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A 71-year-old female with type B3 thymoma developed severe aplastic anemia. Anti-thymocyte globulin was administered with glucocorticoids and cyclosporin A as the treatment for aplastic anemia. Computed tomography scan revealed that thymoma apparently shrank and remained without regrowth for at least 7 months. As previously reported, glucocorticoid has therapeutic effects on thymoma especially with abundant lymphocytes. Anti-thymocyte globulin also depletes peripheral lymphocytes, but its efficacy in the treatment of thymoma is unknown. Anti-thymocyte globulin and glucocorticoids may have cooperated with each other in reducing thymoma in our case. More cases should be accumulated to elucidate the effects of anti-thymocyte globulin on thymoma.

Recent studies indicate that inhibition of the efflux transporter P-glycoprotein (P-gp) at the blood–brain barrier (BBB) may represent a putative strategy to increase the BBB penetration of several antibiotics. Therefore, the present study aimed to investigate the effect of P-gp inhibition on the transport of ceftriaxone (CFX) across the BBB. Blood and brain microdialysis in rats was used to monitor blood and brain unbound CFX concentrations following intravenous administration (50 mg/kg), with or without pretreatment with one of the P-gp inhibitors, cyclosporin A (6.25, 12.5, 25 mg/kg) or verapamil (5, 10, 20 mg/kg). An inhibitory effect was demonstrated by an increase in the ratio of unbound brain to unbound blood concentration (Kp.uu.brain) of CFX. The concentrations of CFX in blood and brain from 0 to 180 min after intravenous administration (CFX, 50 mg/kg) ranged from 3 to 40 μg/ml and 1 to 10 μg/ml, respectively. The Kp.uu.brain of CFX was 24.74 ± 1.34%. Pretreatment with cyclosporin A increased the brain concentration and the Kp.uu.brain of CFX in a dose-dependent manner. However, pretreatment with verapamil increased the brain concentration of CFX but not the Kp.uu.brain. The present data shows that CFX might be a substrate of P-gp efflux transporter at the BBB and P-gp inhibition might enhance the brain concentration of CFX. Future studies involving more selective P-gp inhibitors or knockout mouse models should be conducted to specifically elucidate the impact of P-gp inhibition on penetration of CFX across the BBB.

Efficient cardiac differentiation of human pluripotent stem cells (hPSCs) is essential for their use in the field of basic research as well as in cell-based therapy. Regulation of the Wnt/β-catenin pathway is a key step in the process of cardiac differentiation of hPSCs. In this study, we reported cyclosporin A (CsA), a strong immunosuppressor, as an inducer of cardiac differentiation in human embryonic stem cells (hESCs). Our results showed that CsA promoted cardiac differentiation in the mesodermal differentiation stage of hESCs and that this effect was mediated by the inhibition of Wnt/β-catenin signaling. Therefore, our results provide novel information about CsA as a Wnt/β-catenin inhibitor as well as a strategy for the cardiac differentiation of hESCs.

Background Extravillous trophoblast (EVT) cells play an essential role in the maternal-fetal interaction. Although abnormal development and function of EVT cells, including impaired migration and invasion capability, are believed to be etiologically linked to severe pregnancy disorders including pre-eclampsia (PE), the associated molecular mechanisms are not clear ascribed to the lack of an appropriate cell model in vitro. Cyclosporine A (CsA) is a macrolide immunosuppressant and is also used in clinic to improve pregnancy outcomes. However, whether CsA has any effects on the function of EVT cells has not been well investigated. Methods In this study, we induced differentiation of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) into EVT cells (hiPSC-EVT and hESC-EVT cells, respectively) by Y27632, NRG1, A83-01 and matrigel, and collected these derived EVT cells by flow cytometry for sorting cells positive for double HLA-G and KRT7, which are EVT markers. We then investigated the effects of CsA on the invasion and migration of these derived EVT cells. Results We found that the hiPSC-EVT and hESC-EVT cells expressed high levels of the EVT markers such as KRT7, ITGA5 and HLA-G but low levels of OCT4, a stem cell marker, and that CsA significantly promoted the invasion and migration of hiPSC-EVT and hESC-EVT cells. Conclusions We successfully generated hiPSC/hESC-derived human EVT cells, which may be applicable for investigating the remodeling process of spiral arteries remodeling and the possible mechanisms of EVT-related diseases in vitro. Furthermore, our findings provide direct evidence that CsA regulates the function of EVT cells and molecular basis by which CsA may be used to treat pregnancy complications in clinic associated with deficient EVT function.

Chandipura virus (CHPV), belonging to the genus Vesiculovirus of the family Rhabdoviridae, has been identified as one of the causes of pediatric encephalitis in India. Currently, neither vaccines nor therapeutic drugs are available against this agent. Considering that the disease progresses very fast with a high mortality rate, working towards the development of potential therapeutics against it will have a public health impact. Although the use of viral inhibitors as antiviral agents is the most common way to curb virus replication, the mutation-prone nature of viruses results in the development of resistance to antiviral agents. The recent development of proteomic platforms for analysis of purified viral agents has allowed certain upregulated host proteins that are involved in the morphogenesis and replication of viruses to be identified. Thus, the alternative approach of inhibition of host proteins involved in the regulation of virus replication could be explored for their therapeutic effectiveness. In the current study, we have evaluated the effect of inhibition of cyclophilin A (CypA), an immunophilin with peptidyl-prolyl cis/trans-isomerase activity, on the replication of CHPV. Treatment with cyclosporin A, used in vitro for the inhibition of CypA, resulted in a 3-log reduction in CHPV titer and an undetectable level of CypA in comparison to an untreated control. An in silico analysis of the interaction of the CHPV nucleoprotein with the human CypA protein showed stable interaction in molecular docking and molecular dynamics simulations. Overall, the results of this study suggest a possible role of CypA in facilitating CHPV replication, thus making it one of the potential host factors to be explored in future antiviral studies. Supplementary information The online version contains supplementary material available at 10.1007/s00705-021-05237-1.

S-adenosylmethionine (AdoMet) predominantly accumulates in tissues and biological fluids of patients affected by liver dysmethylating diseases, particularly glycine N-methyltransferase, S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies, as well as in some hepatic mtDNA depletion syndromes, whose pathogenesis of liver dysfunction is still poorly established. Therefore, in the present work, we investigated the effects of S-adenosylmethionine (AdoMet) on mitochondrial functions and redox homeostasis in rat liver. AdoMet decreased mitochondrial membrane potential and Ca2+ retention capacity, and these effects were fully prevented by cyclosporin A and ADP, indicating mitochondrial permeability transition (mPT) induction. It was also verified that the thiol-alkylating agent NEM prevented AdoMet-induced ΔΨm dissipation, implying a role for thiol oxidation in the mPT pore opening. AdoMet also increased ROS production and provoked protein and lipid oxidation. Furthermore, AdoMet reduced GSH levels and the activities of aconitase and α-ketoglutarate dehydrogenase. Free radical scavengers attenuated AdoMet effects on lipid peroxidation and GSH levels, supporting a role of ROS in these effects. It is therefore presumed that disturbance of mitochondrial functions associated with mPT and redox unbalance may represent relevant pathomechanisms of liver damage provoked by AdoMet in disorders in which this metabolite accumulates.

Leprosy, also known as Hansen’s disease, continues to have a substantial impact on infectious diseases throughout the world. Leprosy is a chronic granulomatous infection caused by Mycobacterium leprae and shows a wide clinical and immunopathological spectrum related to the immune response of the host. This disease affects the skin and other internal organs with a predilection to infect Schwann cells, which play an active role during axonal degeneration, affecting peripheral nerves and promoting neurological damage. This chronic inflammation influences immune function, leading to neuroimmune disorders. Leprosy is also associated with neuroimmune reactions, including type 1 (reverse) and type 2 (erythema nodosum leprosum) reactions, which are immune-mediated inflammatory complications that can occur during the disease and appear to worsen dramatically; these complications are the main concerns of patients. The reactions may induce neuritis and neuropathic pain that progressively worsen with irreversible deformity and disabilities responsible for the immunopathological damage and glial/neuronal death. However, the neuronal damage is not always associated with the reactional episode. Also, the efficacy in the treatment of reactions remains low because of the nonexistence of a specific treatment and missing informations about the immunopathogenesis of the reactional episode. There is increasing evidence that peripheral neuron dysfunction strongly depends on the activity of neurotrophins. The most important neurotrophin in leprosy is nerve growth factor (NGF), which is decreased in the course of leprosy, as well as the presence of autoantibodies against NGF in all clinical forms of leprosy and neuroimmune reactions. The levels of autoantibodies against NGF are decreased by the immunomodulatory activity of cyclosporin A, which mainly controls pain and improves motor function and sensitivity. Therefore, the suppression of anti-NGF and the regulation of NGF levels can be attractive targets for immunomodulatory treatment and for controlling the neuroimmune reactions of leprosy, although further studies are needed to clarify this point.

