Immunocytochemical Methods Research Articles

The Effect of Bitter Melon Extract (Momordica Charantia) On CASPASE-9 and Bcl-2 Proteins Expression

Breast cancer has a significant morbidity and death rate worldwide, thus various therapeutic strategies, including the phytopharmaca approach, must be researched further. Bitter melon (Momordica charantia) extract is predicted to have anticancer properties due to its influence on Caspase-9 and BcL2 proteins, which will be shown in this in vitro investigation utilizing MCF-7 cell culture. This study was an in vitro laboratory experimental study with a post-test-only control group design. The doses of Momordica charantia extract used were 125 μg/ml, 500 μg/ml, and 1,000 μg/ml. Caspase-9 and BcL2 protein expression in MCF-7cell culture was measured by immunocytochemistry method, Shapiro Wilk, Levene Test, One Way Anova, and Post Hoc LSD. Administration of Momordica charantia extract at a dose of 500 μg/ml and 1,000 μg/ml significantly increased the average expression of Caspase-9 protein (p = 0.044 and p = 0.004 respectively), but not at a dose of 125 μg/mL (p = 0.125). Administration of bitter melon extract at doses of 125 μg/ml, 500 μg/ml, and 1,000 μg/ml significantly reduced the average BcL2 protein expression (p < 0.001, p < 0.001, and p = 0.001 respectively). Momordica charantia extract has the potential to be employed as an anti-breast cancer agent due to its ability to increase the average expression of Caspase-9 protein while reducing the average expression of BcL2 protein.

Комплексная морфологическая диагностика аденокарциномы легкого. Наблюдение из практики

A clinical case of diagnosis of advanced lung adenocarcinoma in a 72-year-old woman is presented. Thanks to the use of cytological and immunocytochemical methods in the study of the patient's pericardial fluid, a diagnosis of lung adenocarcinoma was established even before the start of treatment. Further examination using fibrobronchoscopy and a trans-bronchial lung biopsy allowed to obtain material for cytological, histological, immunohistochemical studies, the results of which coincided. The patient was diagnosed with lung adenocarcinoma. Del19ex deletion in the EGFR gene was detected using molecular genetic examination of biopsy material. Taking into account the results of morphological and molecular genetic studies, the patient was prescribed targeted therapy with osimertinib.

The neuro-exocrine secretion: A new type of gland in tapeworms?

The phenomenon of exocrine secretion via nervous cells into the host tissue has been discovered in cestodes. In five cestode species of different orders specialized “cup-shaped” free nerve endings located in the tegument have been found. Their ultrastructure is characterized by the presence of a septate junction, a thin support ring and neurosecretory vesicles 90–110 nm in diameter, which are secreted onto the surface of the tegument through a thin pore. The phenomenon is referred to in this article as the neuro-exocrine secretion. We observed a direct relationship between neurosecretory processes in the deep subtegument and free endings in a series of ultrathin sections in two species. The peripheral neurosecretory neurons of species studied are characterized by similar ultrastructural features: size and location; diameter of neurosecretory granules; absence of microtubules and mitochondria in the neurites. The size of neurosecretory granules has been found to decrease from perikaryon towards neurosecretory terminals that lead to the tegument. In two species, we examined the neurosecretion during incubation in the host's blood serum. Depending on the time of incubation we have shown the changes a) in the diameter of the cup-shaped endings, b) in the number of secretory vesicles in the endings; c) changes in number and diameter of neurosecretory vesicles in the processes of neurosecretory neurons in the subtegument. The detected changes differ in D.dendriticus and L.interrupta and, taken together, indirectly confirm the secretory specialization of the cup-shaped endings. Supposed targets for the neurosecretory neurons in the studied cestodes are the following: (a) eccrine frontal gland ducts, especially their terminal regions involved in the release of secretory products; (b) longitudinal and circular muscles in the subtegument region; (c) the basal membrane of the tegument. Besides the discovered secretion vesicles through the cup-shaped terminals, we observed vacuoles derived from the basal membrane of the tegument containing extracellular substances released into the host tissue. Their possible role in the release of neurosecretory substances is discussed. Considering the data acquired via immunocytochemical methods, an assumption about involvement of FMRFamide-like related peptides (FaRPs) in the neuro-exocrine secretion is proposed. Possible functions of the neuro-exocrine secretion are discussed in the context of host-parasite interactions.

