Anti-Allergic Effects of Myrciaria dubia (Camu-Camu) Fruit Extract by Inhibiting Histamine H1 and H4 Receptors and Histidine Decarboxylase in RBL-2H3 Cells

Although Myrciaria dubia (camu-camu) has been shown to exert anti-oxidant and anti-inflammatory effects in both in vitro and in vivo studies, its use in allergic responses has not been elucidated. In the present study, the anti-allergic effect of 70% ethanol camu-camu fruit extract was tested on calcium ionophore (A23187)-induced allergies in RBL-2H3 cells. The RBL-2H3 cells were induced with 100 nM A23187 for 6 h, followed by a 1 h camu-camu fruit extract treatment. A23187 sanitization exacerbated mast cell degranulation; however, camu-camu fruit extract decreased the release of histamine and β-hexosaminidase, which are considered as key biomarkers in cell degranulation. Camu-camu fruit extract inhibited cell exocytosis by regulating the calcium/nuclear factor of activated T cell (NFAT) signaling. By downregulating the activation of mitogen-activated protein kinase (MAPK) signaling, camu-camu fruit extract hindered the activation of both histamine H1 and H4 receptors and inhibited histidine decarboxylase (HDC) expression by mediating its transcription factors KLF4/SP1 and GATA2/MITF. In A23187-induced ROS overproduction, camu-camu fruit extract activated nuclear factor erythroid-2-related factor 2 (Nrf2) to protect mast cells against A23187-induced oxidative stress. These findings indicate that camu-camu fruit extract can be developed to act as a mast cell stabilizer and an anti-histamine. This work also “opens the door” to new investigations using natural products to achieve breakthroughs in allergic disorder treatment.

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in Toxoplasma gondii

Fundamental processes of obligate intracellular parasites, such as Toxoplasma gondii and Plasmodium falciparum are controlled by a set of plant-like calcium dependent kinases (CDPKs), the conserved cAMP- and cGMP-dependent protein kinases (PKA and PKG), secondary messengers and lipid signalling. While some major components of the signalling networks have been identified, how these are connected is largely not known. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels to activate PKG or CDPKs, respectively. We show that both these inducers trigger near identical signalling pathways and provide evidence for a positive feedback loop involving CDPK3. We measure phospho- and lipid signalling in parasites treated with the Ca2+ ionophore A23187 in a sub-minute timecourse and show CDPK3-dependent regulation of diacylglycerol levels and increased phosphorylation of four phosphodiesterases (PDEs), suggesting their function in the feedback loop. Disruption of CDPK3 leads to elevated cAMP levels and inhibition of PKA signalling rescues the egress defect of ΔCDPK3 parasites treated with A23187. Biochemical analysis of the four PDEs identifies PDE2 as the only cAMP-specific PDE among these candidates while the other PDEs are cGMP specific; two of which are inhibited by the predicted PDE inhibitor BIPPO. Conditional deletion of the four PDEs supports an important, but non-essential role of PDE1 and PDE2 for growth, with PDE2 controlling A23187-mediated egress. In summary we uncover a positive feedback loop that potentiates signalling during egress and links several signalling pathways together.

R-Phycoerythrin from Colaconema formosanum (Rhodophyta), an Anti-Allergic and Collagen Promoting Material for Cosmeceuticals

R-phycoerythrin (R-PE), a pigment complex found in red algae, was extracted and purified from a newly identified red alga, Colaconema formosanum, and its bioactivities were examined. It was revealed that R-PE treatment resulted in high cell viability (>70%) to the mammalian cell lines NIH-3T3, RBL-2H3, RAW264.7, and Hs68, and had no effect on cell morphology in NIH-3T3 cells. Its suppression effect was insignificant on the production of IL-6 and TNF-α in lipopolysaccharides-stimulated RAW264.7 cells. However, calcium ionophore A23187-induced β-hexosaminidase release was effectively inhibited in a dose-dependent manner in RBL-2H3 cells. Additionally, it was revealed to be non-irritating to bionic epidermal tissues. Notably, procollagen production was promoted in Hs68 cells. Overall, the data revealed that R-PE purified from C. formosanum exhibits anti-allergic and anti-aging bioactivities with no observed consequential toxicity on multiple mammalian cell lines as well as epidermal tissues, suggesting that this macromolecule is a novel material for potential cosmetic use.

