Comparison of VEGF-A levels in women with threatened abortion, early pregnancy loss and uncomplicated healthy pregnancies.

INTRODUCTION: To estimate the possible role of VEGF-A in predicting poor early pregnancy outcomes including threatened abortion and early pregnancy loss. METHODS: We conducted a prospective case-control study with three groups of pregnant women diagnosed with threatened abortion, early pregnancy loss, and uncomplicated healthy pregnancies between 01 March 2023 and 15 March 2023. Maternal serum VEGF-A concentration was measured using the Sandwich-ELISA method in accordance to the commercial kit's instructions. There were 30 patients in each 3 group and the gestational age of the patients was between 6 and 14 weeks. The Kruskal-Wallis test was performed for comparing the median values between the groups. Mann-Whitney U test was conducted for pairwise comparisons. RESULTS: VEGF-A levels were compared between 3 groups and a statistically significant difference was found (p = 0.007). There was a moderately significant correlation between VEGF-A levels and poor early pregnancy outcomes. For poor early pregnancy outcomes, the area under the curve (AUC) was 0.75 (95% CI: 0.64-0.85). The best balance of sensitivity/specificity in ROC curves was 0.60 (63.3% sensitivity, 74.3% specificity). DISCUSSION: In conclusion, this study pointed out the increased VEGF concentrations in pregnant women with threatened miscarriage and early pregnancy loss. VEGF-A may be a potential biomarker for the indication of poor early pregnancy outcomes.

Discovery of Potent Cholinesterase Inhibition-Based Multi-Target-Directed Lead Compounds for Synaptoprotection in Alzheimer's Disease.

Drug development efforts that focused on single targets failed to provide effective treatment for Alzheimer's disease (AD). Therefore, we designed cholinesterase inhibition (ChEI)-based multi-target-directed ligands (MTDLs) to simultaneously target AD-related receptors. We built a library of 70 compounds, sequentially screened for ChEI, and determined σ1R, σ2R, NMDAR-GluN2B binding affinities, and P2X7R antagonistic activities. Nine fulfilled in silico drug-likeness criteria and did not display toxicity in three cell lines. Seven displayed cytoprotective activity in two stress-induced cellular models. Compared to donepezil, six showed equal/better synaptic protection in a zebrafish model of acute amyloidosis-induced synaptic degeneration. Two P2X7R antagonists alleviated the activation state of microglia in vivo. Permeability studies were performed, and four did not inhibit CYP450 3A4, 2D6, and 2C9. Therefore, four ChEI-based lead MTDLs are promising drug candidates for synaptic integrity protection and could serve as disease-modifying AD treatment. Our study also proposes zebrafish as a useful preclinical tool for drug discovery and development.

[The Role of Vitamin D3 on the Immune Responses of Monocytes]. / Vitamin D3'ün Monositlerin Immün Yaniti Üzerindeki Rolü.

The active form of vitamin D (Vit D), 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), is important for cell functions and immunity, as well as its role in bone metabolism. Monocytes/macrophages initiate innate immune response, and is considered to be the cell that first comes into contact with the pathogen. They play effective roles in innate immune and inflammatory responses by intercellular relations and inflammatory mediator secretion. Human THP-1 leukemia cells are frequently used for the in vitro determination of the signal pathways, and the functions of monocyte/macrophages. Nuclear factor-kappa B (NF-κB) are complex networks of signaling pathways that regulate many important cellular behaviors, especially in inflammation, cell death, cell differentiation or proliferation. Midkine (MK) is a cytokine and growth factor that is one of the regulators of inflammatory processes, immune cell functions, proliferation and autoimmunity. The effects of Vit D3 on inflammation and MK secretion in hyperglycemia is still unknown. In this study, it was aimed to determine the dose-dependent effects of Vit D3 on the lipopolysaccharide (LPS) stimulated pro/ anti-inflammatory cytokine, NF-κB and MK responses of THP-1 monocyte cells under normo and hyperglycemic conditions. For this purpose, THP-1 monocyte cells stimulated with LPS (Escherichia coli, 0111, 1 µg/ml) under normoglycemic (glucose 100 mg/dl)/hyperglycemic (glucose 500 mg/dl) conditions, were incubated for 24 hours in the presence and absence of 10-50-100 IU/ml Vit D3. MK, TNF-α, IL-8, IL-10 cytokine levels in the supernatants collected from the wells at the end of the incubation periods, and NF-κB levels in the obtained cell lysates were detected by ELISA method. LPS stimulation induced higher levels of TNF-α, IL-8 and MK responses in hyperglycemic conditions. IL-10 secretions were found to be decreased under hyperglycemia. Vit D3 modulates TNF-α, IL-10 and MK secretions in hyperglycemic conditions. The MK and TNF-α levels were determined to be correlated with NF-κB and IL-10. The results obtained in the study showed that Vit D3 can play a role in immune modulation by regulating NF-κB and cytokine/ chemokine-like molecule MK suppression and proinflammatory/anti-inflammatory cytokine balance. The mechanism of the action of Vit D3 under different conditions should be examined in detail.