Investigation of Neuroprotective Effects of Sulforaphane and Allyl Isothiocyanate in an in vitro Alzheimer’s Disease Model

Background This study aimed to establish an in vitro model of Alzheimer’s Disease (AD) to investigate the neuroprotective activities of allyl isothiocyanate (AITC) and sulforaphane (SFN). Materials and Methods Human neuroblastoma cell lines (SKNAS) were used for the in vitro model of AD after amyloid-β25−35 (Aβ25–35) treatment. Cytotoxicity analysis was performed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Indirect immunocytochemical methods were used to assess the tau protein, alpha-synuclein (α-synuclein), and β-amyloid distribution in the in vitro model of AD and SKNAS cells. Results An in vitro AD model was induced by treatment of SKNAS cells with 1 µM of Aβ25–35 for 48 h. AITC and SFN were applied for 48 h, and the optimal concentrations were determined as 50 µM AITC and 15 µM SFN. Reduced tau immunoreactivity was shown after AITC and SFN administration in SKNAS cells and in vitro models, demonstrating that AITC and SFN prevented amyloid plaque production in the in vitro AD model control group by reducing the β-amyloid level, α-synuclein levels were similar in control and in vitro AD model cells. Reduced α-synuclein levels were observed after SFN treatment in the AD model cells and AITC treatment in the control cells. Conclusion It could be concluded that AITC and SFN are potential components as neuroprotective agents against AD.

Combined Dextran-Graft-Polyacrylamide/Zinc Oxide Nanocarrier for Effective Anticancer Therapy in vitro.

Cancer chemotherapy faces two major challenges - high toxicity of active substances and tumor resistance to drugs. Low toxic nanocarriers in combination with anticancer agents can significantly increase the effectiveness of therapy. Modern advances in nanotechnology make it easy to create materials with the necessary physical and chemical properties. Two hybrid nanosystems of dextran-polyacrylamide/ zinc oxide nanoparticles (D-PAA/ZnO NPs) were synthesized in aqueous solution with zinc sulphate (D-PAA/ZnO NPs (SO42-)) and zinc acetate (D-PAA/ZnO NPs (-OAc)). The light absorption, fluorescence, dynamic light scattering and transmission electron microscopy for nanocomposite characterization were used. MTT, neutral red uptake and scratch assays were selected as fibroblasts cytotoxicity assays. Cytotoxicity was tested in vitro for normal fibroblasts, MAEC, prostate (LNCaP, PC-3, DU-145) and breast (MDA-MB-231, MCF-7) cancer cells lines. Immunocytochemical methods were used for detection of Ki-67, p53, Bcl-2, Bax, e-cadherin, N-cadherin and CD44 expression. Acridine orange was used to detect morphological changes in cells. The radius of ZnO NPs (SO42-) was 1.5 nm and ZnO NPs (-OAc) was 2 nm. The nanosystems were low-toxic to fibroblasts, MAEC. Cells in the last stages of apoptosis with the formation of apoptotic bodies were detected for all investigated cancer cell lines. Proapoptotic proteins expression in cancer cells indicates an apoptotic death. Increased expression of E-cadherin and N-cadherin was registered for cancer cells line LNCaP, PC-3, DU-145 and MCF-7 after 48 h incubation with D-PAA/ZnO NPs (SO42-). The nanosystems were low-toxic to fibroblasts, MAEC. The D-PAA/ZnO NPs nanosystem synthesized using zinc sulphate demonstrates high cytotoxicity due to destruction of various types of cancer cells in vitro and potentially increases adhesion between cells. Thus, our findings indicate the selective cytotoxicity of D-PAA/ZnO NPs against cancer cells and can be potentially used for cancer treatment.