A macrocyclic peptide scaffold with well-established structure-property relationship is desirable for tackling undruggable targets. Here, we adopted a natural macrocycle, cyclosporin O (CsO) and its derivatives (CP1-3), and evaluated the impact of conformation on membrane permeability, cyclophilin A (CypA) binding, and the pharmacokinetic (PK) profile. In nonpolar media, CsO showed a similar conformation to cyclosporin A (CsA), a well-known chameleonic macrocycle, but less chameleonic behavior in a polar environment. The weak chameleonicity of CsO resulted in decreased membrane permeability; however, the more rigid conformation of CsO was not detrimental to its PK profile. CsO exhibited a higher plasma concentration than CsA, which resulted from minimal CypA binding and lower accumulation in red blood cells and moderate oral bioavailability (F = 12%). Our study aids understanding of CsO, a macrocyclic peptide that is less explored than CsA but with greater potential for diversity generation and rational design.

Cyclosporin a (CsA) was characterized by a narrow therapeutic window and high interindividual pharmacokinetic variability. In this study, we aimed to identify the association of CYP3A4, ABCB1, ABCC...

Acetaminophen (APAP)-induced hepatotoxicity is the most common cause of acute liver failure in worldwide. N-acetyl cysteine (NAC) is used as the APAP antidote. Cyclosporin A (CsA) is suppressed mitochondrial damage by binding cyclophilin, a mitochondrial pore transport component. The study aimed to evaluate the effects of NAC, CsA, and NAC+CsA treatments on APAP-induced hepatotoxicity in mice. Mice were randomly divided into five groups (n = 6). 400 mg/kg/ip/single dose APAP, 1200 mg/kg/i.p/single dose NAC and 50 mg/kg/i.p/single dose CsA were performed. Light and electron microscopic alterations were investigated in liver samples. Levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and liver glutathione (GSH) were analyzed. 3-nitrotyrosine and cytochrome c immunoreactivities were evaluated in liver tissue. Here, we found that APAP leads to histopathological and ultrastructural changes in mice liver. Also, APAP increased cytochrome c and 3-nitrotyrosine immunopositive staining. Besides, a significant decrease in liver GSH and an increase in serum AST and ALT levels were detected in the APAP group. Interestingly, NAC+CsA treatment improved histological alterations, cytochrome c, and 3-nitrotyrosine immunoreactivities and liver GSH, serum AST/ALT levels caused by APAP. We suggest that the combination of NAC and CsA reduces acetaminophen-induced hepatotoxicity in mice.

2021 marks the 30th anniversary of the revelation that cyclosporin A and FK506 act in a way previously not seen-as "molecular glues" that induce neo-protein-protein associations. As a torrent of new molecular-glue probes and medicines are fueling interest in this field, I explore the arc of this story.

Inflammatory response regulation is a mechanism through which human umbilical cord mesenchymal stem cells (HUCMSCs) improve myocardial ischemia reperfusion injury (IRI); however, the timing of HUCMSC delivery to achieve maximum effectiveness is controversial. To investigate the effects of HUCMSC delivery on the acute inflammatory stage of IRI, we transplanted HUCMSCs or HUCMSCs with cyclosporin A (CsA) through the coronary artery simultaneously during ischemia reperfusion in pigs. Ferumoxytol-labeled HUCMSCs (HUCMSC), HUCMSCs with cyclosporin A (HUCMSC+CsA), and PBS (control) groups were investigated to evaluate the homing of transplanted cells and changes in infarct features, cardiac activity, and inflammatory response at three time points post-transplantation. Animals were sacrificed 2 weeks later for histological analysis of the hearts. We detected Prussian blue-dyed granules distributed around T lymphocyte clusters in the infarct area in the HUCMSC group. Infarct size and collagen deposition in the infarct area were lower in the HUCMSC group than in the control and HUCMSC+CsA groups. Cardiac function was mildly impaired in both the control and HUCMSC groups, whereas added CsA had a more severe impact. The levels of proinflammatory markers were lower in the HUCMSC group than in the control group at 24-h follow-up, and the difference was more significant after adding CsA. There were more CD3+ T lymphocytes and Foxp3+ Tregs in the HUCMSC group infarct area than in the other two groups. Proliferation rate of T lymphocytes was higher in the HUCMSC group than in the other two groups. Indirect co-culture experiments in vitro showed that MSCs promoted the generation of CD4+CD25+ Foxp3+Tregs through a paracrine mechanism. These results indicate that immediate intracoronary delivery of HUCMSCs after ischemia reperfusion can reduce acute myocardial IRI and promote myocardial repair, mainly through T lymphocyte interactions to regulate the intense inflammatory response during the acute inflammatory stage.

© 2021, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.Cyclosporine A (CsA) is an immunosuppressive drug commonly used to prevent autoimmune diseases. At the same time, CsA is a calcineurin (CaN) inhibitor. It affects the intracellular calcium signaling pathway. The effect of CsA on breast cancer cells, MDA-MB-231, plasma membrane calcium pump 1 (PMCA1), calmodulin (CaM), calcineurin (CaN), and cMyc, which are proteins that affect calcium signaling, were investigated. CsA inhibited the proliferation of MDA-MB-231 cells but did not affect the migration of the cells. After 24 h of incubation, CsA suppressed the PMCA1 protein, which pumps intracellular calcium out of the cell. At the same time, calcium started to accumulate inside the cell and CaM protein was expressed, while PMCA1 was suppressed. The CaN protein was suppressed 72 h after the administration of CsA, but the cMyc protein was expressed. Interestingly, 24 h incubation when the PMCA1 protein is down-regulated after the duration of time, the cMyc protein is also down-regulated. Although the indirect effect of CaN and cMyc is known, this relationship between PMCA1 and cMyc was not known. As a result, it has been shown that CsA affects the PMCA pump by disrupting the intracellular calcium pathway in breast cancer cells. Graphic Abstract: [Figure not available: see fulltext.]

Neuroprotective strategies in the treatment of stroke have been attracting a great deal of attentions. Our previous clinical and basic studies have demonstrated that protopanaxadiol ginsenoside-Rd (Rd), a monomer compound extracted from Panax ginseng or Panax notoginseng, has neuroprotective effects against ischemic stroke, probably due to its ability to block Ca2+ overload, an usual consequence of the overactivation of NMDA receptor (NMDAR). As an extending study, we explored here whether Rd exerted its neuroprotection as a novel NMDAR blocker. Our whole-cell patch-clamp results showed that Rd reduced NMDAR currents of cultured rat cortical neurons (EC50 = 7.7 μM) dose-dependently by acting on extrasynaptic NMDAR NR2b subunit. However, unexpectedly, cell transfection and radioligand binding assays revealed that Rd did not bind to the NMDAR channel directly. Alternatively, it inhibited the phosphorylation of NR2b at Ser-1303, a target of death associated protein kinase 1 (DAPK1). Moreover, cell-based and cell-free enzymatic assays showed that Rd did not inhibit the activity of DAPK1 directly, but blocked the activity of calcineurin, a key phosphatase for activating DAPK1. Importantly, other protopanaxadiol ginsenosides were also found to have potential inhibitory effects on calcineurin activity. Furthermore, as expected, calcineurin inhibition by cyclosporin A could mimic Rd’s effects and protect against NMDA-, oxygen glucose deprivation- or transient ischemic stroke-induced neuronal injury. Therefore, our present study provided the first evidence that Rd could exert an inhibitive effect on NMDAR-triggered currents and sequential excitotoxicity through mitigation of DAPK1-mediated NR2b phosphorylation by attenuating calcineurin activity.

Cyclosporine-A (CsA) is a widely used immunosuppressant. In this study, we explore the pathway through which CsA suppressed the Porphyromonas gingivalis lipopolysaccharide (P.g-LPS)-induced increase in matrix metalloproteinase (MMP) activities in co-cultured human gingival fibroblasts (HGFs) and THP-1 monocytes. In the co-culture, we found that CsA inhibited the expression of cyclophilin A (CyPA), CD147 and the activities of MMPs, which were all induced by P.g-LPS. We also found that P.g-LPS and recombinant human CyPA increased activation of ERK1/2 and IκB (an NF-κB inhibitory protein), but CsA and the anti-CD147 antibody significantly inhibited these effects. Taken together, CsA in the presence of P.g-LPS might suppress MMP activities by blocking the CyPA/CD147 interaction that results in the inhibition of ERK1/2 and NF-κB signaling by interfering with the phosphorylation of ERK1/2 and IκB.