Comparison of VEGF-A values between pregnant women with COVID-19 and healthy pregnancies and its association with composite adverse outcomes.

The aim is to compare VEGF-A values between pregnant women with coronavirus disease 2019 (COVID-19) and healthy controls. Furthermore, the association of inflammation parameters, disease severity, and obstetric complications with VEGF-A was investigated. This prospective case-control study was conducted on pregnant women who were admitted to Ankara City Hospital between June 14, 2020 and August 28, 2020. Pregnant women with COVID-19 (n = 95) were compared with a control group of healthy pregnant women (n = 92) with similar clinical and demographic characteristics. Demographic features, clinical characteristics, laboratory test results, VEGF-A values were compared between the groups. A correlation analysis was performed between VEGF-A levels, inflammation parameters, and clinical characteristics of the cases for pregnant women with COVID-19. VEGF-A levels were also compared between patients with composite adverse outcome and patients without any complication in the COVID-19 group. The two groups were similar except for obstetric complications (p > .05). The obstetric complication rate was higher in the COVID-19 group (p =.02). The two groups were comparable in terms of neutrophil to lymphocyte ratio and VEGF-A values. VEGF-A values were slightly different between the trimesters. A negative moderate statistically significant correlation was found between the neutrophil and VEGF-A values (r = -0.231, p =.02). VEGF-A values were similar between patients with and without composite adverse outcomes (p > .05). VEGF-A values were similar between pregnant women with COVID-19 and healthy controls.

[Investigation of cytokine and midkine responses of human THP-1 leukemia cells induced by phorbol-12-Myristate-13-Acetate (PMA) at different concentrations and times]. / Degisik konsantrasyonlarda ve sürelerde Phorbol-12-Myristate-13-Acetate ile uyarilan insan THP-1 lösemi hücrelerinin sitokin ve midkin yanitlarinin arastirilmasi.

Macrophages are accepted as cells that initially contact with the pathogens and initiate the innate immune response. They play effective roles in innate immune and inflammatory responses by intercellular relations and inflammatory mediator secretion. Human THP-1 leukemia cells are frequently used for the in vitro determination of the signal pathways, and the functions of macrophages. Phorbol-12-Myristate-13-Acetate (PMA) is commonly used to induce macrophage differentiation of monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. Midkine acts as a cytokine and growth factor which organizes proliferation, differentiation, survival, adhesion and migration of immune cells. The aim of this study was to observe the differences in the secretion of midkine, TNF-α, IL-10 and IFN-γ of macrophages differentiated from monocytes when stimulated with different doses of PMA for different durations. For this purpose, THP-1 monocytic cells were proliferated by PMA at 24, 48 and 72 hours by using the concentrations of 10 ng/ml, 20 ng/ml, 40 ng/ml and 60 ng/ ml. Midkine, TNF-α, IL-10 and IFN-γ cytokine levels were determined by ELISA in the supernatants of the cells collected at the end of incubation times. PMA stimuli initiated changes that were indicative of differentiation in the cell morphology. Differentiation of cells by PMA induced midkine, TNF-α, IL-10 and IFN-γ secretions in monocytic cells even at the lowest dosage (10 ng/ml). PMA caused cytotoxicity in the cells when the dosages were increased (> 20 ng/ml). THP-1 cells have a basal secretion of midkine and are increased by dosage dependent with PMA stimulation. Midkine secretion has shown changes dependent with dosage and time. A difference was also observed in the cytokine profile of PMA stimulated cells at different doses. The results indicated that the differentiation of THP-1 monocytes to macrophages requires optimization to ensure that this in vitro macrophage model more precisely reflects real in vivo physiologic conditions. As a conclusion the results have shown that a modified PMA differentiation protocol (20 ng/ml and 48 hours incubation) might enhance macrophage differentiation of THP-1 cells without induced cell death (viability 92.2%) and cytokine secretion and midkine responses were the important discriminators of the level of macrophage differentiation.