Demineralized freeze-dried bovine bone xenograft enhance osteoblast viability and proliferation for jaw regeneration materials

Objectives The aim of this study was to analyze the viability and proliferation properties of the DFDBBX in MC3T3-E1 cell line Material and Methods Bovine was milled to reach nano sizes of 100-200 nm. We used 2 mg/mL, 4 mg/mL and 8 mg/mL to incubate MC3T3-E1 cells in the following experiment. Immunocytochemistry methods were performed using a Live/Dead cell imaging kit to investigate the viability of MC3T3-E1. To investigate the proliferation of MC3T3-E1, ICC was performed using an anti-Ki67 antibody. Using a confocal laser scanning microscope (CLSM), the cells were observed at LSIH Brawijaya University. Statistical analysis was performed using the one-way analysis of variance (ANOVA) at p < 0.05 Results The highest intensity of living cells was DFDBBX 8 mg/ml on day 7 (739.46 A.U.; p < 0.05), indicating that DFDBBX 8 mg/ml had an effect of increasing cell viability two times compared to control. Ki67 intensity in MC3T3-E1 cell culture was highest on day seven and treated DFDBBX 8 mg/ml (186.04 A.U.; p< 0.05), which indicated the proliferation of MC3T3-E1 cells increased nine times compared to the first day and two times greater than the control group Conclusion DFDBBX have great viability and proliferation properties, which shows potentially as biomaterials to repair bone loss

The Role of Aloe vera in Inhibiting the P53 Protein Expression and Enhancing the HT29 Colon Cancer Cells Proliferation

Background: Aloe vera is one of the most widely recognized herbs considered a valid treatment option for its wound healing and anti-inflammatory properties. However, there is insufficient evidence regarding the effectiveness of its anti-tumor activity on colon cancer. Objectives: In this study, therefore, we investigated the effect of Aloe vera on the proliferation and survival of the HT29 colon cancer cell line. Methods: The effect of processed Aloe vera gel (PAG) on colon carcinogenesis was examined using low glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% serum and 1% penicillin-streptomycin in the HT29 cells. Then, we obtained the expression levels of P53 and BCL2 using the immunocytochemical (ICC) method. To determine the viability of the HT29 cells, we also performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The MTT assay showed that the HT29 cells were viable in 5%, 10%, 15%, and 20% of Aloe vera gel (AVG). However, these cells had a more obvious proliferation in the 10% Aloe vera group than in the control group. In the 10% concentration group, mitotic cells were evident in the center of the colonies. The P53 protein expression decreased, but the BCL2 protein expression remained unchanged in the 10% group compared to the control group. Conclusions: The results, therefore, indicated that in the 10% Aloe vera concentration, the expression levels of P53, known as a tumor suppressor protein in colon cancer, decreased in HT29. Our results also showed that the application of Aloe vera for its anti-tumor activity in colon cancer should be further evaluated.

Genome Instability of Hippocampal and Bone Marrow Cells in Male Mice after the Action of Immobilization and Pheromonal Stressor

Different stressors, affecting the cells of target organs, can lead to genomic instability and even disintegration, which can play a role in the formation of post-stress pathologies. We studied the effect of psycho-emotional stressors (immobilization and mouse stress pheromone – 2,5-dimethylpyrazine) on the DNA integrity of hippocampal and bone marrow cell in male mice of CD1, CBA and C3H strains. Cytogenetic and immunocytochemical methods (alkaline comet assay, ana-telophase analysis of mitotic disturbances and analysis of γH2AX foci) were used. It is shown that the classic mouse stressor (immobilization or restraint), similar as 2,5-dimethylpyrazine, damages the genome of the cells of both organs studied. The destabilization of the cell genome of various organs is considered as an essential stage in the development of a stress response, which is an attempt of the organism to adapt to extreme environmental influences.

