CO2 Incubator Research Articles

An Etiology of Sputum Culture Contamination and their Drugs Susceptibility Patterns among TB-suspected Patients at Epicentre Mbarara Research Centre, Uganda

Aims: Contamination in TB cultures is problematic as it allows the growth of non-target bacterial or fungal species in sputum samples, potentially concealing M. tuberculosis. This study aimed to determine the prevalence, characterization, and drug susceptibility patterns of contaminants in sputum cultures from TB-suspected patients. Methods: A laboratory-based cross-sectional study was conducted from October 2023 to June 2024 at the Epicentre Mbarara Research Centre, involving 81 sputum samples from TB-suspected patients. Conventional decontamination procedures were used, and both solid Lowenstein Jensen (LJ) and liquid Mycobacterium Growth Indicator Tube (MGIT) culture methods were applied. Standard culture techniques and physicochemical analyses were employed. Bacteria were incubated at 37°C in O2 and Co2 incubators, with growth observed within 24 to 48 hours and up to three days for yeast contaminants. Sabouraud Dextrose Agar cultures were monitored for two weeks for filamentous fungi. Antibiograms for bacterial isolates were determined using the Kirby-Bauer, while broth microdilution was used for fungal isolates. Results: Of 81 samples, 14(17.28%) were contaminated. The most common bacterial contaminants were Staphylococcus aureus, Bacillus subtilis, Streptococcus viridans, Staphylococcus epidermidis, and Escherichia coli. Fungal contaminants included Aspergillus flavus, Penicillium species, Candida albicans, Cryptococcus neoformans, and non-albican Candida species. High resistance to erythromycin (73%) and tetracycline (50%) was noted among bacterial isolates, with sensitivity to vancomycin (67%), gentamycin (60%), and chloramphenicol (58%). Yeast isolates showed (87.5%) susceptibility to itraconazole and (75%) to amphotericin B but (50%) resistance to 5-flucytosine. Mold isolates exhibited (100%) susceptibility to itraconazole, amphotericin B, and 5-flucytosine but complete resistance to caspofungin. Conclusion: The level of contamination in TB cultures was noted, and Staphylococcus aureus and Candida albicans were observed as the most common contaminants. Bacterial isolates resisted erythromycin and tetracycline, while fungal isolates were generally susceptible to itraconazole and amphotericin B. We recommend Strengthening decontamination and monitoring common resistant contaminants.

Comparison of Urine Specimen with Recto-Vaginal Swab to establish carriage of Group B Streptococcus Colonization in Pregnant Females

Objective: To determine maternal vaginal colonization with Group B Streptococcus using both recto-vaginal swab and mid-stream urine in order to determine which specimen type can give better results. Study Design: Cross-sectional study. Place and Duration of Study: Armed Forces Institute of Pathology, Rawalpindi Pakistan, Department of Gynecology and Obstetrics, Combined Military Hospital and Pak Emirates Military Hospital, Rawalpindi Pakistan, from Jan to Jun 2022. Methodology: Recto-vaginal swabs and mid-stream urine were taken from 194 pregnant women between 36th and 37th weeks of pregnancy. Swabs were inoculated on blood and MacConkey agar while urine specimen was inoculated on blood and CLED agar. All plates were incubated at 37°C in either ambient air or CO₂ incubator. Identification of Group B Streptococcus was made on the basis of colony morphology (β-haemolytic colonies on blood agar), Gram stain, catalase test and confirmation were done by means of latex agglutination tests. Results: In comparison to urine, a Group B Streptococcus carriage rate of 8.2% was detected in recto-vaginal swab. Urine specimen of only 3 patients yielded growth of Group B Streptococcus giving a urine carriage rate of 1.5%. Conclusion: Recto-vaginal swab gives a better insight to the presence of Group B Streptococcus as compared to mid-stream urine.

A human ex vivo skin model breaking boundaries

Ex vivo human skin models are valuable tools in skin research due to their physiological relevance. Traditionally, standard cultivation is performed in a cell culture incubator with a defined temperature of 37 °C and a specific atmosphere enriched with CO2 to ensure media stability. Maintaining the model under these specific conditions limits its flexibility in assessing exposures to which the skin is exposed to in daily life, for example changes in atmospheric compositions. In this study we demonstrated that the foreskin-derived skin model can be successfully cultured at room temperature outside a CO2 incubator using a CO2-independent, serum-free media. Over a cultivation period of three days, the integrity of the tissue and the preservation of immune cells is well maintained, indicating the model’s stability and resilience under the given conditions. Exposing our Medical University of Graz – human Organotypic Skin Explant Culture (MUG-hOSEC) model to cytotoxic and inflammatory stimuli results in responses analyzable within the supernatant. Besides the common analysis of released proteins upon treatment, such as cytokines and enzymes, we have included extracellular vesicle to obtain a more comprehensive picture of cell communication.