Background New therapeutic drugs are urgently needed against visceral leishmaniasis because current drugs, such as pentavalent antimonials and miltefosine, produce severe side effects and development of resistance. Whether cyclosporine A (CsA) and its derivatives can be used as therapeutic drugs for visceral leishmaniasis has been controversial for many years. Methods In this study, we evaluated the efficacy of CsA and its derivative, dihydrocyclosporin A (DHCsA-d), against promastigotes and intracellular amastigotes of Leishmania donovani. Sodium stibogluconate (SSG) was used as a positive control. Results Our results showed that DHCsA-d was able to inhibit the proliferation of L. donovani promastigotes (IC50: 21.24 μM and 12.14 μM at 24 h and 48 h, respectively) and intracellular amastigotes (IC50: 5.23 μM and 4.84 μM at 24 and 48 h, respectively) in vitro, but CsA treatment increased the number of amastigotes in host cells. Both DHCsA-d and CsA caused several alterations in the morphology and ultrastructure of L. donovani, especially in the mitochondria. However, DHCsA-d showed high cytotoxicity towards cells of the mouse macrophage cell line RAW264.7, with CC50 values of 7.98 μM (24 h) and 6.65 μM (48 h). Moreover, DHCsA-d could increase IL-12, TNF-α and IFN-γ production and decrease the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. On the contrary, CsA decreased IL-12, TNF-α, and IFN-γ production and increased the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. The expression of L. donovani cyclophilin A (LdCyPA) in promastigotes and intracellular amastigotes and the expression of cyclophilin A (CyPA) in RAW 264.7 cells were found to be significantly downregulated in the CsA-treated group compared to those in the untreated group. However, no significant changes in LdCyPA and CyPA levels were found after DHCsA-d or SSG treatment. Conclusions Our findings initially resolved the dispute regarding the efficacy of CsA and DHCsA-d for visceral leishmaniasis treatment. CsA showed no significant inhibitory effect on intracellular amastigotes. DHCsA-d significantly inhibited promastigotes and intracellular amastigotes, but it was highly cytotoxic. Therefore, CsA and DHCsA-d are not recommended as antileishmanial drugs.

One of the most common causes of cancer mortality worldwide is hepatocellular carcinoma (HCC). Extracellular signal-regulated kinase (ERK1/2) pathway has been shown to play an important role in the development and progression of HCC. Here, we demonstrate that the immunosuppressive agent cyclosporin A (CsA) has the ability to increase the cellular growth in HCC (HepG2 cells) via activation of ERK1/2 signaling cascade. It was found that ERK1/2 phosphorylation induced by CsA was highly reduced in the presence of the reactive oxygen species (ROS) scavenger polyethylene glycol-superoxide dismutase (PEG-SOD). Furthermore, it was observed that inhibition of metalloproteinase activity using TAPI-2 prevents ERK1/2 activation by CsA. Moreover, a disintegrin and metalloproteinase domain 17 (ADAM-17) activity was found to be critical for ERK phosphorylation by CsA. In addition, CsA-induced ERK phosphorylation was highly reduced in the presence of either neutralizing anti-heparin-binding-epidermal growth factor (HB-EGF) antibody or UO126 (MEK inhibitor). By using the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, it was found that EGFR is critical for ERK phosphorylation induced by CsA. Furthermore, CsA-induced cell proliferation was strongly reduced in the presence of either PEG-SOD or TAPI-2 or neutralizing anti-ADAM17 antibody or neutralizing anti-HB-EGF antibody or AG1478 or UO126. Collectively, these data demonstrate that CsA has the ability to activate ERK1/2 signaling cascade that could be translated into an increase in HepG2 cell proliferation. Furthermore, these data support the role of ROS, ADAM-17, and EGFR in ERK1/2 signaling activation and subsequent cell proliferation induced by CsA in HepG2 cells.

Mycobacterium tuberculosis (MTB) infection induces cytotoxicity to host human macrophages. The underlying signaling mechanisms are largely unknown. Here we discovered that MTB infection induced programmed necrosis in human macrophages, causing mitochondrial cyclophilin-D (CypD)-p53-adenine nucleotide translocator type 1 association, mitochondrial depolarization and lactate dehydrogenase medium release. In human macrophages MTB infection-induced programmed necrosis and apoptosis were largely attenuated by CypD inhibition (by cyclosporin A), silencing and knockout, but intensified with ectopic CypD overexpression. Further studies identified microRNA-1281 as a CypD-targeting miRNA. Ectopic overexpression of microRNA-1281 decreased CypD 3'-untranslated region activity and its expression, protecting human macrophages from MTB-induced programmed necrosis and apoptosis. Conversely, microRNA-1281 inhibition in human macrophages, by the anti-sense sequence, increased CypD expression and potentiated MTB-induced cytotoxicity. Importantly, in CypD-KO macrophages miR-1281 overexpression or inhibition was ineffective against MTB infection. Restoring CypD expression, by an untranslated region-depleted CypD construct, reversed miR-1281-induced cytoprotection against MTB in human macrophages. Collectively, these results show that targeting CypD by miR-1281 protects human macrophages from MTB-induced programmed necrosis and apoptosis.

Oxidative stress is associated with functional disorder of trophoblast cells. Our previous studies have demonstrated that cyclosporin A (CsA) promotes the activity of normal human trophoblast cells. We further investigated the role and mechanism of CsA on oxidative stress in trophoblast cells. JEG-3 cells were co-cultured with H2O2 and CsA. Cell viability and morphology were measured by MTT assay and inverted microscope. Reactive oxygen species (ROS) was analyzed by fluorescence microscopy. Cell mitochondrial membrane potential (MMP) was determined by flow cytometric analysis. Malondialdehyde (MDA) production, superoxide dismutase (SOD) and catalase (CAT) activities were examined using colorimetric assays. The expression and phosphorylation of FAK and Src kinase proteins were examined by western blotting. CsA increased JEG-3 cell viability and reduced the morphologic injury induced by H2O2 treatment. CsA decreased ROS and MDA production, increased SOD and CAT activities, and restored the MMP of H2O2 treated JEG-3 cells. CsA administration suppressed H2O2-induced reduction of FAK and Src phosphorylation. Blocking the activation of FAK or Src attenuated the protective effect of CsA on JEG-3 cells in H2O2-induced oxidative injury. CsA protects JEG-3 cells from H2O2-induced oxidative injury, and the FAK/Src signaling pathway plays an important role in this process.

Despite the important role that canine skin mast cells play in IgE-mediated allergic inflammation, clinically useful compounds for modulating mediator release from these cells or for suppressing cell response are lacking in the dog. The ability of five compounds to inhibit histamine release induced by non immunological (calcium ionophore A23187 and substance P) and IgE-dependent (concanavalin A) stimuli were compared. Sodium cromoglycate, a mast cell stabilizer, and dexamethasone, a glucocorticoid, failed to inhibit histamine release from isolated skin mast cells following any kind of stimulation. Salbutamol, a β-adrenergic agonist, exhibited inhibitory activity (46.0%) only after concanavalin A activation. In contrast, rolipram, a selective phosphodiesterase IV inhibitor and cyclosporin A, an immunosuppressor, showed potent anti allergic actions, inhibiting both IgE-dependent and -independent stimuli. Rolipram inhibited 42.8%, 44.7% and 19.2% of the mediator release induced by ionophore A23187, substance P and concanavalin A, respectively. Similarly cyclosporin A induced 85.9%, 14.9% and 67.3% inhibition after ionophore A23187, substance P and concanavalin A stimulation, respectively. These results suggest that rolipram and cyclosporin A merit to be clinically tested as agents for the treatment of chronic allergic diseases in the dog.

Mitochondrial permeability transition is caused by the opening of the Cyclosporin A (CSA) dependent calcium-induced large pore, known as the Permeability Transition Pore (PTP). PTP activation is believed to be a central event in stress-induced cell death. However, the molecular details of PTP opening remain incompletely understood. PTP opening makes mitochondrial inner membrane permeable to the molecules up to 1.5 kDa in size. Solute equilibration with the media in combination with swelling due to the PTP opening make mitochondria optically transparent - a phenomenon that has been widely used as a bona fide "light-scattering" PTP detection method in isolated mitochondria. Here, we utilized holographic microscopy imaging to monitor mitochondrial optical density changes that occur during solute equilibration between matrix and cytoplasm and thus enabled us to assess PTP induction in living cells. This approach relies on label-free, real-time mitochondrial visualization due to refractive index (RI) differences between the mitochondrial matrix and cytoplasm in the intact cells. PTP activation was detected as the decrease in mitochondrial RI. These measurements were done in parallel with measurements of the mitochondrial membrane potential, using the fluorescent probe TMRM. In intact HAP 1 cells, we found that calcium stress caused CSA-sensitive depolarization of the mitochondrial inner membrane. Unexpectedly, high-conductance PTP did not occur until after nearly complete mitochondrial membrane depolarization. In cells lacking c and {delta}; subunits of the ATP synthase, we observed calcium-induced and CSA-sensitive depolarization but not high-conductance PTP. We demonstrate that holographic imaging is a powerful novel tool with unique capabilities that allow measurement of PTP in living cells with high temporal and spatial resolution. We conclude that contrary to the widely accepted view, in living cells, high-conductance PTP is not the cause of calcium-induced membrane depolarization. Further, we provide direct evidence that ATP synthase is essential for high-conductance PTP, but not for calcium-induced CSA-sensitive membrane depolarization. We propose that PTP activation occurs as a two-phase process, where the first phase of the initial membrane depolarization is followed by the second phase of large pore opening that results in high-amplitude membrane permeabilization.