Effect of dexamethasone therapy on factors of adhesiveness and coagulation in acute lower limb ischemia

Leukocyte-platelet adhesion during hypoxia, tissue damage, activation of inflammation and coagulation is associated with the expression of ICAM-1 membrane molecules and integrins by blood and tissue cells. At the same time, platelet adhesion receptors determine their adhesion to the endothelium and recruited lymphocytes. The role of platelets in the pathogenesis of ischemic cardiovascular diseases also consists in their ability to modulate both hemostasis and inflammatory reactions, which is accompanied by the secretion of inflammatory mediators and factors that promote the recruitment of leukocytes to tissue damage sites. Purpose of the study: to study the effect of the synthetic glucocorticoid dexamethasone on the expression of adhesion receptors CD18+ and CD54+ on leukocytes, the content of platelets and fibrinogen in the blood of patients with ALLI, the relationship of these indicators with the severity and outcome of the disease.To study the effect of anti-inflammatory therapy, a group of 32 patients treated with dexamethasone was formed; the comparison group was represented by 71 patients with basic therapy, the control group consisted of 15 volunteers. After revascularization, all patients received antiplatelet and anticoagulant therapy. Dexamethasone infusions were carried out in a course of 4 to 6 days after reconstructive surgery. In all patients, the content of C-reactive protein in the blood, the content of platelets and fibrinogen were determined. The number of lymphocytes expressing adhesion molecules ICAM-1 (CD54+) and integrins (CD18+) was counted using the immunocytochemical method. Studies were performed before surgery and on days 1, 3, 7, and 10 after surgery.With exacerbation of ischemia and damage to the endothelium, the accumulation of cytolysis products, the expression of adhesion molecules increases both on endotheliocytes and on inflammatory effector cells – leukocytes and platelets. Adhesion molecules conduct an activation signal inside the cell, which promotes adhesion of leukocytes and platelets to the endothelium, lymphocytic-platelet adhesion, the formation of a parietal thrombus, and possible occlusion of damaged vessels. Increased expression of adhesion molecules is associated with the activation of metabolism, inflammation, coagulation and oxidative stress, stimulates all hematopoietic lineages, including platelets. The level of involvement of cellular reactions in the pathogenesis of the disease affects the effectiveness and duration of treatment, the risk of recurrent thrombosis and death. Anti-inflammatory therapy with dexamethasone contributed to earlier remission, a decrease in the proportion of infectious complications, such as wound suppuration from 10% to 6%, the number of necessary amputations from 32% to 16%, the frequency of deaths from 31% to 6%, and a reduction in hospital stay from 13 days to 10.Inflammation, adhesiveness of effector cells and thrombosis are important factors in the pathogenesis of acute lower limb ischemia. Therapy with dexamethasone helps to reduce the level of systemic inflammatory response, the number of necessary amputations, the number of complications and adverse outcomes in the treatment of ALLI, and reduce the length of stay in the hospital.

Genetically Engineered Artificial Microvesicles Carrying Nerve Growth Factor Restrains the Progression of Autoimmune Encephalomyelitis in an Experimental Mouse Model.

Multiple sclerosis (MS) is an incurable, progressive chronic autoimmune demyelinating disease. Therapy for MS is based on slowing down the processes of neurodegeneration and suppressing the immune system of patients. MS is accompanied by inflammation, axon-degeneration and neurogliosis in the central nervous system. One of the directions for a new effective treatment for MS is cellular, subcellular, as well as gene therapy. We investigated the therapeutic potential of adipose mesenchymal stem cell (ADMSC) derived, cytochalasin B induced artificial microvesicles (MVs) expressing nerve growth factor (NGF) on a mouse model of multiple sclerosis experimental autoimmune encephalomyelitis (EAE). These ADMSC-MVs-NGF were tested using histological, immunocytochemical and molecular genetic methods after being injected into the tail vein of animals on the 14th and 21st days post EAE induction. ADMSC-MVs-NGF contained the target protein inside the cytoplasm. Their injection into the caudal vein led to a significant decrease in neurogliosis at the 14th and 21st days post EAE induction. Artificial ADMSC-MVs-NGF stimulate axon regeneration and can modulate gliosis in the EAE model.