Investigation of Possible Protective Effects of Resveratrol on Oxidative Stress and Ferroptosis in PFOA Exposure in HepG-2 Cells

Aim: Exposure to perfluorooctanoic acid (PFOA) is linked to adverse health effects, including cancer and hepatic diseases. PFOA induces reactive oxygen species generation in human hepatic cells, causing oxidative stress and cell death. Resveratrol (RSV) has garnered attention for its protective effects against xenobiotic-induced damage, yet its impact on PFOA-induced oxidative stress and ferroptosis in the liver remains understudied. This study investigates RSV's protective mechanisms against oxidative stress and ferroptosis in HepG2 cells exposed to PFOA. Method: HepG2 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 incubator at 37°C. PFOA was added to the cells at concentrations ranging from 0 to 450 µM and incubated at 37°C for 24 hours. The IC50 was determined to be 457 μM. To examine RSV's protective effects, cells were treated with 60 μM RSV. Following treatment with PFOA, RSV, and the combination of PFOA+RSV, cell lysates were prepared for analysis. Oxidative stress and ferroptosis parameters were measured spectrophotometrically using ELISA. Results: In the PFOA+RSV group, antioxidant capacity increased, and ferroptosis was suppressed compared to the control. Conversely, the PFOA group showed decreased antioxidant capacity, increased oxidant capacity, and induced ferroptosis compared to the control and RSV-treated groups. Conclusion: PFOA exposure heightens oxidative stress and ferroptosis, whereas RSV treatment significantly reduces hepatic oxidative stress and protects against ferroptosis during PFOA exposure.

Carbonation of one-part alkali-activated materials incorporated by MgO: Phase characteristics and micromechanical properties

The role of magnesium oxide (MgO) in the carbonation of cementitious materials, particularly alkali-activated materials (AAM), which is a promising low-carbon alternative to ordinary Portland cement (OPC), has garnered significant attention. However, there remains a lack of clear understanding regarding its underlying mechanisms and interrelation with calcium oxide (CaO) content. This study aims to understand the influence of MgO on carbonation mechanisms, carbonation products, and (C, N)–A–S–H gel micromechanical properties in AAM. Four different binder formulations were examined by varying contents of slag, class F fly ash, and light-burned MgO. A powder-type sodium metasilicate was used as an alkaline activator. CO2 curing was conducted in a CO2 incubator with 1 % concentration and under atmospheric pressure for up to 56 days using pulverized paste binders. The carbonation mechanism, polymorphs of carbonation products, and the micromechanical properties of the (C, N)–A–S–H gel matrix were characterized by conducting laboratory tests including pH measurement, XRD, TGA, DSC, nitrogen adsorption, and nanoindentation at various carbonation stages. A noticeable pattern of pH initially dropping and then rising was observed. Integrated XRD-TGA demonstrated the evolution, polymorphism, and amount of calcium carbonate phases. Higher MgO content in AAM binders favored the formation of aragonite and hydrotalcite. Extensive nanoindentation results and their statistical analysis revealed binder-specific trends in the pozzolanic matrix stiffness before and after carbonation. The findings of this study can be used in developing AAM binders as building materials and to provide a deeper understating of the carbonation reactions in AAM binders.

