Significant Decrease In Cell Count Research Articles

Abstract TP244: Formation Of Neutrophil Extracellular Traps During In Vitro Stroke Facilitates Endothelial Cytotoxicity

Background: Stroke is one of the leading causes of death and disability. Some aspects of inflammation are known to be detrimental and arresting these aspects may improve stroke outcomes. Neutrophils are critical neuroinflammatory mediators and among the first immune cells to respond to the onset of stroke. As the stroke evolves, neutrophils may expel their intracellular contents, referred to as Neutrophil Extracellular Traps (NETs), in a process called NETosis. NETs increase microvascular occlusion and lead to further progression of stroke. However, the interaction of NETs with the cerebral microvascular endothelial cells and the effect of this interaction on stroke outcomes is poorly understood. This project aims to investigate this relationship. Methods: Leukocytes and plasma from C57BL/6 wildtype mice were isolated from whole blood. The leukocytes were then plated in 24-well plates and divided into control and Oxygen Glucose Deprivation (OGD) simulating a stroke environment in vitro. Thereafter, leukocytes were subjected to immunofluorescent staining for neutrophils (Ly6G), and the NETosis markers citrullinated histones (CitH3) and dsDNA. Media from both groups was assayed for Neutrophil Elastase, IL-6, TNF-α, and lactate dehydrogenase (LDH). The remaining media was used to treat primary microvascular endothelial cells to assess inflammation and cell death. Results: Ly6G antibody staining verified that most of the leukocytes plated were neutrophils. The CitH3 and dsDNA immunostaining confirmed induction of NETosis in the OGD group. We also found a significant increase in IL-6 and LDH levels suggesting significant inflammation and cell death in OGD treated leukocytes. Exposure of endothelial cells with NETs enriched media obtained from OGD exposed leukocytes resulted in significant decrease in cell count and increase in IL-6 and LDH levels, suggesting that NETs promoted inflammation and death in the endothelial cells. Nuclear staining of the endothelial cells also exhibited significant decrease in cell count in cohorts treated with the OGD exposed leukocyte media. Conclusion: These pilot data suggest that NETosis may contribute to the cytotoxicity of cerebral endothelial cells during stroke and serve as a potential therapeutic target.

Temozolomide modulates the expression of miRNAs in colorectal cancer

Cancer is the second leading cause of death globally, where nearly 1 in 6 deaths is due to cancer, with 70% of all deaths from cancer occur in low- and middle-income countries. The overall lifetime risk of developing colorectal cancer is 1 in 22 in men and 1 in 24 in women. In this work, we aimed to evaluate the role of temozolomide (TMZ) in controlling colon cancer cells (CRC) via regulating the miRnome. For this purpose, CRC cells (CaCo-2) were treated with 50 µM of TMZ for 48 h. Cell count using trypan test and cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were carried out, and the obtained results indicated a significant decrease in cell count (p = 0.029), and in the cell viability (p = 0.0019). Cell cycle analysis was performed using flow cytometer, and results showed that TMZ arrested CRC cells at G2/M phase. A total of 84 miRNAs were profiled using real time PCR, and the results indicated that TMZ treatment upregulated 15 of 84 miRNAs panel profiled and downregulated the rest. The TMZ-upregulated/downregulated miRNAs were predicted to interact with many epigenetic-related proteins i.e., DNMTs, EZH2, and SUV31H1. This study shed some light on the role of TMZ in regulating the miRnome of CRC and hence in different types of cancers.

LncRNA- RP11-156p1.3, novel diagnostic and therapeutic targeting via CRISPR/Cas9 editing in hepatocellular carcinoma

We aim to characterize the expression of RNA panel in HCC. We assessed the expression of HCC-associated mRNA, miRNA and lncRNA network by real time PCR in sera and tissue samples. In a proof-of-principle approach, CRISPR cas9 mediated knock out for lncRNA- RP11-156p1.3 was performed in HEPG2 cell line to validate the role of the chosen RNA in HCC pathogenesis. The differential expression of RFTN1 mRNA, lncRNA- RP11-156p1.3 and miRNA-4764-5p was statistically different among the studied groups. After CRISPR cas9 mediated knockout of lncRNA- RP11-156p1.3 in HEPG2 cells, there was significant decrease in cell count and viability with reversal of the expression of the chosen RNAs. The chosen RNAs play a significant role in HCC pathogenesis and may be potential diagnostic and therapeutic targets.