Generation of induced pluripotent stem cell lines from helper and cytotoxic T cells of healthy individuals

T lymphocytes are the most abundant mononuclear blood cells and can serve as a source for generating induced pluripotent stem cells (iPSCs) for disease modeling or drug development. Here, we report the derivation of two iPSC lines from CD4+ helper T cells and CD8+ cytolytic T cells, respectively. The reprogramming was performed using Sendai virus encoding Klf-4, c-Myc, Oct-4 and Sox-2. Both iPSC lines displayed typical embryonic stem cell-like morphology and normal karyotype. Pluripotency was confirmed using immunocytochemistry methods and teratoma formation assay.

Irregularities in Meiotic Prophase I as Prerequisites for Reproductive Isolation in Experimental Hybrids Carrying Robertsonian Translocations

The basic causes of postzygotic isolation can be elucidated if gametogenesis is studied, which is a drastically different process in males and females. As a step toward clarifying this problem, we obtained an experimental inbred lineage of the eastern mole vole Ellobius tancrei, whose founder animals were animals with identical diploid numbers 2n = 50 but with different Robertsonian translocations (Rb), namely 2Rb4.12 and 2Rb9.13 in the female and 2Rb.2.18 and 2Rb5.9 in the male. Here, we analyzed strictly inbred hybrids (F1, fertile and F10, sterile) using immunocytochemical methods in order to study spermatocytes during the meiotic prophase I. Previously, the presence of trivalents was assumed to have no significant effect on spermatogenesis and fertility in hybrids, but we demonstrated that spermatogenesis might be disturbed due to the cumulative effects of the retarded synapses of Rb bivalents as well as trivalents and their associations with XX sex bivalents. Alterations in the number of gametes due to the described processes led to a decrease in reproductive capacity up to sterility and can be examined as a mechanism for reproductive isolation, thus starting speciation.

CDCP1 (CUB domain containing protein 1) is a potential urine-based biomarker in the diagnosis of low-grade urothelial carcinoma.

Urine-based cytology is non-invasive and widely used for clinical diagnosis of urothelial carcinoma (UC), but its sensitivity is less than 40% for low-grade UC detection. As such, there is a need for new diagnostic and prognostic biomarkers of UC. CUB domain containing protein 1 (CDCP1) is a type I transmembrane glycoprotein highly expressed in various cancers. Using tissue array analysis, we demonstrated that CDCP1 expression in UC patients (n = 133), especially in those with low-grade UC, was significantly higher than in 16 normal persons. In addition, CDCP1 expression in urinary UC cells could also be detected by using immunocytochemistry method (n = 11). Furthermore, in 5637-CD cells, overexpression of CDCP1 affected the expression of epithelial mesenchymal transition-related markers and increased matrix metalloproteinase 2 expression and migration ability. Conversely, the knockdown of CDCP1 in T24 cells had the opposite effects. Using specific inhibitors, we demonstrated the involvement of c-Src/PKCδ signaling in the CDCP1-regulated migration of UC. In conclusion, our data suggest that CDCP1 contributes to the malignant progression of UC and may have the potential as a urine-based biomarker for detecting low-grade UC. However, a cohort study needs to be conducted.

Lossless Immunocytochemistry Based on Large-Scale Porous Hydrogel Pellicle for Accurate Rare Cell Analysis.

