Murine Red Blood Cells Lack Ligands for B Cell Siglecs, Allowing Strong Activation by Erythrocyte Surface Antigens.

CD22 and sialic acid-binding Ig-like lectin (Siglec)-G are members of the Siglec family of inhibitory coreceptors expressed on B cells that participate in enforcement of peripheral B cell tolerance. We have shown previously that when a BCR engages its cognate Ag on a cell surface that also expresses Siglec ligands, B cell Siglecs are recruited to the immunological synapse, resulting in suppression of BCR signaling and B cell apoptosis. Because all cells display sialic acids, and CD22 and Siglec-G have distinct, yet overlapping, specificities for sialic acid-containing glycan ligands, any cell could, in principle, invoke this tolerogenic mechanism for cell surface Ags. However, we show in this article that C57BL/6J mouse RBCs are essentially devoid of CD22 and Siglec-G ligands. As a consequence, RBCs that display a cell surface Ag, membrane-bound hen egg lysozyme, strongly activate Ag-specific B cells. We reasoned that de novo introduction of CD22 ligands in RBCs should abolish B cell activation toward its cognate Ag on the surface of RBCs. Accordingly, we used a glyco-engineering approach wherein synthetic CD22 ligands linked to lipids are inserted into the membrane of RBCs. Indeed, insertion of CD22 ligands into the RBC cell surface strongly inhibited B cell activation, cytokine secretion, and proliferation. These results demonstrate that the lack of Siglec ligands on the surface of murine RBCs permits B cell responses to erythrocyte Ags and show that Siglec-mediated B cell tolerance is restricted to cell types that express glycan ligands for the B cell Siglecs.

Comparison of caudal ropivacaine-morphine and paravertebral catheter for major upper abdominal surgery in infants.

BACKGROUND: The caudal epidural block is one of the most commonly used regional anesthetic techniques in children. Administration of morphine via caudal injection enables analgesia, even for upper abdominal surgery. The thoracic paravertebral block has also been successfully used to treat perioperative pain during upper abdominal procedures in pediatric patients. AIM: In the current study, we compared the two regional techniques for upper abdominal surgery in infants to determine whether one of them was preferable to the other. METHODS: Consecutive patients under 12 months of age who underwent upper abdominal surgery were retrospectively divided according to the chosen postoperative analgesia: Group C, caudal ropivacaine-morphine; Group P, paravertebral catheter. We analyzed the following outcomes: requirement for additional analgesics, pain scores, need for mechanical ventilation and oxygen dosage, postoperative blood pressure and heart rate, time to pass first stool, time until first full meal, and complications. RESULTS: Twenty-one consecutive patients were included: 10 in Group C and 11 in Group P. Median age at surgery was 80 (47.5-270.0) and 84.5 (34.3-287.5) days, respectively. No difference was found between the two groups in requirement for additional analgesics at 24 h after surgery (median 1 in Group C vs 1 in Group P, P = 0.288, 95% CI: -2 to 1). BOPS pain scores were only lower in Group P when compared to Group C at 24 h after surgery (median 1 vs 2, P = 0.041, 95% CI: -2 to 0). None of the patients had perioperative complications. CONCLUSIONS: In this small series, there was no significant difference between caudal ropivacaine-morphine and paravertebral catheter for postoperative care in infants undergoing upper abdominal surgery. Further prospective studies are needed to compare the efficacy and incidence of complications of caudal block and paravertebral catheter for postoperative analgesia.

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death.

Myocardial ischemia-reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of mitochondrial fission; and (2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.

Evolution of human H3N2 influenza virus receptor specificity has substantially expanded the receptor-binding domain site.

Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2-3-linked sialosides and must adapt to glycans with α2-6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize human α2-6-sialoside receptors that are elongated through addition of poly-LacNAc. We show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors while continuously selecting for strains maintaining this phenotype. This change in receptor engagement is accompanied by an extension of the traditional receptor-binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.

Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin.

The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

Ketamine enhances human neural stem cell proliferation and induces neuronal apoptosis via reactive oxygen species-mediated mitochondrial pathway.