Acute antibody-mediated rejection (AAMR) is an important cause of cardiac allograft dysfunction, and more effective strategies need to be explored to improve allograft prognosis. Interleukin (IL)-6/IL-6R signaling plays a key role in the activation of immune cells including B cells, T cells and macrophages, which participate in the progression of AAMR. In this study, we investigated the effect of IL-6/IL-6R signaling blockade on the prevention of AAMR in a mouse model. We established a mouse model of AAMR for cardiac transplantation via presensitization of skin grafts and addition of cyclosporin A, and sequentially analyzed its features. Tocilizumab, anti-IL-6R antibody, and recipient IL-6 knockout were used to block IL-6/IL-6R signaling. We demonstrated that blockade of IL-6/IL-6R signaling significantly attenuated allograft injury and improved survival. Further mechanistic research revealed that signaling blockade decreased B cells in circulation, spleens, and allografts, thus inhibiting donor-specific antibody production and complement activation. Moreover, macrophage, T cell, and pro-inflammatory cytokine infiltration in allografts was also reduced. Collectively, we provided a highly practical mouse model of AAMR and demonstrated that blockade of IL-6/IL-6R signaling markedly alleviated AAMR, which is expected to provide a superior option for the treatment of AAMR in clinic.

TASK-1, TWIK-related acid-sensitive potassium channel 1, is a member of the two-pore- domain potassium channel family. It is constitutively active at resting potentials and strongly expressed in the heart. However, little is known about the role of TASK-1 channels in hypoxia. A cellular model of hypoxia and reoxygenation from rat heart-derived H9c2 cells or TASK-1 deficient HEK293T cells was employed to explore the role of TASK-1 channels in cytoprotection against hypoxia. The cell viability assay revealed that TASK-1 expression increased the number of viable cells subjected to 2 h of hypoxia followed by 2 h of reoxygenation (H/R). To dissect the protective role of TASK-1 on mitochondrial function, mitochondrial membrane potential (MMP) was assessed by tetramethylrhodamine fluorescence. It was demonstrated that MMP was significantly decreased by H/R, but it was maintained by TASK-1 expression or pretreatment with cyclosporin A, an inhibitor of mitochondrial permeability transition pore (mPTP). The effect of cyclosporin A on MMP was not further altered by TASK-1 expression. Moreover, TASK-1 expression significantly blocked cytochrome c release induced by H/R. While a small fraction of endogenous TASK-1 was found to colocalize with the mitochondrial marker MitoTracker in H9c2 cells, H/R did not alter the extent of colocalization of TASK-1 with MitoTracker. The total TASK-1 protein level was not significantly affected by H/R. In summary, we provided the evidence that TASK-1 channels confer cytoprotection against hypoxia-reoxygenation injury, possibly by their capacity of maintaining the mitochondrial membrane potential via inhibiting MPTP opening.

Patient: Female, 40-year-old Final Diagnosis: Hemophagocytic Lymphohistiocytosis Symptoms: Chills • fatigue • fever • myalgia • sweating Medication: — Clinical Procedure: — Specialty: Infectious Diseases Objective: Rare disease Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome. It is a result of uncontrolled hyperactivation of the reticuloendothelial system with a release of a huge amount of proinflammatory cytokines in the bloodstream, causing a cytokine storm. It may be primary due to genetic defects and secondary, triggered by viruses, bacteria, parasites, lymphoproliferative, and autoimmune diseases. We present a rare case of HLH triggered by leishmaniasis. HLH accounts for only about 1% of all leishmaniasis cases. Case Report: The patient was a 40-year-old previously healthy woman, who has been diagnosed with secondary HLH caused by leishmaniasis. A chronic Epstein-Barr virus (EBV) infection was initially mistakenly interpreted as a trigger agent, because Leishmania amastigotes, present on the first bone marrow biopsy, were not recognized. The treatment with cyclosporin A and corticosteroids suppressed the cytokine storm and prevented the development of complications. Two months later, because of a reactivation of the disease, the patient was referred to a hematologist. A second bone marrow aspiration was performed, in which numerous Leishmania parasites were finally identified. Conclusions: The aim of this case report is to provide more information about the rare phenomenon of secondary HLH triggered by leishmaniasis. Early recognition of the syndrome and its triggering agents will improve disease outcomes and prevent unnecessary treatment with immunosuppressive drugs.

Monothiol glutaredoxins are important regulators of iron homeostasis that play conserved roles in the sensing and trafficking of iron-sulfur clusters. We previously characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis, the interaction with the iron regulator Cir1, and virulence in Cryptococcus neoformans. This important fungal pathogen causes cryptococcal meningoencephalitis in immunocompromised individuals worldwide. Here, we demonstrate that Grx4 is required for proliferation at elevated temperatures (both 37°C and 39°C) and under stress conditions. In particular, the grx4Δ mutant was hypersensitive to SDS, calcofluor white (CFW), and caffeine, suggesting that Grx4 is required for membrane and cell wall integrity (CWI). In this context, we found that Grx4 regulated the phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) of the CWI pathway in cells grown at elevated temperature or upon treatment with CFW, caffeine, or SDS. The grx4Δ mutant also displayed increased sensitivity to FK506 and cyclosporin A, two inhibitors of the calcineurin pathway, indicating that Grx4 may influence growth at higher temperatures in parallel with calcineurin signaling. Upon thermal stress or calcium treatment, loss of Grx4 also caused partial mis-localization of Crz1, the transcription factor that is a calcineurin substrate. The phenotypes of the grx4Δ, crz1Δ, and cna1Δ (calcineurin) mutants suggest shared contributions to the regulation of temperature, cell wall, and other stresses. In summary, we show that Grx4 is also a key regulator of the responses to a variety of stress conditions in addition to its roles in iron homeostasis in C. neoformans.

The neural precursor cell expressed by developmentally downregulated gene 4-2 (NEDD4-2) is a ubiquitin E3 ligase that has a high affinity toward binding and ubiquitinating glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type subunit 1 (GRIA1, also referred to GluR1 or GluA1). Since dysregulation of GRIA1 surface expression is relevant to the responsiveness to AMPA receptor (AMPAR) antagonists (perampanel and GYKI 52466) in chronic epilepsy rats, it is likely that NEDD4-2 may be involved in the pathogenesis of intractable epilepsy. However, the role of NEDD4-2-mediated GRIA1 ubiquitination in refractory seizures to AMPAR antagonists is still unknown. In the present study, both AMPAR antagonists recovered the impaired GRIA1 ubiquitination by regulating protein phosphatase 2B (PP2B)-extracellular signal-regulated kinase 1/2 (ERK1/2)-serum and glucocorticoid-regulated kinase 1 (SGK1)-NEDD4-2 signaling pathway in responders (whose seizure activities are responsive to AMPAR), but not non-responders (whose seizure activities were uncontrolled by AMPAR antagonists). In addition, cyclosporin A (CsA, a PP2B inhibitor) co-treatment improved the effects of AMPAR antagonists in non-responders, independent of AKT signaling pathway. Therefore, our findings suggest that dysregulation of PP2B-ERK1/2-SGK1-NEDD4-2-mediated GRIA1 ubiquitination may be responsible for refractory seizures and that this pathway may be a potential therapeutic target for improving the treatment of intractable epilepsy in response to AMPAR antagonists.

Inhibitors of calcineurin phosphatase activity (CNIs) such as cyclosporin A (CsA) are widely used to treat tissue transplant rejection and acute graft-versus-host disease (aGVHD), for which inhibition of gene expression dependent on nuclear factor of activated T cells (NFAT) is the mechanistic paradigm. We recently reported that CNIs inhibit TCR-proximal signaling by preventing calcineurin-mediated dephosphorylation of Lck(S59), an inhibitory modification, raising the possibility of another mechanism by which CNIs suppress immune responses. Here we used T cells from mice that express Lck(S59A), which cannot accept a phosphate at residue 59, to initiate aGVHD. Although CsA inhibited NFAT-dependent gene upregulation in allo-aggressive T cells expressing either Lck(WT) or Lck(S59A), it was ineffective in treating disease when the T cells expressed Lck(S59A). Two important NFAT-independent T cell functions were found to be CsA-resistant in Lck(S59A) T cells: upregulation of the cytolytic protein perforin in tissue-infiltrating CD8(+) T cells and antigen-specific T/DC adhesion and clustering in lymph nodes. These results demonstrate that effective treatment of aGVHD by CsA requires NFAT-independent inhibition of TCR signaling. Given that NFATs are widely expressed and off-target effects are a major limitation in CNI use, it is possible that targeting TCR-associated calcineurin directly may provide effective therapies with less toxicity.