Rare cells, such as circulating tumor cells or circulating fetal cells, provide important information for the diagnosis and prognosis of cancer and prenatal diagnosis. Since undercounting only a few cells can lead to significant misdiagnosis and incorrect decisions in subsequent treatment, it is crucial to minimize cell loss, particularly for rare cells. Moreover, the morphological and genetic information on cells should be preserved as intact as possible for downstream analysis. The conventional immunocytochemistry (ICC), however, fails to meet these requirements, causing unexpected cell loss and deformation of the cell organelles which may mislead the classification of benign and malignant cells. In this study, a novel ICC technique for preparing lossless cellular specimens was developed to improve the diagnostic accuracy of rare cell analysis and analyze intact cellular morphology. To this end, a robust and reproducible porous hydrogel pellicle was developed. This hydrogel encapsulates cells to minimize cell loss from the repeated exchange of reagents and prevent cell deformation. The soft hydrogel pellicle allows stable and intact cell picking for further downstream analysis, which is difficult with conventional ICC methods that permanently immobilize cells. The lossless ICC platform will pave the way for robust and precise rare cell analysis toward clinical practice.

Determination of Autophagy in Human Cervicovaginal Smears by Cytological and İmmunocytochemical Methods.

Autophagy is a catabolic process whereby organelles and long-lived proteins are recycled through lysosomes to maintain cellular homeostasis. This process is being widely studied using culture techniques and animal models; however, cervicovaginal smears have not been used to detect autophagy. Our study aims to detect and evaluate autophagy in normal, malignant, infectious, and atypical cells in cervicovaginal smears by using cytological and immunocytochemical methods. Papanicolaou-stained 200 cervicovaginal smears were examined and 55 of 200 (27.5%) smears containing negative for intraepithelial lesion or malignancy (NILM) with identifiable infections and/or reactive/reparative changes (INF); briefly, NILM-INF (n = 31, 56.4%), atypical (n = 4, 7.3%), and malignant cells (n = 20, 36.3%) were evaluated as a study group. One hundred forty-five of 200 (72.5%) normal smears were accepted as the NILM without any identifiable infections (control group). The autophagy marker protein Microtubule-associated protein 1 light chain 3 A (MAP1LC3A) was used for immunocytochemical examination. The staining intensity of the MAP1LC3A protein and autophagy positivity were lower in the malignant cells; however, they were higher in the NILM-INF and atypical cells. A statistically significant correlation between the malignant and normal cells was obtained for the autophagy positivity (P = 0.012). In view of the staining intensity of MAP1LC3A protein by the H-score method, a significant correlation was found between the NILM-INF and the normal cells (P = 0.015). Autophagy was detected in various cervicovaginal smears for the first time in this study. Our findings indicate that an autophagy process is essential in infectious cells as well as in the transformation of atypical cells into malignant cells in carcinogenesis.

Reproducibility of cytological diagnoses in evaluating liquid cervical smears and immunocytochemical co-expression of p16/Ki-67 using manual and automatic methods

Aim. To assess the reproducibility of cytological diagnoses in evaluating liquid cervical smears and immunocytochemical co-expression of p16/Ki-67 using manual and automatic methods.
 Materials and methods. Cytological smears prepared using the liquid cytology method on the Becton Dickinson device (SurePath technology) were studied. An immunocytochemical study was carried out using a Ventana BenchMark Ultra automatic immunostainer with a commercial CINtec kit (determination of p16/Ki-67 co-expression). In total, 100 cytological slides (50 pairs of Pap-smears and immunocytochemical slides) were studied. The diagnostic kit was reviewed by five cytologists independently, and the cytologic slides were evaluated using four categories according to the Bethesda system (2014) and according to the categories of normal/abnormal. The co-expression of p16/Ki-67 was assessed per the manufacturer's recommendations (Roche) using the manual method (light microscope) and the automatic Vision Cyto Pap ICC system. Statistical processing of the results was performed using the SPSS software package version 26.0.0.0 with the calculation of the reproducibility indices of Cohen's kappa and Fleiss' kappa.
 Results. When assessing the reproducibility of four categories of cytological diagnoses according to the Bethesda system (2014), Cohen's kappa was 0.0480.265. The overall Fleiss' kappa between all cytologists was 0.103. When only two categories (normal/abnormal) were used, the reproducibility ranged from 0.058 to 0.377. When assessing the co-expression of p16 and Ki-67, Cohen's kappa reproducibility was from 0.196 to 0.574, while the overall Fleiss' kappa was 0.407. When comparing the evaluation results of each of the cytologists with the neural network, Cohen's kappa reproducibility ranged from 0.103 to 0.436.
 Conclusion. The reproducibility of cytological diagnoses according to the Bethesda system (2014) and two categories (normal/abnormal) based on the Pap smear study was low. Such results are primarily due to a large number of abnormal smears in the study. The immunocytochemical method has diagnosis reproducibility three times higher, indicating the need to measure the co-expression of p16 and Ki-67 to increase the sensitivity and specificity of the cytological method. Similar reproducibility when comparing the manual and automatic evaluation of the "double label" suggests that the neural network algorithm can currently help in decision support rather than replace the cytologist at the diagnostic stage.