Regulation of Dual Specificity Phosphatases by Fibroblast Growth Factor signaling pathways in bovine granulosa cells

Controling the duration and amplitude of mitogen activated protein kinase (MAPK) signaling is an important element in deciding cell fate. One group of intracellular negative regulators of MAPK activity is a subfamily of the dual specificity phosphatase (DUSP) superfamily, of which up to 16 members have been described in ovarian granulosa cells. Growth factors stimulate proliferation of granulosa cells through MAPK, PKC and AKT pathways, although it is not known which pathways control DUSP expression in these cells. The aim of the present study was to identify which pathways are involved in the regulation of DUSP expression using a well-established serum-free culture system for bovine granulosa cells. Stimulation of cells with FGF2 increased DUSP1, DUSP5 and DUSP6 mRNA abundance in a time and dose-dependent manner, and increased DUSP5 and DUSP6 protein accumulation. None of the other eleven DUSP measured were regulated by FGF2. Pharmacological inhibition of MAPK3/1 signaling decreased FGF2-stimulated DUSP1, DUSP5 and DUSP6 mRNA levels (p < 0.05) whereas inhibition of PKC did not affect the expression of these three DUSPs. Abundance of FGF2-dependent DUSP6 mRNA was reduced by inhibition of PLC or by chelating calcium, but DUSP5 mRNA abundance was not affected. Abundance of basal DUSP1 and DUSP6, but not DUSP5, mRNA was increased by the addition of the calcium ionophore A23187. We conclude that FGF2 stimulation of DUSP5 abundance requires MAPK3/1 whereas DUSP6 mRNA accumulation is dependent on calcium signaling as well as MAPK3/1 activation, suggesting complex regulation of physiologically important DUSPs in the follicle.

3D holotomographic monitoring of Ca++ dynamics during ionophore-induced Neospora caninum tachyzoite egress from primary bovine host endothelial cells

Neospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca++ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca++ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions.

Calcium Ionophore (A23187) Rescues the Activation of Unfertilized Oocytes After Intracytoplasmic Sperm Injection and Chromosome Analysis of Blastocyst After Activation

Calcium is a crucial factor in regulating the biological behavior of cells. The imbalance of calcium homeostasis in cytoplasm will cause abnormal behavior of cells and the occurrence of diseases. In intracytoplasmic sperm injection (ICSI) cycle, the dysfunction of oocyte activation caused by insufficient release of Ca2+ from endoplasmic reticulum is one of the main reasons for repeated fertilization failure. Calcium ionophore (A23187) is a highly selective calcium ionophore, which can form stable complex with Ca2+ and pass through the cell membrane at will, effectively increasing intracellular Ca2+ levels. It has been reported that calcium ionophore (A23187) can activate oocytes and obtain normal embryos. However, there are few studies on unfertilized oocytes after calcium ionophore (A23187) rescue activation in ICSI cycle. The purpose of this study was to analyze the effects of calcium ionophore (A23187) rescue activation on the activation of unfertilized oocytes, embryonic development potential, embryonic development timing and chromosomal aneuploidy, and to compare and analyze the clinical data of patients with calcium ionophore (A23187) activation in clinical application. The results showed that a certain proportion of high-quality blastocysts with normal karyotype could be obtained after calcium ionophore (A23187) rescue activation of unfertilized oocytes, and it did not have a significant effect on the timing of embryo development. In clinical practice, direct activation with calcium ionophore (A23187) after ICSI was better than rescue activation the next day. In conclusions, the studies on the effectiveness and safety of calcium ionophore (A23187) rescue activation for oocytes with ICSI fertilization failure can enable some patients to obtain usable, high-quality embryos during the first ICSI cycle.