BACKGROUND: Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models, leading to a serious concern regarding the safety of pediatric anesthesia. However, if and how ketamine induces human neural cell toxicity is unknown. Recapitulation of neurogenesis from human embryonic stem cells (hESCs) in vitro allows investigation of the toxic effects of ketamine on neural stem cells (NSCs) and developing neurons, which is impossible to perform in humans. In the present study, we assessed the influence of ketamine on the hESC-derived NSCs and neurons. METHODS: hESCs were directly differentiated into neurons via NSCs. NSCs and 2-week-old neurons were treated with varying doses of ketamine for different durations. NSC proliferation capacity was analyzed by Ki67 immunofluorescence staining and bromodeoxyuridine assay. Neuroapoptosis was analyzed by TUNEL staining and caspase 3 activity measurement. The mitochondria-related neuronal apoptosis pathway including mitochondrial membrane potential, cytochrome c distribution within cells, mitochondrial fission, and reactive oxygen species (ROS) production were also investigated. RESULTS: Ketamine (100 µM) increased NSC proliferation after 6-hour exposure. However, significant neuronal apoptosis was only observed after 24 hours of ketamine treatment. In addition, ketamine decreased mitochondrial membrane potential and increased cytochrome c release from mitochondria into cytosol. Ketamine also enhanced mitochondrial fission as well as ROS production compared with no-treatment control. Importantly, Trolox, a ROS scavenger, significantly attenuated the increase of ketamine-induced ROS production and neuronal apoptosis. CONCLUSIONS: These data for the first time demonstrate that (1) ketamine increases NSC proliferation and causes neuronal apoptosis; (2) mitochondria are involved in ketamine-induced neuronal toxicity, which can be prevented by Trolox; and (3) the stem cell-associated neurogenesis system may provide a simple and promising in vitro model for rapidly screening anesthetic neurotoxicity and studying the underlying mechanisms as well as prevention strategies to avoid this toxic effect.

Glyco-engineered MDCK cells display preferred receptors of H3N2 influenza absent in eggs used for vaccines.

Evolution of human H3N2 influenza viruses driven by immune selection has narrowed the receptor specificity of the hemagglutinin (HA) to a restricted subset of human-type (Neu5Acα2-6 Gal) glycan receptors that have extended poly-LacNAc (Galß1-4GlcNAc) repeats. This altered specificity has presented challenges for hemagglutination assays, growth in laboratory hosts, and vaccine production in eggs. To assess the impact of extended glycan receptors on virus binding, infection, and growth, we have engineered N-glycan extended (NExt) cell lines by overexpressing ß3-Ν-acetylglucosaminyltransferase 2 in MDCK, SIAT, and hCK cell lines. Of these, SIAT-NExt cells exhibit markedly increased binding of H3 HAs and susceptibility to infection by recent H3N2 virus strains, but without impacting final virus titers. Glycome analysis of these cell lines and allantoic and amniotic egg membranes provide insights into the importance of extended glycan receptors for growth of recent H3N2 viruses and relevance to their production for cell- and egg-based vaccines.

[Case report: a normal dose of rocuronium achieved the desired effect in a short time after the administration of sugammadex during reoperation].

A 69-year-old man with normal renal function underwent resection of a parotid tumor under general anesthesia. For tracheal intubation, rocuronium 0.6 mg x kg(-1) was administered, and for facial nerve stimulation, sugammadex 2 mg x kg(-1) was administered immediately after intubation. The operation time was 3 h. At the end of the surgery, sugammadex 2 mg x kg(-1) was administered again. Bleeding occurred 6 h after the surgery. During the second operation, rocuronium 0.6 mg x kg (-1) was administered for tracheal intubation. Maximal suppression was achieved 1 min 42 s after the administration of rocuronium, and the recovery time was 44 min. The times for both maximal suppression and recovery are similar to those when the same dose of rocuronium was used without sugammadex. The half-life of sugammadex is about 2 h. From the observations in this case, we think that after the completion of approximately 3 half-lives, a normal dose of rocuronium can produce the desired effect without the influence of residual sugammadex present in the plasma.