Longitudinal study on clinical and microbial analysis of periodontal status in pregnancy.

This study was aimed to provide a longitudinal overview of the subgingival bacterial microbiome using fluorescence in situ hybridization (FISH) technique, in women in the second trimester of pregnancy (between 14 and 24 weeks), and 48 h and 8 weeks postpartum. Of 31 women evaluated during pregnancy, 24 returned for the 48-h and 18 for their 8-week exams postpartum. Probing depth (PD), bleeding on probing, clinical attachment level, and presence of calculus were recorded. Subgingival plaque samples were collected, and FISH was used to identify the numbers of eight periodontal pathogens. Friedman test was used to compare differences between follow-up examinations, followed by a multiple comparison test for a post hoc pairwise comparison. Clinically, a significantly greater number of teeth with PD = 4-5 mm were found during pregnancy than on postpartum examinations. Microbial analysis showed a statistically significant decrease in cell count over the study period for Prevotella nigrescens. P. intermedia, Campylobacter rectus, and Porphyromonas gingivalis also decrease, although not significantly, and Aggregatibacter actinomycetemcomitans increased. No significant changes were found for Fusobacterium nucleatum, Treponema denticola, or Tannerella forsythia. Our data demonstrate a change in the subgingival microbiota during pregnancy, at least for P. nigrescens.

Mitotic disruption and reduced clonogenicity of pancreatic cancer cells in vitro and in vivo by tumor treating fields

ObjectivesTumor Treating Fields (TTFields) are a non-invasive cancer treatment modality approved for the treatment of patients with recurrent glioblastoma. The present study determined the efficacy and mechanism of action of TTFields in preclinical models of pancreatic cancer. MethodsThe effect of TTFields in vitro was assessed using cell counts, clonogenic assays, cell cycle analysis and analysis of mitotic figures. The effect in vivo effect was studied in the PC1-0 hamster pancreatic cancer model. ResultsApplication of TTFields in vitro showed a significant decrease in cell count, an increase in cell volume and reduced clonogenicity. Further analysis demonstrated significant increase in the number of abnormal mitotic figures, as well as a decrease in G2-M cell population. In hamsters with orthotopic pancreatic tumors, TTFields significantly reduced tumor volume accompanied by an increase in the frequency of abnormal mitotic events. TTFields efficacy was enhanced both in vitro and in vivo when combined with chemotherapy. ConclusionsThese results provide the first evidence that TTFields serve as an effective antimitotic treatment in preclinical pancreatic cancer models and have a long term negative effect on cancer cell survival. These results make TTFields an attractive candidate for testing in the treatment of patients with pancreatic cancer.

Antiproliferative activity of Tinospora cordifolia determined by cell count and trypan blue dye exclusion method in mcf-7 cells

An in-vitro study was performed in mammary tumor cell line MCF-7 to find out the antiproliferative activity of aqueous and hydro-alcoholic extracts of Guduchi Tinospora cordifolia , each at three different doses viz., 200µg/ml, 400µg/ml and 600µg/ml. Their effects on the proliferation of cells were analyzed by cell count assay and cell viability was detected by using trypan blue dye exclusion method. Both of the extracts produced significant decrease in cell count and cell viability, with maximum effect being noticed at the dose level of 600µg/ml. This result suggest that aqueous and hydro-alcoholic extracts of Tinospora cordifolia could reduce cell count and cell viability in MCF-7 cell line and act as effective anti-proliferative agent in mammary tumor.

Evaluation of Light-Emitting Diode (LED-660 Nm) Application over Primary Osteoblast-Like Cells on Titanium Surfaces: An In Vitro Study