P-045 Deciphering meiotic chromatin organization by SCP3

Abstract Study question What’s the pattern of genome-wide chromatin occupancy of SCP3 during meiotic prophase, and how its interaction with chromatin is regulated? Summary answer We document SCP3 enriched at active chromatin regions with largely inherited from leptotene to pachytene stage, which facilitated by transcription and condensate assembly in spermatocytes. What is known already Meiosis is a critical step during the process of gametogenesis to form haploid cells which finally develop into functional gametes. The synaptonemal complex (SC) is a meiosis-specific protein assembly which bridges homologous chromosomes to establish the architecture of meiotic chromosome. Genetic ablation of genes encoding SC components impedes meiosis and gametogenesis in mice and mutation of these genes is frequently found in infertile patients. SCP3 localization changes with chromosome morphology in autosomes and is involved in maintaining the association of sex chromosomes. Male mice with null mutation of SCP3 failed to form SCs, resulting in developmental arrest during meiotic prophase. Study design, size, duration This study included male mice at post-natal day 10 (P10), 4 weeks or adult (8 weeks or older) were maintained on C57BL/6J background. Spermatocytes were performed modified CUT&Tag using antibodies. ChIP-seq data was analyzed together with WGBS and ATAC-seq data. Participants/materials, setting, methods Spermatocytes were obtained by a discontinuous BSA density gradient. Cell pellet was resuspended in culture medium (MEM with 10% FBS) at a concentration of 105 cells/ml and cultured at 37 °C in a 5% CO2 incubator. Spermatocytes were cultured with or without 1,6-hexanediol or α-amanitin. Spermatocytes with or without drug treatment were collected at indicated time points for further examination and then modified CUT&Tag and co-IP using anti-SCP3 and anti-POL II antibodies. Main results and the role of chance Chromatin occupancy of SCP3 in leptotene and pachytene spermatocytes, SCP3-Core sites retained upon treatment of 1,6-hexanediol which dissolves SCP3 condensate, and SCP1 occupancy at meiotic chromatin in pachytene spermatocytes. When we determined inter-peak distances and width of peaks, we observe that: (1) SCP3 occupancy in leptotene stage had greater inter-peak distances and narrower peak width than SCP3 occupancy in pachytene stage, demonstrating gradual accumulated SCP3 at chromatin during meiotic progression; (2) SCP3-Core sites have broader peak width than SCP3-NonCore sites, in agreement with stability of SCP3 occupancy at SCP3-Core sites; (3) SCP1 occupancy had greater inter-peak distances and narrower peak width than SCP3 occupancy in pachytene stage, supporting that SCP1 peaks are a subset of SCP3 peaks and SCP1’s major function in connecting LE and CE respectively. Generally, at pachytene-stage meiosis, SCP3 mainly occupies chromatin regions including transcriptionally active sites, Deu and Alu elements, while SCP1 mainly co-localizes with SCP3 at a subset of regions with open chromatin and high cohesin enrichment to connect meiotic chromosome axes with CE. In this way, a local active chromatin environment is created at meiotic chromosome axes, facilitating homologous chromosome recombination. Limitations, reasons for caution Our test was not successful to examine SCP3 occupancy with as few as less than 1000 spermatocytes using our protocol. It is improvement of efficiency of tagmentation or development of more efficient antibodies against SCP3 will be helpful for clinical sample detection. Wider implications of the findings SCP3 occupancy was largely inherited from leptotene to pachytene stage, facilitated by transcription and condensate assembly, and enriched at specific SINE repeats. Further exploration using knockout mouse models of meiotic axial proteins and epigenetic modifiers will provide valuable information on meiotic chromatin architecture and crosstalk among meiotic chromatin events. Trial registration number N/A

Study of the influence of the extract of pipsissewa on cell cultures

The development of new diuretics of plant origin is an actual direction. Chimaphila umbellata (L.) is a perennial herb with diuretic, astringent, analgesic and other effects; and it can treat various conditions such as edema, dropsy, etc. Pipsissewa herb helps the removal of nitrogenous and chloride salts from the body due to the content of arbutin glycoside, tannins (up to 5 %). The aim. Evaluation of the effect of pipsissewa extract on L929 cell culture. Materials and methods. Cell line L929 (fibroblasts of mouse adipose tissue) was obtained in the low-temperature bank of the Institute of Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine. Cells were cultured in DMEM medium (Bio West, France) enriched with 10 % FBS (Lonza, Germany) with 1 % antibiotic-antimycotic (Bio West, France), in a CO2 incubator (Thermo Fisher Scientific, USA) at 37 ºС in an atmosphere with 5 % CO2. Determination of the minimum toxic concentration at which the cells remained alive was evaluated by morphological features (shape, monolayer integrity, adhesion to plastic). The study of the effect of pipsissewa extract on various cell functions was determined by the following methods: the ability to preserve morphological integrity - by the phase-contrast microscopy method, energy exchange - by the MTT test method, pinocytotic function - by the neutral red absorption method, migratory function - by the scratch test method, proliferative activity - by the doubling calculation method population Results. It is proposed to use concentrations of 0.05, 0.02, 0.01, 0.005 % of pipsissewa extract for further research. After carrying out the MTT reaction, the transition of MTT to formazan was confirmed microscopically in the negative control (native cells), at PE concentrations of 0.01 % and below, and the absence of a reaction in the positive control (cells killed by ethanol) at PE concentrations above 0.02 %. When recording the parameters of the NP absorption reaction, it was determined that PE at a concentration of 0.02 % and higher sharply suppresses pinocytotic activity, despite the partial preservation of cell adhesion, reducing the concentration by two times no longer affects mitochondria. A concentration of 0.01 % reduces proliferative activity, and at a concentration of 0.005 %, no difference with the control values ​​was found. Conclusions. When studying the assessment of the effect of pipsisewa extract on L929 cell culture, a toxic effect on these cells was established when added to the culture medium at a concentration above 0.01 %. The toxic effect had a threshold effect. Migratory and proliferative functions were the most sensitive, energy, pinocytosis and preservation of morphological integrity of cells were less sensitive

Bringing cell biology into classroom: tips to culture and observe skeletal muscle cells in high school and college

Watching living cells through a microscope is much more exciting than seeing pictures of cells in high school and college textbooks. However, bringing cell cultures into the classroom is challenging for biology teachers since culturing cells requires sophisticated and expensive instruments such as a CO2 incubator and an inverted phase-contrast microscope. Here, we describe easy and affordable methods to culture and observe skeletal muscle cells using the L-15 culture medium, tissue culture flask, standard dry incubator, standard upright microscope, and modified Smartphone microscope. Watching natural living cells in a “Do-It-Yourself (DIY)” way may inspire more students’ interest in cell biology.