Honokiol is a pharmacologically active small molecule with multifunctional antitumor effects. Although plenty of literature is available on honokiol-triggered apoptosis and programmed necrosis, few studies have investigated the potential existence of death mode transition from apoptosis to programmed necrosis. In the current study, we demonstrated that the necrotic cell population (PI-positive) gradually increased and the early-stage apoptotic cell population (PI-negative and AV-positive) decreased in a dose- and time-dependent manner following honokiol treatment. Furthermore, we demonstrated that these PI-positive cells were under necrotic cell death, since no late-apoptosis characteristics including conspicuous chromatin condensation or DNA ladder patterns were detected. These results demonstrated that cells suffered death mode transition from early-stage apoptosis to programmed necrosis with the increase of honokiol dose or treatment time. The protein expression of RIP3 markedly increased in parallel with HNK-triggered death mode transition, while the expression of RIP1 decreased. Cyclophilin D expression increased during cell death mode transition, and inhibition of cyclophilin D by cyclosporin A clearly blocked HNK-triggered programmed necrosis. These data indicated that honokiol-induced programmed necrosis and death mode transition are potentially RIP3‑dependent, cyclophilin D-regulated. Further results showed that blocked cyclophilin D by cyclosporin A inhibited HNK-induced necrosis, but did not affect HNK-induced RIP3 overexpression. This indicated that cyclophilin D was a potential modulator at downstream of RIP3. In conclusion, honokiol triggers a potential RIP3-dependent cell death mode transition from early-stage apoptosis to programmed necrosis, which is highly regulated by cyclophilin D.

Cyclosporin A (CsA) is a well-known and effective drug that is commonly used in autoimmune diseases and allotransplantation. However, kidney toxicity and cardiotoxicity limit its use. Circular RNAs (circRNAs) play a crucial role in disease, especially cardiovascular disease. We aimed to explore the circRNA expression profiles and potential mechanisms during CsA-induced cardiotoxicity. Sixty male adult Wistar rats were randomly divided into two groups. The CsA group was injected with CsA (15 mg/kg/day body weight) intraperitoneally (ip) for 2 weeks, whereas the control group was injected ip with the same volume of olive oil. We assessed CsA-induced cardiotoxicity by light microscopy, transferase-mediated dUTP nick-end labeling (TUNEL) staining, and electron microscopy. Microarray analysis was used to detect the expression profiles of circRNAs deregulated in the heart during CsA-induced cardiotoxicity. We confirmed the changes in circRNAs by quantitative PCR. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the microarray data were performed. A conventional dose of CsA induced cardiotoxicity in rats. We identified 67 upregulated and 37 downregulated circRNAs compared with those in the control group. Six of 12 circRNAs were successfully verified by quantitative real-time polymerase chain reaction (qRT-PCR). GO analyses of the differentially expressed circRNAs indicated that these molecules might play important roles in CsA-induced cardiotoxicity. KEGG pathway analyses showed that the differentially expressed circRNAs in CsA-induced cardiotoxicity may be related to autophagy or the Hippo signaling pathway. We identified differential circRNA expression patterns and provided more insight into the mechanism of CsA-induced cardiotoxicity. CircRNAs may serve as potential biomarkers or therapeutic targets of CsA-mediated cardiotoxicity in the future.

Ultraviolet (UV) irradiation causes cellular oxidative stress. Under redox imbalance, Keap1-dependent Nrf2 degradation is minimal. In this study, we examined the role of Ca2+ in Nrf2 homeostasis after UVB irradiation using human dermal fibroblasts. UVB irradiation stimulates 12-lipoxygenase and the product 12-hydroxyeicosatetraenoic acid then activates TRPV1 increasing the cell's cytosolic Ca2+ concentration. UVB irradiation induced reactive oxygen species generation and apoptosis are inhibited in the absence of Ca2+ or in the presence of either a 12-lipoxygenase inhibitor or a TRPV1 inhibitor during and after UVB irradiation. Thus, the Ca2+ increase via TRPV1 is a critical factor in UVB irradiation induced oxidative stress. UVB irradiation induces a Ca2+ dependent Nrf2 degradation and thus activation of TRPV1 with 12-hydroxyeicosatetraenoic acid also decreasing Nrf2 levels. UVB irradiation induced Nrf2 degradation is inhibited by co-treatment of cells with W-7, cyclosporin A, SB-216763 or MG-132, which are inhibitors of calmodulin, calcineurin, GSK3β and the proteasome, respectively. Furthermore, UVB irradiation in parallel induces GSK3β dephosphorylation in a Ca2+ dependent manner. Co-immunoprecipitation showed that UVB irradiation induces an increase in Nrf2 phosphorylation, an increase in the binding of β-TrCP and Nrf2, and an increase in Nrf2 ubiquitination; these effects are all Ca2+ dependent. These findings suggest that UVB irradiation induced GSK3β activation in a Ca2+ dependent manner, which then stimulates the phosphorylation and ubiquitination of Nrf2 via β-TrCP. Indeed, silencing of β-TrCP was found to inhibit UVB irradiation-induced oxidative stress, Nrf2 degradation and apoptosis, while it had no effect on the Ca2+ increase. Taken together, our results suggest that a Ca2+ influx via TRPV1 is responsible for UVB irradiation-induced Nrf2 degradation and that modulation of the Ca2+-calmodulin-calcineurin-GSK3β-Nrf2-β-TrCP-Cullin-1 pathway may explain Ca2+ dependent Nrf2 degradation.

Cyclosporin A (CsA) does not only exert a toxic effect on kidney parenchymal cells, but also protects them against necrotic cell death by inhibiting opening of mitochondrial permeability transition pore. However, whether CsA plays a role in hydrogen peroxide-induced kidney proximal tubular cell death is currently unclear. In the present study, treatment with CsA further increased apoptosis and necrosis in HK-2 human kidney proximal tubule epithelial cells during exposure to hydrogen peroxide. In addition, hydrogen peroxide-induced p53 activation and BH3 interacting-domain death agonist (BID) expression were higher in CsA-treated cells than those in non-treated cells, whereas hydrogen peroxide-induced activation of mitogen-activated protein kinases including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase and activation of protein kinase B were not significantly altered by treatment with CsA. In oxidant-antioxidant system, reactive oxygen species (ROS) production induced by hydrogen peroxide was further enhanced by treatment with CsA. However, expression levels of antioxidant enzymes including manganese superoxide dismutase, copper/zinc superoxide dismutase, and catalase were not altered by treatment with hydrogen peroxide or CsA. Treatment with CsA further enhanced mitochondrial membrane potential induced by exposure to hydrogen peroxide, although it did not alter endoplasmic reticulum stress based on expression of glucose-regulated protein 78 and 94. Taken together, these data suggest that CsA can aggravate hydrogen peroxide-induced cell death through p53 activation, BID expression, and ROS production.

The alteration of thyroxine (T(4)) cellular uptake by an environmental chemical can serve as a contributing factor in thyroid hormone (TH) disruption. This study describes a non-radiolabeled (LC-MS/MS) oil-filtration technique designed to characterize the mechanism(s) responsible for T(4) cellular uptake in cryopreserved rat hepatocyte suspensions. The environmental chemicals perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) that are known thyroid-disrupting agents were evaluated for their potential effect on T(4) cellular uptake. At 37°C, hepatic T(4) uptake demonstrated saturable kinetics with increasing T(4) concentrations (0.1–15 μM), while a linear uptake rate was detected at 4°C that is consistent with passive diffusion. Michaelis-Menten analysis of carrier-mediated (enzymatic) T(4) uptake (37–4°C) displayed a V(max) value of 3.06 pmoles/10(6) cells/sec and a K(M) value of 2.93 μM and was determined to be the predominant uptake process (>70%) versus passive diffusion. Cyclosporin A (CsA) chemically inhibited T(4) uptake rates with no discernable effect on passive diffusion, whereas PFOA/PFOS showed no inhibitory effect on T(4) uptake. However, T(4) uptake assays conducted with PFOA/PFOS and the T(4) serum carrier protein transthyretin (TTR) displayed a dose-response increase in hepatic T(4) uptake rates correlating with increased T(4) free fraction values. Our findings indicate the kinetic determinant of T(4) hepatic uptake was predominantly carrier-mediated as demonstrated by an enhanced first-order rate of T(4) uptake compared to passive diffusion. These in vitro findings provide new mechanistic and physiological insight regarding decreased T(4) serum concentrations (hypothyroxinemia) previously observed within in vivo rodent studies following perfluorinated chemical exposure.