Proinflammatory biomarkers' level and functional genetic polymorphisms in periprosthetic joint infection.

OBJECTIVE: The aims of this study were 1) to identify the level of inflammatory biomarkers interleukin (IL)-1α, IL-1ß, IL-6, IL-8, IL-17, C-reactive protein (CRP), granulocyte colony-stimulating factor (GCSF), ferritin, and tumor necrosis factor (TNF)-α in serum and synovial fluid samples of patients who underwent revision arthroplasty surgery; 2) to establish the relationship between serum and synovial fluid levels; 3) to determine if any of the 11 genetic polymorphisms of TNFα, IL-1, IL-6, IL-8, IL-17, and GCSF on the encoding genes was associated with periprosthetic joint infection (PJI). METHODS: Synovial fluid and serum was collected from 88 patients who underwent revision arthroplasty surgery. The Musculoskeletal Infection Society definition was used to classify these patients into 2 groups: 36 PJIs and 52 aseptic failures. Synovial fluid and serum samples were tested for 9 biomarkers using a micro enzyme-linked immunosorbent assay. Genetic polymorphisms were evaluated with polymerase chain reaction and restriction endonuclease analysis. RESULTS: Synovial fluid-derived IL-1α, IL-1ß, IL-8, IL-17, CRP, GCSF, TNFα, and serum-derived IL-6, IL-17, ferritin, CRP were found suitable to classify PJI and aseptic failure. In addition, IL-17 and CRP levels demonstrated a positive correlation between synovial fluid and serum. TNFα-238, IL6-174, GCSF3R, and IL1 RN-VNTR genetic polymorphisms occurred more frequently in individuals with septic failure. CONCLUSION: Significant differences between the two groups were observed in the functional polymorphisms of the genes encoding the cytokines investigated. These differences could be interpreted as indicating that there is an association between PJI and genetic polymorphisms. LEVEL OF EVIDENCE: Level III, diagnostic study.

[Investigation of cytokine responses and variations in the expression of beta-defensin-3 of Caco-2 human colon epidermal adenocarsinoma and THP-1 human leukemia monocyte cell lines in response to Toxoplasma gondii under various conditions]. / Caco-2 insan kolon epidermal adenokarsinom ve THP-1 insan lösemi monosit hücre dizilerinin çesitli kosullarda Toxoplasma gondii'ye karsi sitokin yanitlarinin ve insan beta-defensin-3 ekspresyon degisikliklerinin arastirilmasi.