A Pilot Study of Aerosolization of InfectiousMurine Norovirus in an Experimental Setup.

Human norovirus is transmitted mainly via the faecal-oral route, but norovirus disease outbreaks have been reported in which airborne transmission has been suggested as the only explanation. We used murine norovirus (MNV) as a surrogate for human norovirus to determine the aerosolization of infectious norovirus in an experimental setup. A 3-l air chamber system was used for aerosolization of MNV. Virus in solution (6 log10 TCID50/ml) was introduced into the nebulizer for generating aerosols and a RAW 264.7 cell dish without a lid was placed in the air chamber. Cell culture medium samples were taken from the dishes after the aerosol exposure time of 30 or 90min, and the dishes were placed in a 37°C, 5% CO2 incubator and inspected with a light microscope for viral cytopathic effects (CPEs). We determined both the infectious MNV TCID50 titre and used an RT-qPCR assay. During the experiments, virus infectivity remained stable for 30 and 90min in the MNV solution in the nebulizer. Infectious MNV TCID50 values/ml of 2.89 ± 0.29 and 3.20 ± 0.49 log10 were measured in the chamber in RAW 264.7 cell dish media after the 30-min and 90-min exposure, respectively. The MNV RNA loads were 6.20 ± 0.24 and 6.93 ± 1.02 log10 genome copies/ml, respectively. Later, a typical MNV CPE appeared in the aerosol-exposed RAW cell dishes. We demonstrated that MNV was aerosolized and that it remained infectious in the experimental setup used. Further studies required for understanding the behaviour of MNV in aerosols can thus be performed.

Effect of lactate supplementation to skeletal muscle cells on fiber type transition and cellular bioenergetics

It is well-known that lactate (LAC) is the primary cellular end-product of glycolysis. However, during intense exercise and transient hypoxic conditions, the intracellular LAC levels are significantly elevated. Recently, our in vitro biochemical and biophysical studies have revealed that LAC avidly binds to oxy-myoglobin (oxy-Mb) in acidic conditions and rapidly releases oxygen (O2). Based on these results, we hypothesize that LAC plays a significant role in tissue O2 management combating hypoxic conditions in high demanding exercising tissues, such as Mb rich skeletal muscles type I oxidative fibers. Here, we have investigated three types of mouse C2C12 cell line models, i.e. Wild-type (WT) cells, Mb knock-out (MbKO) cells, and Empty vector (EV) cells. 5-day old differentiated C2C12 cells were supplemented with varying concentrations (3 mM, 5 mM and 8 mM) of sodium lactate added to the DMEM growth medium. Thereafter, cells were incubated in temperature regulated CO2 incubator for varying time periods (3 h, 6 h, and 24 h) to evaluate the early and prolonged effect on tissue fiber type transition and cellular bioenergetics. Metabolic assays such as hexokinase activity, LAC dehydrogenase (LDH) activity, and glucose uptake were performed using the intracellular cell lysate whereas extracellular LAC levels were measured using LAC assay kit. Seahorse experiments were conducted to measure the cellular glycolytic and mitochondrial ATP production rates. Our findings revealed that, there was no significant difference in myotube width in C2C12 cells either with or without LAC supplementation across all the test conditions. Intriguingly, at lower LAC supplementation (1.8 mM) conditions, the extracellular LAC levels were found to be significantly increased in WT cells compared to MbKO cells. However, at mimicking hyperlactatemia (5 mM LAC) and lactic acidosis (8 mM LAC) conditions, distinct extracellular LAC levels were recorded with WT and MbKO cells. Our results clearly suggests that Mb containing cells effciently utilize LAC for cellular bioenergetics and other biochemical processes. Similarly, in response to increasing LAC supplementation levels, LDH activity was also found to higher in WT cells compared to MbKO cells. Furthermore, glucose uptake in MbKO cells was significantly decreased. Finally, significantly lower rates of glycolytic ATP production was observed when compared to mitochondrial ATP production in WT cells with similar overall ATP production rates in all treatment conditions and incubation periods. However, marginal decrease in both glycolytic and mitochondrial ATP production rates were observed in MbKO cells. Overall, this study clearly demonstrated that external supplementation of LAC to C2C12 cells had no significant effect on fiber type transition but reveals that Mb plays a significant role in LAC utilization. USDA-ARS project 6026-51000-012-06S, Sturgis Foundation Pilot Grant G1-54750-01 and ACRI-ABI Investigator Initiated Grant Award GR037175-4450S ABI. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Morphofunctional changes in ventral and dorsal hippocampus in adult rats after chronic mild stress: a preclinical experimental study

