findings suggest a potential subclinical or asymptomatic infection and underdiagnosis in non-endemic areas. Notably, only 43% of the ELISA-positive samples were confirmed by virus neutralization tests indicating that not all ELISA positives are true TBEV infections. Additionally, 137 samples of patients presenting with symptoms of CNS infections from a non-endemic area were included. Of these samples, 11 ELISA-positive samples were analyzed for cross-reactivity among flaviviruses. Cross-reactivity was detected with Dengue virus, West Nile Virus, and non-specific reactions. This underscores the importance of using multiple diagnostic tests to confirm TBEV infections. None of the patients with CNS infection was found to be TBE positive, and in the whole cohort, we found a low TBEV seroprevalence of 0.7%.
- MeSH
- Child MeSH
- Adult MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Encephalitis, Tick-Borne * blood diagnosis epidemiology MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Neutralization Tests MeSH
- Child, Preschool MeSH
- Antibodies, Viral * blood MeSH
- Retrospective Studies MeSH
- Sensitivity and Specificity MeSH
- Seroepidemiologic Studies MeSH
- Encephalitis Viruses, Tick-Borne MeSH
- Cross Reactions MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Norway MeSH
Background: A hallmark of cancer is the presence of an immunosuppressive tumor microenvironment (TME). Immunosuppressive M2 macrophages (MΦs) in the TME facilitate escape from immune surveillance and promote tumor growth; therefore, TME-induced immunosuppression is a potent immunotherapeutic approach to treating cancer. Methods: Cancer cell-secreted proteins were detected by using liquid chromatography-mass spectrometry (LC-MS). Neutralizing antibodies (nAbs) were used to assess which proteins were involved in MΦs polarization and differentiation. The protein-protein interaction was characterized using co-immunoprecipitation and immunofluorescence assays. Cancer-secreted heat shock protein 70 (Hsp70) protein was quantified using an enzyme-linked immunosorbent assay (ELISA). MΦ polarization and tumor growth were assessed in vivo with subcutaneous LLC-GFP tumor models and toll-like receptor 2 (TLR2) knockout mice; in vitro assessments were conducted using TLR2 knockout and both LLC-GFP and LN227 lentiviral-mediated knockdown (KD) cells. Results: Cancer cells released a secreted form of Hsp70 that acted on MΦ TLR2 to upregulate Mer receptor tyrosine kinase (MerTK) and induce MΦ M2 polarization. Hsp70 nAbs led to a reduction in CD14 expression by 75% in THP-1 cells in response to Gli36 EMD-CM. In addition, neutralizing TLR2 nAbs resulted in a 30% and 50% reduction in CD14 expression on THP-1 cells in response to MiaPaCa-2 and Gli36 exosome/microparticle-depleted conditioned media (EMD-CMs), respectively. Hsp70, TLR2, and MerTK formed a protein complex. Tumor growth and intra-tumor M2 MΦs were significantly reduced upon cancer cell Hsp70 knockdown and in TLR2 knockout mice. Conclusions: Cancer-secreted Hsp70 interacts with TLR2, upregulates MerTK on MΦs, and induces immunosuppressive MΦ M2 polarization. This previously unreported action of secreted Hsp70 suggests that disrupting the Hsp70-TLR2-MerTK interaction could serve as a promising immunotherapeutic approach to mitigate TME immunosuppression in solid cancers.
- Publication type
- Journal Article MeSH
BACKGROUND: The neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and serum as a marker of neuronal damage may be a potential biomarker of neuropsychiatric involvement in SLE (NPSLE). METHODS: 80 patients with SLE were included.We obtained paired serum and CSF samples from 48 patients (NPSLE n=32, non-NPSLE n=16) and 31 controls. The serum and CSF levels of NfL were determined using ELISA. RESULTS: Patients with NPSLE demonstrated significantly higher levels of serum NfL compared with the non-NPSLE group (mean 31.68±36.63 pg/mL vs mean 16.75±12.48 pg/mL, respectively, p<0.05) and with controls (mean 10.74±4.36 pg/mL, p<0.01). Notably, CSF NfL concentrations in patients with NPSLE showed an upward trend (mean 1600±2852 pg/mL) in contrast to non-NPSLE patients (mean 393.4±191.9 pg/mL) and controls (mean 509.7±358.5 pg/mL). Furthermore, a positive correlation was observed between serum and CSF NfL levels in patients with NPSLE (R=0.8686, p<0.01). Elevated serum triacylglycerol concentrations, C reactive protein and organ damage were linked to increased serum (p=0.002; p<0.001; p=0.036) and CSF (p=0.008; p=0.007; p<0.001) NfL concentrations. In addition, we established a significant correlation between intrathecal NfL concentrations and interleukin-6 levels in the CSF of patients with NPSLE (R=0.5118, p<0.05). CONCLUSION: The serum NfL levels may be a readily available marker of neuropsychiatric involvement in SLE.