The goal of this study was to evaluate the behavior of neonatal rat calvarial osteoblast-like cells cultured on different implant surfaces and exposed once or three times to a 660-nm light-emitting diode (LED). An LED with a 660-nm wavelength was applied once or three times to cultured cells on standard and modified sandblasted acid-etched surfaces (SLA and SLActive; Straumann, Basel, Switzerland). To analyze the effect of the LED on cell proliferation, numbers, and viability, cells were cultured on titanium discs, and measurements were taken after 72 h. Cell proliferation rates were assessed using a bromodeoxyuridine immunohistochemical technique. Cell morphologies were evaluated by scanning electron microscopy (SEM). Osteoblast-like cells proliferated on all tested surfaces, with differences among groups in cell counts and DNA synthesis values. The application of one LED treatment caused a significant increase in cell count in the SLActive group in comparison with the SLA group (p = 0.001), whereas the application of three LED treatments caused a significant decrease in cell count in the SLA group compared with the SLActive group (p < 0.001). After 72 h, the number of cells was highest in the SLActive group exposed once to the LED. One LED application in the SLActive group resulted in significantly increased cell numbers. However, these findings were not exactly compatible with the SEM findings, which demonstrated fewer cells and weak attachments between cells and to the surface. Thus, further studies using different LED application times are needed to clarify the reason for the increased number of cells that are apparently incapable of attaching to the titanium surfaces after 72 h.

Pressure Effects on the Growth of Human Scar Fibroblasts

Although pressure therapy is the mainstay of treatment for hypertrophic scars, its actual mechanism remains unknown. An in vitro study was designed to investigate the effects of positive pressure on the growth of human scar-derived fibroblasts through its transforming growth factor beta1 (TGF-beta1) secretion. A pneumatic pressure system connecting to a cell culture chamber was designed. Six-well cultured plates with fibroblasts implanted were treated with different pressure settings. Cells were treated with constant pressure 20 mm Hg above atmosphere pressure (group A n = 18) or with 40 mm Hg above atmosphere pressure (group B n = 18) daily for nine successive days. Cells without pressure were treated as the control study (group C n = 6). Each experimental group was divided into daily pressure applied at 24 hours (n = 6), 18 hours (n = 6), and 12 hours (n = 6). Cell counting was performed on the 2nd, 4th, 7th, 9th, 11th, and 14th day after implantation. On day 4, the concentration of transforming growth factor beta1 was measured, and cell doubling time was calculated. Compared with the control group, there was a significant decrease in cell count and the concentration in the 18-hour and 24-hour 20 mm Hg or 40 mm Hg pressure treated group. The cell doubling time was significantly increased in the 24-hour 20 mm Hg or 40 mm Hg pressure treated groups, and the 18-hour 40 mm Hg pressure treated group. (P < .05) Pressure inhibits the growth and activity of human scar fibroblasts, and a higher pressure application can shorten the daily application period. There should be an optimal pressure level corresponding to a daily application period to achieve the most effective results on pressure therapy for scars.

The influence of viscoelastic substances on the corneal endothelial cell population during cataract surgery: a prospective study of cohesive and dispersive viscoelastics

To compare the ability of cohesive and dispersive ophthalmic viscoelastic devices (OVDs) to protect the corneal endothelium following in-the-bag phacoemulsification with implantation of a foldable posterior chamber intraocular lens (IOL). In a prospective single-masked randomized study, 60 eyes of 60 cataract patients were assigned to three groups of 20 patients each, according to which OVD was used: Celoftal, Vitrax or Healon. The corneal response to surgery was evaluated by measuring the endothelial cell loss, the variation in mean cell area of the endothelial cells (CV), the frequency of hexagonal cells, and the central corneal thickness. Data were recorded preoperatively and 3 months postoperatively. Preoperatively, no significant difference was observed in cell count, CV, hexagonal pattern or pachymetry among groups. Postoperatively, all three groups had a significant decrease in cell count, but the decrease was significantly less in the Vitrax group (6.97%) than in the Celoftal (18.03%) and Healon groups (18.46%). No changes in CV, hexagonality or corneal thickness were observed within any of the three groups or among the groups. There was an equal and significant increase in visual acuity. Phacoemulsification with implantation of a posterior chamber lens is known to affect the density and morphology of corneal endothelial cells. Viscoelastics facilitate cataract surgery and protect the corneal endothelium during the procedure. Choosing a dispersive hyaluronate OVD during the phaco procedure may allow for protection of the endothelial cells while suppressing the formation of free radicals. This may be the reason for the superior protective effect on the corneal endothelial cells of Vitrax compared with Celoftal and Healon.