Background. Stressful influences, depending on their severity and duration, can cause the development of pathological conditions. Repeated episodes of stress cause functional and structural changes in the central nervous system and can cause the development of depressive conditions. Depression is one of the leading mental illnesses. One of the most stress-sensitive brain structures is the hippocampus. Objective. To study is to evaluate structural changes in the hippocampus, which is considered as a heterogeneous structure with separate dorsal and ventral regions, to evaluate the expression of inducible nitric oxide synthase, endothelial nitric oxide synthase, serine racemase, synaptophysin in a mild stress model.Methods. A study of the effects of mild stress was carried out on 16 adult male Wistar rats (age 12 months, body weight 350–400 g). After acclimatization, the rats were divided into two equal groups (n = 8): intact (control) and stressed. When keeping animals, modeling and removing them from the experiment, we were guided by the Regulations for Carrying Out Work Using Laboratory Animals and the Declaration of Helsinki. Experimental modeling of depression in animals was induced by mild stress exposure for 7 days (30 minutes daily). Euthanasia was performed in a CO2 incubator. The brain was fixed in neutral buffered 10% formalin. Paraffin sections were made in the frontal plane, stained with hematoxylin and eosin, thionin using the Nissl method and examined at a level from –2.40 to –3.96 mm relative to bregma using an Axio Lab A1 microscope (Carl Zeiss Microscopy GmbH, Germany). Photo documentation was carried out with an AxioCam 105 color camera (Carl Zeiss Microscopy GmbH, Germany). Using the Image Analysis module of the ZEN 1.1.2.0 program (Carl Zeiss Microscopy GmbH, Germany) in the pyramidal layer of the hippocampus. Statistical analysis was performed with Microsoft Office Excel 2016 (Microsoft, USA) and Prism 6 (GraphPad Software Inc., USA). Comparisons of two conditions were made by nonparametric Mann-Whitney-U test to avoid a statistical bias of unequal data distribution. The level of significance was set at p < 0.05. The summarized data were presented as a as mean ± standard error of mean.Results. Functional research methods and assessment of pathological changes in hippocampal neurons are presented. An increase in the relative number of wrinkled hyperchromatic pyramidal neurons in the dorsal cornu ammonis field 3 in stressed rats was noted by 23.6% (p < 0.05) compared to the control. There was an increase in the relative number of inducible nitric oxide synthase-immunopositive neurons in the dorsal cornu ammonis field 3 by 40% ( p < 0.05) and the relative area of inducible nitric oxide synthase-immunoreactive material by 35% (p < 0.05) in the pyramidal layer of cornu ammonis field 3 in stressed rats. A decrease in the relative area of synaptophysin-immunopositive material in stressed rats was found in the ventral cornu ammonis field 3 compared to the control group by 16.8% (p < 0.05); decrease in the relative area of serine racemase-immunopositive material in dorsal cornu ammonis field 3 by 4.3% (p < 0.05) and ventral cornu ammonis field 3 by 7.8% (p < 0.05).Conclusion. The results of the study demonstrate that mild stress is an adequate model of depression in rats. In animals exposed to mild stress, pronounced morphological signs of damage to hippocampal neurons were revealed; motor and indicative exploratory activity decreases. Differences were found in morphofunctional changes in the dorsal and ventral parts of the hippocampus under the influence of mild stress. In cornu ammonis field 3 of the dorsal hippocampus, in contrast to the ventral section, more pronounced signs of damage to pyramidal layer neurons were observed. The increase in the relative number of inducible nitric oxide synthase-immunopositive neurons and the relative area of inducible nitric oxide synthase-immunoreactive material in the cornu ammonis field 3 pyramidal layer in stressed rats indicates an increase in nitric oxide production and the participation of nitrooxide-dependent free radical mechanisms of damage to hippocampal neurons. The decrease in the relative area of synaptophysin-immunoreactive material in stressed rats may contribute to changes in synaptic plasticity. A decrease in the relative area of serine racemase-immunoreactive material in the dorsal and ventral parts of cornu ammonis field 3 is considered to be a sign of a possible decrease in N-methyl-D-aspartate-dependent neurotransmission in the hippocampus under stress.