Hepatitis B virus (HBV) infection, which increases the risk of cirrhosis and hepatocellular carcinoma and requires lifelong treatment, has become a major global health problem. However, host factors essential to the HBV life cycle are still unclear, and the development of new drugs is needed. Cells derived from the human hepatoma cell line HepG2 and engineered to overexpress sodium taurocholate cotransporting polypeptide (NTCP: a receptor for HBV), termed HepG2/NTCP cells, are widely used as the cell-based HBV infection and replication systems for HBV research. We recently found that human hepatoma cell line Li23-derived cells overexpressing NTCP (A8 cells subcloned from Li23 cells), whose gene expression profile was distinct from that of HepG2/NTCP cells, were also sensitive to HBV infection. However, the HBV susceptibility of A8 cells was around 1/100 that of HepG2/NTCP cells. Since we considered that plural cell assay systems will be needed for the objective evaluation of anti-HBV reagents, as we previously demonstrated in hepatitis C virus research, we here attempted to develop a new Li23 cell-derived assay system equivalent to that using HepG2/NTCP cells. By repeated subcloning of A8 cells, we successfully established a new cell line (A8.15.78.10) exhibiting high HBV susceptibility equal to that of HepG2/NTCP cells. Characterization of A8.15.78.10 cells revealed that the increase of HBV susceptibility was correlated with increases in the protein and glycosylation levels of NTCP, and with decreased expression of STING, a factor contributing to innate immunity. Finally, we performed a comparative evaluation of HBV entry inhibitors (cyclosporin A and rosiglitazone) by an HBV/secNL reporter assay using A8.15.78.10 cells or HepG2/NTCP cells. The results confirmed that cyclosporin A exhibited anti-HBV activity in both cell lines, as previously reported. However, we found that rosiglitazone did not show the anti-HBV activity in A8.15.78.10 cells, although it worked in HepG2/NTCP cells as previously reported. This suggested that the difference in anti-HBV activity between cyclosporin A and rosiglitazone was due to the different types of cells used for the assay. In conclusion, plural assay systems using different types of cells are required for the objective and impartial evaluation of anti-HBV reagents.

Background: Subcellular expression of P-glycoprotein (P-gp) may play an essential role in multidrug resistance (MDR) in many cancers. However, the mitochondria expression and functional activity of P-gp in ovarian cancer are still unclear. In this study, we isolated mitochondria from A2780 cell line and its paclitaxel-resistant subline A2780T and investigated the expression and function of mitochondria P-gp. Methods: Immunocytochemistry was used to evaluate P-gp expression and subcellular localization in cancer cells. Immunofluorescence and laser confocal microscopy were used to detect the co-localization of P-gp and mitochondria both in ovarian cancer tissues and in cell lines. Western blotting (WB), transmission electron microscopy and JC-1 kit were used to evaluate the purity, integrity and activity of the isolated mitochondria. P-gp expression in the whole cell and the isolated mitochondria was evaluated by WB. Flow cytometry was used to evaluate the efflux function of mitochondria P-gp. Results: P-gp expression was detected at the membrane, cytoplasm and nuclei of the A2780T cells, but not in the A2780 cells. Co-localization of P-gp and mitochondria was observed in the A2780T cell line and ovarian cancer tissues, but not in A2780 cells. The purity, integration and activity of the isolated mitochondria are high. P-gp was highly expressed in the A2780T cells and the isolated mitochondria, but was not found in A2780 cells. Rho123 efflux rate was significantly increased in isolated A2780T mitochondria compared to those in A2780 (43.2% vs 9.6%), but it was partly reversed by cyclosporin A (CsA, a P-gp inhibitor). Conclusion: P-gp is highly expressed in mitochondria of taxol-resistant ovarian cancer cells and ovarian cancer tissues and mediates the drug efflux, which probably protect cancer cells from chemotherapy.

Cyclosporin A (CSA) is a widely used drug to prevent the immune cell function. It is well known that CSA blocks transcription of cytokine genes in activated T cells. The connection between T cells and CSA has been well established. However, the effect of CSA on natural killer (NK) cells is not thoroughly understood. Therefore, in the present study, splenocytes and peripheral blood mononuclear cells (PBMCs) were treated with CSA in the presence of concanavalin A (Con A) or interleukin-2 (IL-2). CSA at higher concentrations induces apoptosis and inhibition of proliferation, while lower concentrations showed synergistically enhanced proliferation in splenocytes and PBMCs. Further, CSA favored the in vitro conversion of CD3+CD161+ cells. Splenocytes and PBMC were found to have synergistic proliferation with Con A, and PBMC exhibited significantly higher expression of NKp30, NKp44, and granzyme B along with enhanced cytotoxicity against K-562 cells in CSA-treated animals. Proliferation assay also showed that proliferation of CD161+ cells was higher in CSA-treated animals. Collectively, our results suggest that CSA differentially influences the population, function, and expression of the NK cell phenotype.

Objectives During fasting, hepatic gluconeogenesis is induced to maintain energy homeostasis. Moreover, abnormal dysregulation of hepatic glucose production is commonly observed in type 2 diabetes. However, the signaling components controlling hepatic glucose production to maintain normal glucose levels are not fully understood. Here, we examined the physiological role of Down syndrome critical region 1–4 (DSCR1-4), an endogenous calcineurin signaling inhibitor in the liver that mediates metabolic adaptation to fasting. Methods We assessed the effect of cyclosporine A, an inhibitor of calcineurin signaling on gluconeogenic gene expression in primary hepatocytes. DSCR1-4 expression was examined in diet- and genetically-induced mouse models of obesity. We also investigated the metabolic phenotype of a single extra copy of DSCR1-4 in transgenic mice and how DSCR1-4 regulates glucose homeostasis in the liver. Results Treatment with cyclosporin A increased hepatic glucose production and gluconeogenic gene expression. The expression of DSCR1-4 was induced by refeeding and overexpressed in obese mouse livers. Moreover, transgenic mice with a single extra copy of DSCR1-4 exhibited pyruvate intolerance and impaired glucose homeostasis. Mechanistically, DSCR1-4 overexpression increased phosphorylation of the cAMP response element-binding protein, which led to elevated expression levels of gluconeogenic genes and, thus, enhanced hepatic glucose production during fasting. Conclusion A single extra copy of DSCR1-4 results in dysregulated hepatic glucose homeostasis and pyruvate intolerance. Our findings suggest that nutrient-sensitive DSCR1-4 is a novel target for controlling hepatic gluconeogenesis in diabetes., Highlights • DSCR1 mRNA and protein levels are increased in livers upon nutrient availability. • DSCR1-4 is overexpressed in diet- or genetically induced obesity. • DSCR1-4 trisomy mice exhibit impaired glucose homeostasis and pyruvate intolerance. • Trisomy of DSCR1-4 leads to increased hepatic glucose production.

A clinical case of development of secondary hemophagocytic syndrome is presented on the background of sepsis caused by Serratia proteamaculans, in a child of monthly age, culminating in recovery. Hemophagocytic syndrome in the child developed on the 32nd day of life and was accompanied by fever, hepatosplenomegaly, anemia, thrombocytopenia, elevated blood levels of triglycerides and ferritin. Blood culture was positive for Serratia proteamaculans. Isolate of Serratia proteamaculans was resistant to aminopenicillins (including protected ones), 1–4 generations of cephalosporins and aminoglycosides but sensitive to ciprofloxacin and moderately sensitive to imipenem and meropenem. The duration of fever in the child was seven days, thrombocytopenia, hyperfertritinemia and hypertriglyceridemia – 28 days. The normalization of the indices of ferritin, triglycerides and platelets coincided with the reduction of bacteremia and high values of the C-reactive protein. The duration of bacteremia was three weeks: the isolation of Serratia proteamaculans from the blood occurred three times. Elevated levels of C-reactive protein in the blood were noted for 30 days. Reduced hemoglobin and red blood cells counts were noted for eight weeks. Positive dynamics of the hemophagocytic syndrome occurred as a result of measures aimed at treating sepsis – antibiotic therapy with imipenem in combination with ciprofloxacin, appointed by the decision of the medical commission on the basis of the sensitivity of Serratia proteamaculans to antibiotics, intravenous immunoglobulin, and infusion therapy. Glucocorticosteroids and cytotoxic agents (etoposide, cyclosporin A) have not been used in this case.