- MeSH
- Biomarkers * blood cerebrospinal fluid MeSH
- Adult MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Interleukin-6 blood cerebrospinal fluid MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Neurofilament Proteins * blood cerebrospinal fluid MeSH
- Cross-Sectional Studies MeSH
- Case-Control Studies MeSH
- Lupus Vasculitis, Central Nervous System * blood cerebrospinal fluid MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
INTRODUCTION: Lyme borreliosis (LB), an infection caused by Borrelia burgdorferi sensu lato (Bbsl), is the most common tick-borne disease in Europe. To further characterize the LB burden in the Czech Republic, we conducted a seroprevalence study and estimated the incidence of symptomatic Bbsl infections. METHODS: Anti-Bbsl IgM and IgG antibodies were detected in sera collected from the adult population in 2011 -2012 by enzyme-linked immunosorbent assay and immunoblot tests at the National Reference Laboratory. The incidence of symptomatic Bbsl infections was estimated from the seroprevalence results and the symptomatic proportion and duration of persistence of anti-Bbsl IgG antibodies in Bbsl-infected individuals. Surveillance under-detection of symptomatic Bbsl infections was estimated by comparing surveillance-reported and seroprevalence-based incidence. RESULTS: Samples from 1996 adults were tested; the median age (range) was 45 (18 -87) years; 1037 (52.0 %) were female. The prevalence (with 95 % confidence interval) of anti-Bbsl IgG, and IgM and/or IgG (IgM/IgG) antibodies was 6.3 % (5.3 -7.5 %), and 9.5 % (8.3 -10.9 %), respectively. The IgM/IgG prevalence was 7.8 % (6.5 -9.2 %) in Bohemia and 15.3 % (12.2 -19.0 %) in Moravia. There were an estimated 30,563 (26,550 -34,962) symptomatic incident Bbsl infections in adults in the Czech Republic in 2012, for an incidence of 352.2 (306.0 -402.9) symptomatic Bbsl infections per 100,000 adults per year. There were an estimated 11 (10 -13) symptomatic Bbsl infections for each surveillance-reported LB case in the Czech Republic in 2012. CONCLUSIONS: There is high incidence of symptomatic Bbsl infections in the Czech Republic, particularly in Moravia. Interventions are needed to address the substantial burden of LB in the Czech Republic.
- MeSH
- Borrelia burgdorferi Group * immunology isolation & purification MeSH
- Adult MeSH
- Enzyme-Linked Immunosorbent Assay * MeSH
- Immunoglobulin G * blood MeSH
- Immunoglobulin M * blood MeSH
- Incidence MeSH
- Middle Aged MeSH
- Humans MeSH
- Lyme Disease * epidemiology microbiology blood MeSH
- Adolescent MeSH
- Young Adult MeSH
- Prevalence MeSH
- Antibodies, Bacterial * blood MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Seroepidemiologic Studies MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
This study aimed to establish a rat model of chronic wounds to observe the effects of hyperbaric oxygen (HBO) on chronic wound repair and pyroptosis and explore the potential role of pyroptosis in the pathogenesis of chronic wounds. Sprague-Dawley (SD) rats were randomly divided into acute wound group (control group), chronic wound group (model group), chronic wound + HBO treatment group (HBO group), and chronic wound + VX-765 (IL-converting enzyme/Caspase-1 inhibitor) treatment group (VX-765 group). After 7 days of respective interventions, the wound healing status was observed, and wound tissue specimens were collected. Hematoxylin and eosin (HE) staining was used to observe the pathological changes in wound tissues. Transmission electron microscopy was used to observe the changes in cellular ultrastructure. Immunofluorescence was used to observe the expression and localization of vascular endothelial growth factor A (VEGF-A) and the N-terminal domain of gasdermin D (GSDMD-N). Western blot was conducted to detect the expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), cysteine-requiring aspartate protease-1 (Caspase-1), VEGF-A, and GSDMD-N proteins in wound tissues. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of NLRP3, Caspase-1, and GSDMD genes. Enzyme-linked immunosorbent assay (ELISA) was performed to observe the expression of the inflammatory cytokines interleukin-1 beta (IL-1beta) and IL-18. The results showed that the HBO group had a faster wound healing rate and better pathology improvement compared to the model group. The expression level of VEGF-A was higher in the HBO group compared to the model group, while the expression levels of NLRP3, Caspase-1, GSDMD, IL-1beta, and IL-18 were lower than those in the model group. HBO can effectively promote the healing of chronic wounds, and the regulation of pyroptosis may be one of its mechanisms of action. Keywords: Hyperbaric oxygen, Pyroptosis, Chronic wounds, Inflammatory.