Profile of miRNAs in small extracellular vesicles released from glioblastoma cells treated by boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a tumor cell-selective particle-radiation therapy. In BNCT, administered p-boronophenylalanine (BPA) is selectively taken up by tumor cells, and the tumor is irradiated with thermal neutrons. High-LET α-particles and recoil 7Li, which have a path length of 5-9μm, are generated by the capture reaction between 10B and thermal neutrons and selectively kill tumor cells that have uptaken 10B. Although BNCT has prolonged the survival time of malignant glioma patients, recurrences are still to be resolved. miRNAs, that are encapsulated in small extracellular vesicles (sEVs) in body fluids and exist stably may serve critical role in recurrence. In this study, we comprehensively investigated microRNAs (miRNAs) in sEVs released from post-BNCT glioblastoma cells. Glioblastoma U87 MG cells were treated with 25 ppm of BPA in the culture media and irradiated with thermal neutrons. After irradiation, they were plated into dishes and cultured for 3 days in the 5% CO2 incubator. Then, sEVs released into the medium were collected by column chromatography, and miRNAs in sEVs were comprehensively investigated using microarrays. An increase in 20 individual miRNAs (ratio > 2) and a decrease in 2 individual miRNAs (ratio < 0.5) were detected in BNCT cells compared with non-irradiated cells. Among detected miRNAs, 20 miRNAs were associated with worse prognosis of glioma in Kaplan Meier Survival Analysis of overall survival in TCGA. These miRNA after BNCT may proceed tumors, modulate radiation resistance, or inhibit invasion and affect the prognosis of glioma.

Resveratrol and tetrahydroisoquinoline effects on neutrophil sensitivity to NETosis formation in low-risk essential thrombocythemia patients.

Recent studies scrutinize how NETosis (a unique cell death mechanism of neutrophil), impacts thrombosis patients with essential thrombocythemia (ET). This research evaluates the susceptibility of ET neutrophils to form NETs and tests two potential inhibitors, resveratrol (RSV) and tetrahydroisoquinoline (THIQ), in vitro. Platelet-rich plasma from low-risk ET patients was used, along with neutrophils from both patients and controls. NET formation assays, with or without RSV and THIQ treatment after LPS stimulation, were conducted in a CO2 incubator. Evaluation included flow cytometry and fluorescence microscopy for NET formation and ELISA for TNFα, IL8, and vWF:Ag levels in patient and control plasma. Neutrophils from ET patients released more NETs than controls, confirmed by flow cytometry and fluorescence microscopy. Additionally, patients had significantly higher plasma levels of IL8 and TNFα compared to controls, while RSV was more effective than THIQ in reducing NETosis rates in these patients. In ET patients, a platelet counts over 1 million indicates the need for preventive treatment against thrombotic events. Similarly, in this study, RSV and THIQ significantly reduced the rate of NETosis in ET patients with higher platelet counts, and this role was more prominent in the case of the second inhibitor (RSV).

Abstract 6334: Metabolic reprogramming enhances expansion and potency of CAR T cells

Abstract Cell therapies, such as Chimeric Antigen Receptor (CAR) T cell therapy, have revolutionized the treatment of hematological malignancies, but their use in solid tumors remains a significant challenge for the field. This is due, in part, to immunosuppressive mechanisms in the tumor microenvironment (TME), which include reduced oxygen tension, high interstitial pressure, and an abundance of immunosuppressive proteins, resulting in mitochondrial dysfunction, exhaustion, and depletion of adoptively transferred cells. Metabolic manipulation of therapeutic cells could overcome these barriers to enable cell therapy success in solid tumors. To this end, we have employed AVATAR technology (Xcellbio, San Francisco, CA), an incubation system that enables precise control of oxygen tension and hyperbaric pressure on cells in culture to improve the manufacture of adoptive T cell therapies. Using a CD19 CAR T model system, we have shown that transduction and expansion of CAR T cells under reduced oxygen and hyperbaric pressure conditions yield greater percentages and total numbers of lentiviral-transduced cells. While cells transduced in a standard CO2 incubator yielded 10-20% CD19 CAR+ cells, transduction in the AVATAR system with increased pressure yielded 15-40% CD19 CAR+ cells. Similarly, T cell cultures performed under pressurized AVATAR conditions generated ~2X more viable cells after 10 days. Furthermore, these metabolically reprogrammed cells exhibit enhanced potency, with improved anti-tumor cytotoxic activity in vitro. We have extended these studies to measure the ability of AVATAR-expanded CD19 CAR T cells to control the growth of CD19-expressing NALM6-Luc tumor cells in vivo compared to CAR T cells grown in a conventional CO2 incubator. Mice (female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratories, Bar Harbor, ME) dosed with the AVATAR-expanded cells exhibited good tumor control and increased persistence of CAR T cells in relevant organs, along with alterations in cell trafficking to the bone marrow and spleen. The transferred cells maintained a less differentiated phenotype, with central memory and effector memory populations, as measured by expression of the markers CD45RA and CD62L, dominating the T cell compartment. This work showcases the benefits of metabolic reprogramming to improve the yield and functional potency of CAR T cells, which has direct applications to reducing the cost and improving the efficacy of these lifesaving treatments. The AVATAR technology is currently being translated into a closed system bioreactor for use in GMP cell therapy manufacturing. Citation Format: Yewei Xing, Shannon Eaker, Yelena Bronevetsky, Candy Garcia, Hadia Lemar, Evan Massi, Albert Wong, Sheri Barnes, Derrik Germain, Scott Wise, James Lim. Metabolic reprogramming enhances expansion and potency of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6334.