Background The efficacy and anti-infective effect of high-dose intravenous immunoglobulin (HDIVIG) in severe or very severe aplastic anemia children were evaluated. Patients and methods In total, 61 patients who underwent immunosuppressive therapy were retrospectively reviewed. The non-IVIG group (30 cases) received rabbit-antithymocyte protein (R-ATG, 3 to 5 mg/kg/d, for 5 consecutive days)+cyclosporin A (CSA), and the HDIVIG group (31 cases) underwent R-ATG+CSA+immunoglobulin (1 g/kg/d, for 2 consecutive days, once a month, 6 times). Results The early effective rate was higher in the HDIVIG group (P=0.020). However, the long-term effective rate and the 5-year overall survival rates difference were not statistically significant (P=0.717, 0.419). The infection rate and severe infection rate in the HDIVIG group were lower (P=0.003, 0.008). The infection-related mortality differences were not statistically significant after ATG application (P>0.05). In the HDIVIG group, 9 patients were nonresponders. Among the nonresponders, 8 patients' first-dose IVIG was given within 7 days before ATG. Conclusions HDIVIG may increase the early effective rate and reduce early infection and serious infection for aplastic anemia children, but failed to reduce the infection-related mortality.

Glycosylation is ubiquitous throughout the central nervous system and altered following spinal cord injury (SCI). The glial scar that forms following SCI is composed of several chondroitin sulfate proteoglycans, which inhibit axonal regrowth. Cyclosporin-A (CsA), an immunosuppressive therapeutic, has been proposed as a potential treatment after SCI. We investigated CsA treatment in the spinal cord of healthy, contusion injured, and injured CsA-treated rats. Lectin histochemistry using fluorescently labeled lectins, SBA, MAA, SNA-I, and WFA, was performed to identify the terminal carbohydrate residues of glycoconjugates within the spinal cord. SBA staining decreased in gray and white matter following spinal cord injury, whereas staining was increased at the lesion site in CsA-treated animals, indicating an increase in galactose and N-acetylgalactosamine terminal structures. No significant changes in MAA were observed. WFA staining was abundant in gray matter and observed to increase at the lesion site, in agreement with increased expression of chondroitin sulfate proteoglycans. SNA-I-stained blood vessels in all spinal cord regions and dual staining identified a subpopulation of astrocytes in the lesion site, which expressed ¿-(2,6)-sialic acid. Glycosylation were altered in injured spinal cord treated with CsA, indicating that glycosylation and alteration of particular carbohydrate structures are important factors to consider in the examination of the environment of the spinal cord after injury. This work was supported by the EU FP7 programme in support of GlycoHIT (Grant no. 260600) and Science Foundation Ireland (Grant no. 13/RC/2073) in support of the Centre for Research in Medical Devices (CÚRAM). M.K. is grateful to the Royal Society of Chemistry Analytical Chemistry Trust Fund (ACTF) for the ACTF Fellowship 2018. The authors acknowledge the facilities and scientific and technical assistance (Mark Canney and Dr. Kerry Thompson) of the Centre for Microscopy and Imaging at the National University of Ireland Galway (www.imaging.nuigalway.ie), a facility which is co-funded by the Irish Government’s Programme for Research in Third Level Institutions, Cycles 4 and 5, National Development Plan 2007–2013. peer-reviewed

This study aims to investigate the effects and the molecular mechanism of cyclosporin A (CsA) against oxidative stress injury in cultured neonatal rat cardiomyocytes. Bax/Bcl-2, cl-casp-9/casp-9, cl-casp-3/casp-3, and iNOS/β-actin ratios and p-IκB and IκB levels were analyzed by western blot. IL-1β and TNF-α levels were analyzed by ELISA. CsA effectively improved the cell viability and reduced the extracellular lactate dehydrogenase release in cardiomyocytes after H2O2-induced oxidative damage. CsA significantly increased the superoxide dismutase activity, glutathione production, and catalase activity but decreased the malonaldehyde level. CsA treatment considerably reduced the H2O2-induced intracellular generation of reactive oxygen species, mitochondrial dysfunction, and release of cytochrome c. CsA could act against H2O2-induced ATP reduction, TCA cycle enzymes, mitochondrial complex I enzyme, and complex V enzyme in cardiomyocytes. CsA significantly decreased the Bax/Bcl-2 ratio, cl-casp-9/casp-9, and cl-casp-3/casp-3 in a concentration-dependent manner. CsA also remarkably reduced the cleaved PARP level and DNA fragmentation. NF-κB was closely related to oxidative stress injury. CsA inhibited NF-κB activation, thereby preventing the upregulation of IL-1β, TNF-α, iNOS, and intracellular NO release. CsA protected cardiomyocytes against H2O2-induced cell injury. Hence, CsA may be developed as a candidate drug to prevent or treat myocardial ischemia reperfusion injury.

Immunosuppressive drugs such as cyclosporin A (CsA) can elicit hepatotoxicity by affecting gene expression. Here, we address the link between CsA and large-scale chromatin organization in HepG2 hepatocarcinoma cells. We show the existence of lamina-associated domains (LADs) interacting with lamin A, lamin B, or both. These ‘A-B’, ‘A-only’ and ‘B-only’ LADs display distinct fates after CsA treatment: A-B LADs remain constitutive or lose A, A-only LADs mainly lose A or switch to B, and B-only LADs remain B-only or acquire A. LAD rearrangement is overall uncoupled from changes in gene expression. Three-dimensional (3D) genome modeling predicts changes in radial positioning of LADs as LADs switch identities, which are corroborated by fluorescence in situ hybridization. Our results reveal interplay between A- and B-type lamins on radial locus positioning, suggesting complementary contributions to large-scale genome architecture. The data also unveil a hitherto unsuspected impact of cytotoxic drugs on genome conformation.Abbreviations: ChIP-seq: chromatin immunoprecipitation sequencing; CsA: cyclosporin A; FISH; fluorescence in situ hybridization; ICMT: isoprenylcysteine methyltransferase; LAD: lamina-associated domain; TAD: topologically-associated domain

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated, apical anion channel that regulates ion and fluid transport in many epithelia including the airways. We have previously shown that cigarette smoke (CS) exposure to airway epithelia causes a reduction in plasma membrane CFTR expression which correlated with a decrease in airway surface hydration. The effect of CS on CFTR was dependent on an increase in cytosolic Ca2+. However, the underlying mechanism for this Ca2+-dependent, internalisation of CFTR is unknown. To gain a better understanding of the effect of Ca2+ on CFTR, we performed whole cell current recordings to study the temporal effect of raising cytosolic Ca2+ on CFTR function. We show that an increase in cytosolic Ca2+ induced a time-dependent reduction in whole cell CFTR conductance, which was paralleled by a loss of cell surface CFTR expression, as measured by confocal and widefield fluorescence microscopy. The decrease in CFTR conductance and cell surface expression were both dynamin-dependent. Single channel reconstitution studies showed that raising cytosolic Ca2+ per se had no direct effect on CFTR. In fact, the loss of CFTR plasma membrane activity correlated with activation of calcineurin, a Ca2+-dependent phosphatase, suggesting that dephosphorylation of CFTR was linked to the loss of surface expression. In support of this, the calcineurin inhibitor, cyclosporin A, prevented the Ca2+-induced decrease in cell surface CFTR. These results provide a hitherto unrecognised role for cytosolic Ca2+ in modulating the residency of CFTR at the plasma membrane through a dynamin- and calcineurin-dependent mechanism.

Mitochondrial permeability transition pore (MPTP) has been associated to calcium homeostasis and reactive oxygen species (ROS) generation in several cell types. While extensively investigated in somatic cells, there are few data regarding MPTP phenomenon in gametes. The aim of the present work was to investigate MPTP occurrence in sea urchin female gametes. The protonophores CCCP and FCCP, and the Ca2+ ionophore ionomycin, were used as pore inductors. Pore opening was monitored by mitochondrial potential sensitive probes and cobalt-quenched calcein assay. The pore desensitizer cyclosporin A (CsA) prevented the loss of mitochondrial inner membrane potential (ΔΨm) and pore opening induced by MPTP activators. The disruption of ΔΨm led to an increase in ROS generation, which was completely prevented by CsA. Our data also demonstrated that the increase in ROS production induced by MPTP opening requires extracellular Ca2+. In summary, the current study provides evidence about the occurrence of MPTP in sea urchin eggs in a similar manner as described in vertebrate somatic cells - CsA-sensitive, voltage- and Ca2+-triggered - and shows MPTP as a highly conserved physiological event through the evolution.