Mononuclear phogocytic cells and epithelial cells are effective during the initiation and regulation of the innate immune response. They have an active role in mucosal immune response both mechanically and by interaction with other cells with cytokine release. Defensins are microbicidal peptides that are expressed in various cells and are thought to be effective in the first line defense against pathogens. IL-12 and IL-10, showing proinflamatory and antiinflamatory activities, respectively, are actors of the cellular immunity and limit the infection of the host without causing immunopathology. The aim of this study was to observe the differences in the release of IL-12 and IL-10 and the expression of human beta-defensin-3 (hBD-3) inCaco-2 (human colon epidermal adenocarcinoma cell) and THP-1 (human leukemia monocytic cell) cell lines cultured alone or in co-culture, by the stimulation of Toxoplasma gondii tachyzoites either in direct contact with the cells or separated by an insert filter from the cells. Twenty-four hours after the addition of RH strain tachyzoites to the cells, the supernatants were collected from the experiment wells, and commercial ELISA kits (Invitrogen) were used according to the manufacturers instructions to measure IL-12 and IL-10 levels. HBD-3 expression of cells collected from the experiment wells afterfour and 24 hourswere analyzed by using real time PCR. For this procedure, complementary c-DNA was obtained (Transcriptor High Fidelity cDNA Synthesis Kit, Roche Diagnostics, Germany)after the extraction of RNA with a commercial kit (High pure RNA isolation kit, Roche Diagnostics, Germany). IL-12 was higher than IL-10 in all experiment wells. IL-12 was induced more in the co-culture wells where Caco-2 and THP-1 cells were challenged together, than the wells in which the cells infected with T.gondii tachyzoites alone. No differences in respect to cytokine response were observed between the cells with which tachyzoites were in contact and the cells which were separated from the parasites with an insert. In hBD-3 experiments, Caco-2 and THP-1 cells interacted in co-culture wells when infected with tachyzoites and displayed a higher level of hBD-3 expression than the condition when they were infected alone. This study showed that, IL-12 release and hBD-3 expression, which play a role in innate immunity, are greater when various antigens of T.gondii interacted with stimulated mononuclear cell and epithelial cells.

[Investigation of the bactericidal effect of urinary epithelial cells against different doses of Escherichia coli]. / Uriner epitel hücrelerinin farkli antijenik miktarlardaki Escherichia coli'ye karsi bakterisidal etkisinin arastirilmasi.

Epithelial cells take part in the stimulation of immune system during the conversion of nonspecific immune response to the adaptive one in the mucosal immune system. Epithelial cells also have critical roles in designating immune homeostasis towards immune regulation or tolerance. Its response type is determined according to the dose and structure of the antigen and the way it is presented. In this study, we aimed to evaluate the response of human urinary epithelial cells to different doses of Escherichia coli K12 by means of their bactericidal effect. Urine was collected from 16 healthy volunteers and urinary epithelial cells were prepared. The cells (effector) were stimulated with bacteria (target) in microplates for one hour with different effector/target cell ratios. At the end of the incubation period, the bactericidal effects were calculated and compared with microorganism controls. Mann-Whitney U test was used for statistical analysis. Urinary epithelial cells which were stimulated by E. coli K12 showed dose dependent bactericidal effect, calculated mean value for bactericidal effects were 60.7% at the 1/100.000 effector/target cell ratio and 11.3% 1/1.000.000 effector/target cell ratio, indicating that the bactericidal effect was dose dependent.

[Investigation of bactericidal effect and cytokine response of the oral epithelial cells against different antigenic doses of Streptococcus pyogenes]. / Insan agiz epitel hücresinin farkli antijenik miktarlardaki Streptococcus pyogenes'e karsi bakterisidal etkisinin ve sitokin yanitinin arastirilmasi.