Selective Apoptotic Effects of Resilon and Epiphany Se: In Vitro Investigation in Cell Cultures

Objective: The aim of this investigation is to examine the programmed cell death mechanism using Resilon, a root canal filling material, and Epiphany SE, a root canal sealant, in a microenvironment of cultured epithelial-like cells. Methods: Vero cells were distributed into a 24-well plate at a density of 2 x 105 cells/ml per well. Fol- lowing an incubation period of 18 hours, Resilon was applied to the cell monolayer. The monolayer was combined with Resilon and covered with a pre-warmed medium before being incubated at 37 0 C in a 5% CO2 incubator. Following this, the wells were treated with a 50% diluted eluate of Epiphany SE. Resilon and Epiphany SE were then left to incubate for 0.5, 1, 3, 6, 12, 18 and 24 hours at 37 0C. Data were analyzed in IBM SPSS V23. Statistical analysis involved determining the differences in means of the specimens using a one-way ANOVA, followed by Duncan’s multiple range test to as- sess specific statistical differences across various time periods. Results: It was found that between 0 and 6 hours, it was unclear whether Resilon and Epiphany SE induce apoptosis in Vero cells. Significant differences were observed 12 hours after examination comparing both the Resilon and Epiphany SE groups to the examination time prior to 12 hours (p &lt; 0.05). These cells decreased at 18 and 24 hours. However, necrotic cells increased at each stage of the study period. The increase in the number of necrotic cells was statistically significant at 18 and 24 hours (p &lt; 0.05). Conclusion: It is inferred that necrosis of fibroblastic cells is caused by the cytotoxic effects of both Resilon and Epiphany SE. Hence, it can cause problems with root canal over obturation.

Dual Enzyme Sequential Digestion Protocol for Isolation of Human Primary Chondrocytes From the Articular Cartilage Derived From Knee Osteoarthritis Patients

Introduction: Majority of available protocols for isolation of chondrocytes from articular cartilage tissue rely on the enzymatic digestion of the tissue by collagenase type 2. The yield of chondrocytes in such protocols is low. Herein, we designed a novel indigenous sequential digestion by dual enzyme Pronase and Collagenase Type 1 for isolating human Chondrocytes from articular cartilage. Methods: Articular cartilage of Osteoarthritis (OA) patients undergoing total knee replacement were collected for the isolation of chondrocyte cells and subjected to sequential digestion by Pronase for three hours followed by Collagenase 1 overnight. Pellet of cells collected after digestion was plated on culture flask in 5% CO2 incubator. Results: From day three onwards, round to elongated cells adhered to the flask were visible which developed into elongated cell population of homogenous morphology, expressed Aggrecan (Agg), Collagen 2a (Col2a) and SRY-box transcription factor (Sox9) and had chondrogenic differentiation similar to a commercially available healthy chondrocyte. These cells were negative for Alizarin red stain, thus confirming the purity of chondrocytes. Conclusion: We have successfully established a sequential dual enzyme digestion-based culture technique for isolating the human chondrocytes from the articular cartilage biopsy derived from OA knee joints.

Enhancing Titanium Disk Performance through In-Pack Cold Atmospheric Plasma Treatment.

While titanium dental implants have already been clinically established, ongoing research is continuously being conducted to advance the fields of osseointegration and bacterial resistance, seeking further improvements in these areas. In this study, we introduce an innovative method for treating titanium surfaces within tightly sealed packaging. Specifically, titanium discs, enclosed in surgical-grade packaging, underwent treatment using cold atmospheric plasma (CAP). The surfaces were thoroughly characterized in terms of wettability, crystalline structure, and chemical composition. Hemocompatibility analyses were conducted using blood diluted in sodium citrate (1:9) exposed to titanium discs for 30 min inside a CO2 incubator at 37 °C. Subsequently, various blood parameters were evaluated, including prothrombin time (PT), activated partial thromboplastin time (APTT), and platelet adhesion. Microbiological analyses were also performed using Pseudomonas aeruginosa (ATCC 27853) for 4 h at 37 °C. The treatment with CAP Jet resulted in a reduction in contact angle without causing any changes in the crystalline structure. No statistically significant differences were observed in the blood parameters. The plasma-treated samples exhibited lower PT and APTT values compared to those of the control group. The surfaces treated with CAP Jet showed increased platelet activation, platelet density, and thrombus formation when compared with the untreated samples. Moreover, the treated surfaces demonstrated lower bacterial colony formation compared with other surfaces.

Effect of Temperature on the Development of Stages of Spermatogenesis and the Functionality of Sertoli Cells In Vitro.

Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal spermatogenesis occurs in vivo at 34 °C to 35 °C, and high temperatures are known to cause male infertility. The aim of the present study was to examine the effect of temperature (35 °C compared to 37 °C) on the viability/apoptosis of developed cells, on the development of different stages of spermatogenesis in 3D in vitro culture conditions, and the functionality of Sertoli cells under these conditions. We used isolated cells from seminiferous tubules of sexually immature mice. The cells were cultured in methylcellulose (as a three-dimensional (3D) in vitro culture system) and incubated in a CO2 incubator at 35 °C or 37 °C. After two to six weeks, the developed cells and organoids were collected and examined for cell viability and apoptosis markers. The development of different stages of spermatogenesis was evaluated by immunofluorescence staining or qPCR analysis using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate the functionality of Sertoli cells were assessed by qPCR analysis. The developed organoids were examined by a confocal microscope. Our results show that the percentages and/or the expression levels of the developed pre-meiotic, meiotic, and post-meiotic cells were significantly higher at 35 °C compared to those at 37 °C, including the expression levels of the androgen receptor, the FSH receptor, transferrin, the androgen-binding protein (ABP), and the glial-derived nerve growth factor (GDNF) which were similarly significantly higher at 35 °C than at 37 °C. The percentages of apoptotic cells (according to acridine orange staining) and the expression levels of BAX, FAS, and CASPAS 3 were significantly higher in cultures incubated at 37 °C compared to those incubated at 35 °C. These findings support the in vivo results regarding the negative effect of high temperatures on the process of spermatogenesis and suggest a possible effect of high temperatures on the viability/apoptosis of spermatogenic cells. In addition, increasing the temperature in vitro also impaired the functionality of Sertoli cells. These findings may deepen our understanding of the mechanisms behind optimal conditions for normal spermatogenesis in vivo and in vitro.

Herpes simplex virus infection induces necroptosis of neurons and astrocytes in human fetal organotypic brain slice cultures.

Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro. Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections. Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14days at 37°C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death-especially necroptosis-in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE. We developed a novel human brain culture model in which the viability of the major brain-resident cells-including neurons, microglia, astrocytes and oligodendrocytes-and the tissue architecture is maintained for at least 2weeks in vitro under serum-free culture conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.

Investigation of proapoptotic and cytotoxic effects of 2-aminobenzothiazole on human laryngeal carcinoma cells.

In the present study, we investigated the effects of 2-aminobenzothiazole application on human laryngeal carcinoma cells. Human larynx epidermoid carcinoma (HEp-2) (ATCC® CCL-23™) cells were purchased from American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. 2-aminobenzothiazole was prepared, and further dilutions ranging from 3.13 to 100 μM were prepared in fresh culture DMEM. HEp-2 cells on 96 well plates were incubated with the prepared dilutions of 2-aminobenzothiazole for 24, 48, and 72 hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test performed cytotoxicity evaluation and viability percentages. The annexin-V staining technique detected 2-aminobenzothiazole-triggered apoptosis of HEp-2 cells. The activated caspases 3/7 on HEp-2 cells after 2-aminobenzothiazole exposure were evaluated with flow cytometric analysis. The membrane potential changing of HEp-2 cells was measured following the Muse™ MitoPotential kit manufacturer instructions. MTT cytotoxicity test results showed that the viability of human laryngeal carcinoma cells decreased with an increase in the application of 2-aminobenzothiazole for 24 hours. The highest growth inhibition by 2-aminobenzothiazole for short-term application of 24 hours was detected at the highest concentration of 2-aminobenzothiazole (100 µM). The results underline that the cytotoxic effect of 2-aminobenzothiazole is dose-dependent. Cytotoxicity test results for an application time of 48 hours showed that the cytotoxicity of 2-aminobenzothiazole is dose-dependent on HEp-2 cells. The required dose of 2-aminobenzothiazole to decrease the cell viability to 50 percent has been 9-fold augmented. Annexin-V findings showed that after exposure to IC50 concentration of 2-aminobenzothiazole for 24 hours, HEp-2 cells underwent the early apoptotic stage (25.99%) and late apoptotic (16.69%), whereas 56.93% of the treated cells were alive. Only 0.39% of 2-aminobenzothiazole treated cells were necrotic. All study results showed that 2-aminobenzothiazole triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells 42.62 compared to untreated HEp-2 cells. Caspase 3/7 activation results showed that only 0.65% of control HEp-2 cells were with activated caspase 3/7, and 99.35% live cells. The analysis data from the Muse cell analyzer revealed that the percentage of cells with intact mitochondrial membranes was 21.30 after 2-aminobenzothiazole application, and 79.9% were cells with depolarized mitochondrial membranes. It has been understood that the depolarization of the inner mitochondrial membrane has been considered a dysfunction in mitochondria as a sign of apoptosis and drug toxicity. Based on all study findings, 2-aminobenzothiazole has cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner. That means that it decreased viability via inducing caspase-dependent apoptosis. Consequently, it was concluded that 2-aminobenzothiazole has good potential to lead to cytotoxicity and apoptosis on human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anticancer drugs with high efficiency.