P–279 Effect of Ca2+ ionophore A23187 (calcymicin) on fertilization rates, embryo development and pregnancy in human assisted reproduction cycles

Study question Does Calcymicin improve reproductive outcomes of ICSI cycles in cases of fertilization failure and/or embryo blockage indications? Summary answer The application of the Calcymicin after ICSI improves reproductive outcomes, especially in cases with clinical indication of fertilization failure. What is known already According to the bibliography, deficiencies in the oocyte activation process frequently lead to failed ICSI cycles, and these can be corrected by increasing initial levels of calcium (Ca2+) in the oocyte using assisted oocyte activation techniques (AOA), such as the use of Ca2+ ionophores. Ca2+ Ionophores have been shown to trigger an initial Ca2+ spike in the ooplasm that activates Ca2+/Calmodulin dependent protein kinase II, which initiates the cascade of cellular events leading to oocyte activation. Previous results suggest that Ca2+ ionophore treatment can give live offspring after failed ICSI cycles. Study design, size, duration 270 oocytes collected from 17 patients who presented cycles with low fertilization rates and/or embryo blockage or poor quality embryos (according to ASEBIR’s embryo classification criteria) were retrospectively analyzed. Oocytes were divided into two groups, a control group that underwent conventional IVF/ICSI and another group that underwent an ICSI cycle with AOA. Study groups were defined according to clinical indications and subgroups according to AOA or control. All data were collected from 2017 until 2020. Participants/materials, setting, methods Among the 270 oocytes of the study sample, 142 belonged to the control group and 128 belonged to the AOA group. The AOA group oocytes were activated for 15 minutes immediately after ICSI using a prepared solution containing the Ca2+ ionophore A23187, CultActive© (Gynemed, Germany). Fertilization rate and type, blastocyst formation rate, blastocyst quality, embryo kinetics, and pregnancy rates were analyzed, all of them were compared to FIV/ICSI cycles without oocyte activation (control group). Main results and the role of chance In the analyses of the whole sample of oocytes, the AOA treatment gave a fertilization rate of 72.5%, which was significantly higher compared to 53.8% of the control cycles (p = 0.002). Good quality blastocysts and pregnancy rates were also significantly higher than the control (p = 0.01). In the group with an indication of fertilization failure, a significantly higher fertilization rate was recorded compared to the control (65% and 33%, respectively). A higher rate of abnormal embryos with three pronuclei was also found compared to the control (p &lt; 0.001). There were no significant differences in blastocyst formation rates, quality, or embryo kinetics (p &gt; 0.05). In the group with an indication of embryo blockage/poor embryo quality, a significantly higher rate of good quality blastocysts and lower blastulation time were recorded compared to the control (p &lt; 0.05). Limitations, reasons for caution The safety of the AOA technique with Ca2+ ionophore has not been fully demonstrated. In our study, none of the newborns had malformations, and gestational weeks and birth weights were normal. However, further studies on the safety of this technique are needed to implement it routinely in human reproduction clinics. Wider implications of the findings: According to these findings, an increase in the initial levels of calcium in the oocyte through the application of the Ca2+ ionophore A23187 after ICSI improves the results of failed assisted reproduction cycles, especially in the case of those diagnosed with fertility failure, which is a clear indication for AOA. Trial registration number Not applicable

Changes in Red Blood Cell Membrane Properties: The Role of Metabolic Syndrome Components

Metabolic syndrome (MetS) is a cluster of metabolic, hormonal and hemodynamic disorders that contribute to a change in the structural and functional status of erythrocytes and contribute to dysregulation of their cation transport function, where Ca2+ -dependent potassium channels (KCa channels) play an important role. A MetS model was performed using male Wistar rats, which were divided into control and experimental groups. Rats in the control group were fed standard rat chow. Rats in the experimental group were exposed to a high-fat and high-carbohydrate (HFHC) diet for 12 weeks. The data obtained indicate that the HFHC diet led to obesity, high blood pressure, hyperglycemia, impaired glucose tolerance, and dyslipidemia. The level of glutathione (GSH) decreased in the erythrocytes of rats suffering from MetS, but the level of malondialdehyde (MDA) increased. It was shown that the amplitude of the membrane potential of erythrocytes of rats with MetS changed depending on the acting agent: when stimulated with calcium ionophore A23187 it decreased, when the redox system ascorbat –phenazine methosulfate was used, it increased compared to the control group. The data obtained indicate that a HFHC diet leads to changes in the physical and chemical properties of the erythrocyte membrane.