Isolation of Cells and Exosomes from Glioblastoma Tissue to Investigate the Effects of Ascorbic Acid on the c-Myc, HIF-1α, and Lnc-SNHG16 Genes.

Glioblastoma multiforme (GBM) is incurable with routine treatments. Ascorbic acid (Asc) has antioxidant and anti-cancer properties. However, its specific anti-cancer mechanisms are only partially understood. In this study, the effect of Asc on the c-Myc, HIF-1α, and lnc-SNHG16 genes in GBM cells and their exosomes was investigated. Cells isolated from the tissue were characterized by the immunocytochemistry method (GFAP+). The cell-doubling time was determined, and FBS-free medium supplemented with Asc (5 mM) was added to the cells. The extracted exosomes in the cell culture medium were scanned by electron microscopy, Zetasizer, and BCA assay. The expression of lnc-SNHG16 in the exosomes and c-Myc and HIF-1α in the treated and control cells was evaluated by real-time PCR. The interactions between Asc and the c-Myc and HIF-1α proteins were studied using the molecular docking method. The cells showed 90-100% GFAP+ in passage 4, with a cell-doubling time of 4.8 days. Exosomal vesicles measuring 98.25-105.9 were observed. Zetasizer results showed a sharp pick at 90 nm. Protein quantitation showed 3.812 µg/ml protein in the exosomes. Lnc-SNHG16 expression was reduced (P = 0.041), and c-Myc was upregulated (P = 0.002). The expression of HIF-1α was not significant in the treated cells. Also, Asc was able to interact and affect c-Myc and HIF-1α. Asc exerts its effect by reducing lnc-SNHG16 expression in exosomes, upregulating c-Myc in GBM cells, and interacting with HIF-1α and c-Myc. Further research is necessary to achieve a full understanding of these findings.

Microscopic Manifestations of Maternal Diabetes in Placental Structure

The human placenta is crucial for prenatal development and good pregnancy outcome. Maternal diabetic disorder influences normal intrauterine development of individual and expresses itself in the placenta. Due to the progress in diagnostic methods and methods of metabolic compensation the macroscopic signs associated with maternal diabetes, e.g., placentomegaly, occur nowadays rarely. In the microscopic picture of placenta there are no structural differences specific for maternal diabetes. However, the application of quantitative morphological methods has revealed some differences of normal and diabetic placenta. Stereological studies have shown significantly higher total volume of peripheral villi that distorts shapes and dimensions of pores in the intervillous space, and larger surface areas of peripheral villi and villous capillaries. Methods of confocal microscopy and 3D reconstruction gave the evidence that the fetus reacts on the hypoxia in maternal diabetic environment by enhanced angiogenesis and branching of villous capillaries. The enhanced tissue volumes are conditioned by higher mitotic activity. However, it decreases the proliferative potential of cells taking part in the enlargement of key structures for maternofetal transport at the end of pregnancy. Syncytiotrophoblast produces many factors playing important roles in maternal and fetal regulation and in the placenta itself. Quantitative methods of catalytic histochemistry and immunocytochemistry pointed at the role of maternal diabetes in decreased synthesis of some of those factors (e.g., alkaline phosphatase, SP1 glycoprotein). The presented data show that the quantitative morphological methods contribute to better understanding of the influence of maternal diabetes on fetoplacental unit.