SummaryOver the past 20 years, the emergence of three highly pathogenic coronaviruses (CoV) – SARS-CoV, MERS-CoV, and most recently SARS-CoV-2 – has shown that CoVs pose a serious risk to human health and highlighted the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycle. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens to identify pan-CoV host factors required for the replication of both endemic and emerging CoVs, including the novel CoV SARS-CoV-2. Strikingly, we found that several autophagy-related genes, including the immunophilin FKBP8, TMEM41B, and MINAR1, were common host factors required for CoV replication. Importantly, inhibition of the immunophilin family with the compounds Tacrolimus, Cyclosporin A, and the non-immunosuppressive derivative Alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures that resemble the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

Iron overload injury is considered to be a part of blood stasis syndrome of arthralgia in Traditional Chinese Medicine. The primary therapies are clearing away heat and detoxification, activating blood circulation, and removing blood stasis. Lonicera japonica flos (LJF) has long been known as an excellent antipyretic and antidote. Luteoloside (Lut) is one of the main components of LJF and exhibits antioxidant, anti-inflammatory, and cytoprotection properties. However, the protection of Lut against iron overload injury and its underlying mechanisms remain unclear. Therefore, HUVECs were exposed to 50 μM iron dextran for 48 h to establish an iron overload damage model and Lut’s effects were assessed. Our results showed that 20 μM Lut not only increased cell viability and decreased LDH activity, but also significantly upregulated DDAHⅡexpression and activity, increased p-eNOS/eNOS ratio and NO content, and reduced ADMA content on HUVECs exposed to Iron. Furthermore, Lut significantly attenuated intracellular/mitochondrial ROS generation, improved SOD, CAT, and GSH-Px activities, reduced MDA content, maintained MMP, inhibited mPTP opening, prevented cyt c from mitochondria released into cytoplasm, reduced cleaved-caspase3 expression, and ultimately decreased cell apoptosis induced by iron overload. The effects of Lut were similar to that of L-arginine (ADMA competitive substrate), cyclosporin A (mPTP blocker agent), and edaravone (free radical scavenger) as positive control reagent. However, adding pAD/DDAHⅡ-shRNA adenovirus could reverse the above beneficial effects of Lut. In conclusion, Lut could protect HUVECs against iron overload damage via ROS/ADMA/DDAHⅡ/eNOS/NO pathway. Mitochondria were the target organelles of Lut’s protection.

The rapid emergence of P. falciparum-resistant strains raises an urgent need to find new antimalarial drug candidates. This study reports the rational repositioning of the anti-Hepatitis C Virus drug, Alisporivir, a non-immunosuppressive analog of cyclosporin A (CsA) against multiple, drug-resistant strains of P. falciparum. Alisporivir being non-hemolytic has been proven to be a better drug than CsA. Indeed, our study also demonstrated the same. Alisporivir inhibited chloroquine-sensitive parasite growth with an IC50 of 196.6nM. Alisporivir also inhibited the growth of chloroquine-resistant parasites with an IC50 of 422.1nM. Alisporivir exhibited, anti-malarial activity in in vivo. Further, we exploited the Cyclophilins targeting potential of Alisporivir against artemisinin-resistant malaria parasite owing to the fact that PfCyP-19B is one of the genes that is overexpressed in artemisinin-resistant parasite revealed by a population transcriptomic study. Our semiquantitative real-time transcript and immunofluorescence analysis confirmed the overexpression of PfCyP-19B in Artemisinin-resistant P. falciparum (PfKelch13R539T). Artemisinin resistance is attributed to slow clearance of ring stage parasites. Ring survival assay (RSA) is designed to access the potency of compounds on these dormant slow clearing parasites leading to drug resistance. Thus, the potency of Alisporivir against PfKelch13R539T was evaluated by RSA. A 2.5-fold decrease in parasite survival was detected with Alisporivir. Further, combination of Alisporivir with DHA found to potentiate the efficacy of DHA by 4.55-fold. These results support the hypothesis that targeting of resistance mechanism is a potential approach to deal with resistant parasite. Overall, this study demonstrates the rational reposition of Alisporivir against resistant malaria resistance.

Aim: Reperfusion after myocardial ischemia causes cellular injury, in part due to changes in mitochondrial Ca2+ handling, oxidative stress, and myocyte energetics. We have previously shown that the 18-kDa translocator protein of the outer mitochondrial membrane (TSPO) can modulate Ca2+ handling. Here, we aim to evaluate the role of the TSPO in ischemia/reperfusion (I/R) injury.Methods: Rabbit ventricular myocytes underwent simulated acute ischemia (20 min) and reperfusion (at 15 min, 1 h, and 3 h) in the absence and presence of 50 μM PK11195, a TSPO inhibitor. Cell death was measured by lactate dehydrogenase (LDH) assay, while changes in mitochondrial Ca2+, membrane potential (ΔΨm), and reactive oxygen species (ROS) generation were monitored using confocal microscopy in combination with fluorescent indicators. Substrate utilization was measured with Biolog mitochondrial plates.Results: Cell death was increased by ~200% following I/R compared to control untreated ventricular myocytes. Incubation with 50 μM PK11195 during both ischemia and reperfusion did not reduce cell death but increased mitochondrial Ca2+ uptake and ROS generation. However, application of 50 μM PK11195 only at the onset and during reperfusion effectively protected against cell death. The large-scale oscillations in ΔΨm observed after ~1 h of reperfusion were significantly delayed by 1 μM cyclosporin A and almost completely prevented by 50 μM PK11195 applied during 3 h of reperfusion. After an initial increase, mitochondrial Ca2+, measured with Myticam, rapidly declined during 3 h of reperfusion after the initial transient increase. This decline was prevented by application of PK11195 at the onset and during reperfusion. PK11195 prevented a significant increase in succinate utilization following I/R and succinate-induced forward-mode ROS generation. Treatment with PK11195 was also associated with a significant increase in glutamate and a decrease in leucine utilization.Conclusion: PK11195 administered specifically at the moment of reperfusion limited ROS-induced ROS release and cell death, likely in part, by a shift from succinate to glutamate utilization. These data demonstrate a unique mechanism to limit cardiac injury after I/R.

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway., The nature of the mitochondrial permeability transition pore (PTP) is still under debate. Here, through genetically modified F-ATP synthase, the authors show that PTP formation can be mediated by F-ATP synthase or by adenine nucleotide translocator, suggesting the existence of distinct but related permeability pathways.

Although the mitochondrial permeability transition pore (PTP) is presumably formed by either ATP synthase or the ATP/ADP carrier (AAC), little is known about their differential roles in PTP activation. We explored the role of AAC and ATP synthase in PTP formation in Saccharomyces cerevisiae using bisindolylpyrrole (BP), an activator of the mammalian PTP. The yeast mitochondrial membrane potential, as indicated by tetramethylrhodamine methyl ester signals, dissipated over 2&ndash, 4 h after treatment of cells with 5 &mu, M BP, which was sensitive to cyclosporin A (CsA) and Cpr3 deficiency and blocked by porin1/2 deficiency. The BP-induced depolarization was inhibited by a specific AAC inhibitor, bongkrekate, and consistently blocked in a yeast strain lacking all three AACs, while it was not affected in the strain with defective ATP synthase dimerization, suggesting the involvement of an AAC-associated pore. Upon BP treatment, isolated yeast mitochondria underwent CsA- and bongkrekate-sensitive depolarization without affecting the mitochondrial calcein signals, indicating the induction of a low conductance channel. These data suggest that, upon BP treatment, yeast can form a porin1/2- and Cpr3-regulated PTP, which is mediated by AACs but not by ATP synthase dimers. This implies that yeast may be an excellent tool for the screening of PTP modulators.

Background Failure to clear Aβ from the brain is partly responsible for Aβ brain accumulation in Alzheimer’s disease (AD). A critical protein for clearing Aβ across the blood-brain barrier is the efflux transporter P-glycoprotein (P-gp). In AD, P-gp levels are reduced, which contributes to impaired Aβ brain clearance. However, the mechanism responsible for decreased P-gp levels is poorly understood and there are no strategies available to protect P-gp. We previously demonstrated in isolated brain capillaries ex vivo that human Aβ40 (hAβ40) triggers P-gp degradation by activating the ubiquitin-proteasome pathway. In this pathway, hAβ40 initiates P-gp ubiquitination, leading to internalization and proteasomal degradation of P-gp, which then results in decreased P-gp protein expression and transport activity levels. Here, we extend this line of research and present results from an in vivo study using a transgenic mouse model of AD (human amyloid precursor protein (hAPP)-overexpressing mice; Tg2576). Methods In our study, hAPP mice were treated with vehicle, nocodazole (NCZ, microtubule inhibitor to block P-gp internalization), or a combination of NCZ and the P-gp inhibitor cyclosporin A (CSA). We determined P-gp protein expression and transport activity levels in isolated mouse brain capillaries and Aβ levels in plasma and brain tissue. Results Treating hAPP mice with 5 mg/kg NCZ for 14 days increased P-gp levels to levels found in WT mice. Consistent with this, P-gp-mediated hAβ42 transport in brain capillaries was increased in NCZ-treated hAPP mice compared to untreated hAPP mice. Importantly, NCZ treatment significantly lowered hAβ40 and hAβ42 brain levels in hAPP mice, whereas hAβ40 and hAβ42 levels in plasma remained unchanged. Conclusions These findings provide in vivo evidence that microtubule inhibition maintains P-gp protein expression and transport activity levels, which in turn helps to lower hAβ brain levels in hAPP mice. Thus, protecting P-gp at the blood-brain barrier may provide a novel therapeutic strategy for AD and other Aβ-based pathologies.