- MeSH
- Chronic Disease MeSH
- Gasdermins MeSH
- Wound Healing * physiology MeSH
- Hyperbaric Oxygenation * methods MeSH
- Rats MeSH
- Rats, Sprague-Dawley * MeSH
- NLR Family, Pyrin Domain-Containing 3 Protein metabolism MeSH
- Phosphate-Binding Proteins metabolism MeSH
- Pyroptosis * physiology MeSH
- Vascular Endothelial Growth Factor A metabolism genetics MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Non-immune cells, like innate immune cells, can develop a memory-like phenotype in response to priming with microbial compounds or certain metabolites, which enables an enhanced response to a secondary unspecific stimulus. This paper describes a step-by-step protocol for the induction and analysis of trained immunity in human endothelial and smooth muscle cells. We then describe steps for cell culture with cryopreserved vascular cells, subcultivation, and induction of trained immunity. We then provide detailed procedures for downstream analysis using ELISA and qPCR. For complete details on the use and execution of this protocol, please refer to Sohrabi et al. (2020)1 and Shcnack et al.2.
- MeSH
- Cell Culture Techniques MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Endothelial Cells * MeSH
- Humans MeSH
- Myocytes, Smooth Muscle MeSH
- Trained Immunity * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Background and Objectives: Initial diagnosis of brest cancer (BC) is important for fate and prognosis of the diseases profile, we sought to identify the correlation between Midkine (MK) as a new biomarker with cancer antigen (CA)15-3, liver function test, renal function test, blood cells parameters in individuals with invasive ductal carcinoma.Methods: The serum MK and CA15-3 of all subjects were measured by the ELISA technique, Liver enzymes were measured by colourimetric methods and neutrophils, and lymphocytes were measured by an Electrical Impedance Cell Counting method (automated machine).Results: The results of the correlation among serum MK and other parameters in invasive ductal carcinoma of the breast showed a considerable positive correlation among MK and CA15-3 and measured white blood cells. Moreover, there were a weak correlation with Aspartate Aminotransferase (AST) and RBC, while there is no correlation between serum MK and other liver enzymes or blood parameters. Conclusion: The study results of the correlation between serum MK and other parameters in colorectal carcinoma patients show a significant positive correlation of MK with CA15-3 markers in invasive ductal carcinoma of the breast.
- MeSH
- Carcinoma, Ductal, Breast blood MeSH
- Enzyme-Linked Immunosorbent Assay methods MeSH
- Liver Function Tests MeSH
- Clinical Studies as Topic methods MeSH
- Humans MeSH
- Lymphocytes metabolism MeSH
- Midkine * analysis blood MeSH
- Biomarkers, Tumor * blood MeSH
- Breast Neoplasms * diagnosis blood MeSH
- Neutrophils metabolism MeSH
- Kidney Function Tests MeSH
- Check Tag
- Humans MeSH
Equine neosporosis is an intracellular protozoan disease with a global distribution, affecting a diverse range of warm-blooded animals. Neospora caninum Dubey, Carpenter, Speer, Topper et Uggla, 1988 is associated with foetal loss, neurological disease and abortion in equids. No information was available regarding equine N. caninum infection among equids in Iraq. Thus, the aim of this study was to determine the prevalence rate of N. caninum in equines by using a competitive enzyme-linked immunosorbent assay (c-ELISA). A total of 329 blood samples randomly selected from equines, comprising 268 horses and 61 donkeys were examined. The seroprevalence rate of N. caninum was determined as 46% (28/61) for donkeys and 24% (64/268) for horses. The prevalence of N. caninum indicated a significantly higher risk of infection in donkeys compared to horses (P < 0.001). However, the odds of N. caninum infection in draught equids were 8.2 times greater than other equids with a significant difference (P < 0.001). The current study revealed no significant differences in the prevalence of N. caninum across various genders, breeds, clinical statuses, disease histories and among equids that had contact with dogs. While outdoor feeding and mixed (grazing), showed a significant difference (P = 0.003) and (P = 0.75), respectively, in the presence of antibodies against N. caninum compared to indoor feeding (stable). Moreover, the odds of infection in equids with a history of late abortion were 4.8 times higher than those without such a history of abortion (2.20-10.56) with statistical significance (P < 0.001).
- MeSH
- Enzyme-Linked Immunosorbent Assay * veterinary MeSH
- Equidae * parasitology MeSH
- Coccidiosis * veterinary epidemiology parasitology MeSH
- Horses MeSH
- Horse Diseases * epidemiology parasitology MeSH
- Neospora * isolation & purification MeSH
- Prevalence MeSH
- Antibodies, Protozoan blood MeSH
- Seroepidemiologic Studies MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Iraq MeSH