Ellagic Acid Inhibits TGFβ1/Smad-Induced Renal Fibrosis in Diabetic Kidney Injury

Aim: Free radical formation increases due to hyperglycemia occurring in the pathogenesis of diabetes mellitus (DM), and as a result, oxidative stress occurs. Hyperglycemia-mediated oxidative stress plays an important role in the pathogenesis of diabetic nephropathy. The antihyperglycemic, antioxidative, anti-apoptotic, and anti-inflammatory effects of ellagic acid (EA) have been demonstrated by many studies. In this study, it was aimed to demonstrate the antifibrotic effect of EA on TGFβ1/Smad signaling in rats with streptozotocin induced diabetic nephropathy. Material and Methods: A total of 24 male Sprague Dawley rats, weighing 200-250 g, were used in this study. The animals were divided into four groups as control, EA, DM, and DM+EA. The kidney tissues were used for histological and immunohistochemical procedures. While the collagen density in kidney tissues was revealed by Masson's trichrome staining, the expression levels of fibrotic markers TGFβ1, p-Smad3, and αSMA were determined by the immunocytochemical method. Results: It was shown that the collagen density in the renal tissue of the DM group increased significantly in the intertubular area, while the collagen density in the EA-treated DM group was statistically significantly decreased. When TGFβ1, p-Smad3, and αSMA immunopositivity in kidney tissue sections of all groups were evaluated, the highest staining intensity was in the DM group, while the intensity of staining was close to the control group in the treatment group. It was observed that αSMA, TGFβ1, and p-Smad3 protein expression were down-regulated with EA treatment. Conclusion: EA reduced fibrosis in diabetic nephropathy by returning profibrotic parameters to normal levels.

Novel Self-Nanoemulsifying Drug Delivery System (SNEDDS) of Single Garlic Extract: Bio-accessibility, Cytotoxicity, and Anti-Inflammatory Studies

Single garlic has many health benefits in which the active compound content five times higher than regular garlic. However, the utilization of the active compound in single garlic was not optimal due to its lipophilic characteristics, volatility, and low stability in gastrointestinal fluids. Self-Nanoemulsifying Drug Delivery System (SNEDDS) has the potential to control the release rate of the active compounds and lead to increase bioavailability in the digestive tract. The study aimed to formulate SNEDDS for single garlic extract (SGE) to increase its bioaccessibility and anti-inflammatory potentials. SNEDDS-SGE was formulated with Tween-80, PEG-400, canola oil, and SGE. SNEDDS-SGE was characterized by response tests including emulsification time, emulsion pH, and transmittance; morphology and droplet size of SNEDDS using TEM; and SGE stability in SNEDDS was evaluated by in vitro gastrointestinal simulation. The cytotoxicity of SNEDDS-SGE was tested using MTT assay, and its anti-inflammatory potential on IL-1ß expression was assessed by the immunocytochemistry (ICC) method on TIG-1 cells induced by methylglyoxal. Response test data and characterization of SNEDDS-SGE were analyzed descriptively, bioaccessibility data were analyzed by T-test, cytotoxicity test by linear regression, IL-1β and C/EBPα expression data by One-way ANOVA. The results showed that SNEDDS-SGE had a fast emulsification time of 41.913 seconds, a stable pH of 7.56, and a high transmittance percentage of 98.027%. The SNEDDS-SGE droplet has a uniform shape with a size of less than 100 nm. SNEDDS-SGE was able to increase the bioaccessibility of SGE by 89.34% for allicin and 89.31% for alliin, also non-toxic at concentrations below 1000 µg/ml. SNEDDS was able to significantly increase the effect of SGE in suppressing the expression of IL-1ß at all concentrations of SNEDDS-SGE 62.5 µg/ml, 125 µg/ml, and 250 µg/ml with the lowest decreased expression of 44.82 ± 4.30 AU. Our study suggested that SNEDDS has the potential to enhance the biological effects of SGE, as well as being a promising anti-inflammatory agent.