Epithelial cells (EC) have an important role in the constitution of both innate and acquired immune responses. The aim of this study was to investigate the alterations of bactericidal effects and cytokine production patterns of human oral epithelial cells (OEC) against different doses of Streptococcus pyogenes. For this purpose, OEC have been stimulated with S. pyogenes with an effector/target (E/T) cell ratio of 1/1, 1/100, 1/1.000 and 1/10.000, and bactericidal effects and interleukin (IL)-6, IL-8 and IL-10 levels were detected in the first and sixth hours of incubation. The mean rates of bactericidal effect detected in the first and sixth hours were 38.7% and 54.5%, respectively. The bactericidal effects observed at 1/1 E/T cell ratio in the first hour, and at 1/1 and 1/100 E/T cell ratio in the the sixth hour were found significantly higher then the other cells ratios (p<0.05). Time- and dose-depended differences were detected in the cytokine responses of OEC for different S. pyogenes concentrations. IL-6 levels produced by stimulated OEC were found higher, and IL-8 levels were found lower then the levels which were produced by unstimulated OEC (p<0.05, p<0.001, respectively) in the first hour, while there were no change in IL-10 levels after stimulation with different bacterial concentrations (p>0.05). At the sixth hour there were no differences in the IL-6 levels produced by stimulated and unstimulated cells, while the levels of IL-8 produced by stimulated cells were found lower then the levels produced by unstimulated cells in the E/T cell ratio of 1/100, 1/1000 and 1/10.000 (p<0.05, p<0.01, p<0.01, respectively). Nevertheless IL-10 levels in the E/T cell ratio of 1/100 and 1/1.000 were statistically higher then the levels produced by unstimulated cells (p<0.05, p<0.05). As a result OEC stimulated with S. pyogenes showed dose dependent manner in bactericidal effect and cytokine production. It is suggested that epithelial cells stimulation with different doses of antigen contributed to the immune system activation or tolerance.

[Bactericidal effects of human oral and urinary system epithelial cells against different Escherichia coli strains]. / Insan agiz ve uriner sistem epitel hücrelerinin farkli Escherichia coli suslarina karsi bakterisidal etkisinin arastirilmasi.

The functions of epithelial cells are more than a physical selective barrier for protection from mucosal pathogens. The epithelial response regulated by host-microorganism interaction may change due to histological and physiological differences between sterile and nonsterile sites. In this study we examined the presence of a difference between bactericidal capacities of urinary and oral epithelial cells against different strains of Escherichia coli. Oral epithelial cells were collected from unstimulated saliva and uroepithelial cells were collected from urine of eight healthy volunteers. E. coli K12 and O75 strains and epithelial cells were prepared for the experiment, added to microplates and incubated for one hour. The growth of bacteria was detected by the quantitative colony counting method. Antibacterial effects of the oral and urinary epithelial cells against E. coli K12 and O75 strains were 49.8%, 34.7%, 45.7% and 29.2%, respectively. As a result, it was detected that the antibacterial activity of oral epithelial cells to E. coli K12 and O75 were higher than urinary epithelial cells.

[Herpes simplex virus type 1 in the samples of patients with clinically prediagnosed as herpetic keratitis or keratoconjunctivitis]. / Klinik olarak herpetik keratit veya keratokonjunktivit ön tanisi alan hasta örneklerinde Herpes simpleks tip 1 virusunun arastirilmasi.

The aim of this study was to investigate the presence of Herpes simplex virus type 1, in the specimens from patients clinically prediagnosed as herpetic keratitis or keratoconjunctivitis, by virus isolation and direct immunoperoxidase methods. The samples obtained from a total of 33 patients included ulcerated corneal epithelium scrapings and, swabs from ulcer, corneal epithelium, and lower conjunctivas. For virus isolation, both scraping and swab samples for each patient, were inoculated into monolayered Vero cell lines which have previously cultivated on 24-well plates, and examined for the presence of cytopathic effects typical for HSV-1. At the end of 5-days incubation period, the culture media were discarded, the cells were fixed and stained with peroxidase labeled specific HSV-1 antibodies (direct immunoperoxidase--DIP--method). Simultaneously, the smears were prepared from the samples which were sufficient in amount, and stained with DIP method. As a result, cytopathic effects on cell cultures were observed in 4 (12.1%) of the samples, of which 2 were also positive with DIP method. Following DIP staining of smears prepared from 15 samples, HSV-1 antigen positivity was detected in only 1 of the samples. This sample was negative in cell culture. In contrast, one of the 2 cell culture positive samples yielded negative result in smear examination, while the other could not be examined since the sample was not enough. In conclusion, HSV-1 has been shown as the etiologic agent in 3 (9.1%) of our patients clinically prediagnosed as herpetic keratitis or keratoconjunctivitis, and lesion scrapings were determined as the most appropriate samples for virus isolation.