Effects of Dexamethasone, All-Trans Retinoic Acid, Vitamin D3 and Interferon-α on FO Myeloma Cells

Background: Since multiple myeloma responds poorly to conventional chemotherapy or radiotherapy, new therapeutic approaches are needed. This study investigated the effects of dexamethasone, all-trans retinoic acid (ATRA), the active metabolite of vitamin D₃ [1,25(OH)₂D₃] and interferon-α on FO mouse myeloma cells (non-immunoglobulin-secreting myeloma cell line) in single drug or drug combination groups in vitro. Methods: Apoptosis ratio and change in cell counts in 4 single drug groups (dexamethasone, ATRA, vitamin D₃ and interferon-α) and 6 combination drug groups (dexamethasone + vitamin D_3, dexamethasone + ATRA, dexamethasone + interferon-α, vitamin D₃ + ATRA, vitamin D₃ + interferon-α, interferon-α + ATRA) were compared with the control group. Results: When treatment groups were compared with the control group, there was a significant increase in apoptosis in all, but this was most prominent in the group treated with dexamethasone alone. The apoptosis ratios were 0.10 and 6.82% in the control and dexamethasone-only groups, respectively. We also found that there was a significant decrease in cell count, particularly in the dexamethasone-only, ATRA-only, and ATRA-vitamin D₃ combination groups. Conclusion: ATRA, interferon-α, vitaminD₃ and particularly dexamethasone have significant effects on FO mouse myeloma cells resulting in a decreased cell count and an increased apoptosis ratio. This study should be repeated with human myeloma cell lines for further information.

Partial liquid ventilation with perfluorocarbon improves gas exchange and decreases inflammatory response in oleic acid-induced lung injury in beagles.

The aim of this study was to determine the effect of partial liquid ventilation (PLV) using a perfluorocarbon (PFC) on gas exchange and lung inflammatory response in a canine acute lung injury model. After inducing severe lung injury by oleic acid infusion, beagle dogs were randomized to receive either gas ventilation only (control group, n = 6) or PLV (PLV group, n = 7) by sequential instillation of 10 mL/kg of perfluorodecalin (PFC) at 30 min intervals till functional residual capacity was attained. Measurements were made every 30 min till 210 min. Then the lungs were removed and bronchoalveolar lavage (BAL) (35 mL/kg) was performed on the right lung and the left lung was submitted for histologic analysis. There was significant improvement in PaO2 and PaCO2 in the PLV group compared to the control group (p < 0.05) which was associated with a significant decrease in shunt (p < 0.05). There was no significant difference in parameters of lung mechanics and hemodynamics. There was a significant decrease in cell count and neutrophil percentage in BAL fluid and significantly less inflammation and exudate scores in histology in the PLV group (p < 0.05). We conclude that PLV with perfluorodecalin improves gas exchange and decreases inflammatory response in the acutely-injured lung.

Antitumour activity of 5-fluorouracil, verapamil and hyperthermia against human gastric adenocarcinoma cell (AGS) in vitro

The purpose of this study was to assess the efficacy of verapamil (20 μM) and hyperthermia (42 °C) as modifiers of 5-fluorouracil (5-FU), used at different concentrations, in inhibiting the growth of gastric adenocarcinoma cells. Combined verapamil and hyperthermia treatment showed a significant decrease in cell count when compared to control (72.2%), hyperthermia alone (68.4%), or verapamil alone (65%). At a high concentration of 5-FU (50 μg/ml), verapamil and hyperthermia had an additive growth inhibitory effect over a 4-day period when compared to control. A combination of 5-FU at low concentration (0.5 μg/ml) with verapamil significantly suppressed growth by 31.2% in comparison to control — with this effect being independent of the duration of treatment. The modalities analysed in this study require further investigation and have potential for clinical applicability to gastric cancer therapy in the future.

Effect of mitomycin C, verapamil, and hyperthermia on human gastric adenocarcinoma

The purpose of this study was to assess the efficacy of verapamil (20 microM) and hyperthermia (42 degrees C) as modifiers of mitomycin C (MMC), used at different concentrations, in inhibiting the growth of human gastric adenocarcinoma (AGS) cells. Combined verapamil and hyperthermia treatment resulted in a significant decrease in cell count by 72.2% as compared with the control value. Verapamil drastically enhanced the growth-inhibitory activity of MMC at high concentration against AGS cells by 67.5% and had no effect at intermediate and low concentrations. Hyperthermia did not enhance the effect of MMC on AGS cells. The modalities analyzed in this study require further investigation and may have potential for in vivo studies on gastric cancer therapy in the near future.