Anti-inflammatory effects of a novel phosphodiesterase-4 inhibitor, AA6216, in mouse dermatitis models.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that is commonly treated with corticosteroids. However, these drugs have long-term adverse effects, representing an unmet need for new treatments. AD is associated with dysregulation of phosphodiesterase 4 (PDE4) activity in inflammatory cells and the topical PDE4 inhibitor, crisaborole, is approved by the US FDA for mild-to-moderate AD. In this study, we compared the effects of a novel PDE4 inhibitor, AA6216, with those of crisaborole on skin inflammation. We found that AA6216 is a more potent inhibitor of PDE4 and of cytokine production (TNF-α, IL-12/23p40, IL-4, IL-13, and IFN-γ) by human peripheral blood mononuclear cells (PBMCs) stimulated by phytohemagglutinin (PHA) or anti-CD3 antibodies, with IC50 values ranging from 5.9 to 47 nM. AA6216 also significantly suppressed skin inflammation in three mouse models of dermatitis. In acute and chronic oxazolone-induced dermatitis models, topical AA6216 exhibited stronger inhibitory effects on ear inflammation and cytokine production (TNFα, IL-1ß, and IL-4) in skin lesions compared with crisaborole. In a Dermatophagoides farinae-induced dermatitis model, AA6216 significantly reduced the dermatitis score, based on the development of erythema/hemorrhage, scarring/dryness, edema, and excoriation/erosion, compared with a clinically used topical AD drug, tacrolimus. These results suggest the possibility that AA6216 is a novel and effective topical therapeutic agent for the treatment of dermatitis including AD.

Sialic Acid Ligands of CD28 Suppress Costimulation of T Cells.

Effector T cells comprise the cellular arm of the adaptive immune system and are essential for mounting immune responses against pathogens and cancer. To reach effector status, costimulation through CD28 is required. Here, we report that sialic acid-containing glycans on the surface of both T cells and APCs are alternative ligands of CD28 that compete with binding to its well-documented activatory ligand CD80 on the APC, resulting in attenuated costimulation. Removal of sialic acids enhances antigen-mediated activation of naïve T cells and also increases the revival of effector T cells made hypofunctional or exhausted via chronic viral infection. This occurs through a mechanism that is synergistic with antibody blockade of the inhibitory PD-1 axis. These results reveal a previously unrecognized role of sialic acid ligands in attenuation of CD28-mediated costimulation of T cells.

Successful management of an infant with hypertensive heart failure associated with Wilms' tumor: a case report.

BACKGROUND: Wilms' tumor with hyperreninemia may result in critical cardiovascular decompensation. We report a case of severe hypertensive heart failure followed by tumor resection in a 3-month-old infant with Wilms' tumor. CASE PRESENTATION: A 3-month-old girl was admitted to the intensive care unit for Wilms' tumor with hypertension and hypoxia. Her systolic blood pressure was 110 mmHg, and her SpO2 was 92%. She presented with severe hypertensive heart failure and received mechanical ventilation and antihypertensive therapy for hypertension and heart failure. An alpha 2-adrenergic receptor agonist was used for sedation as part of her antihypertensive therapy. On hospital day 16, nephrectomy with tumor resection was performed under general anesthesia. Her systolic blood pressure did not vary more than 20 mmHg during surgery due to appropriate preoperative management. Hemodynamic collapse did not occur. CONCLUSIONS: The highlight of this case report is the successful management of an infant with Wilms' tumor, particularly with respect to preoperative hemodynamic control and sedation.

Rare Case of Neonatal Cardiac Ischemia Due to Intermittent Aortic Insufficiency.

We report a case of a 23-day-old girl who presented with repeated life-threatening myocardial ischemia from intermittent aortic insufficiency because of fixation of the left coronary cusp against the aortic wall. The patient underwent aortic valve repair with partial commissuroplasty of the left-right and left-noncommissures. After the surgical intervention the patient has been well, with no ischemic event for over 1 year. This diagnosis should be considered as a differential diagnosis in pediatric cases with myocardial ischemia.

64Cu-labeled minibody D2101 visualizes CDH17-positive gastric cancer xenografts with short waiting time.

OBJECTIVE: We previously reported In-labeled anti-cadherin17 (CDH17) IgG visualized CDH17-positive gastric cancer xenografts. Unfortunately, a long waiting time was required to obtain high-contrast images due to long blood retention (blood half-life: 26 h). To accelerate blood clearance, we have developed anti-CDH17 minibody (D2101 minibody) and evaluated the pharmacokinetics in gastric cancer mouse models. METHODS: Two different single chain Fvs (scFvs), D2101 mutant and D2111, were developed from each parental IgG. The binding ability to CDH17 and stability in plasma were evaluated. D2101 minibody, constructed based on D2101 mutant scFv, was labeled with Cu (Cu-D2101 minibody), and the in-vitro and in-vivo properties were evaluated by cell ELISA, biodistribution experiments, and PET imaging in mice bearing CDH17-positive AGS and CDH17-negative MKN74 tumors. RESULTS: D2101 mutant and D2111 scFvs showed similar affinities to CDH17. D2101 mutant scFv was more stable than D2111 scFv in plasma. No loss of binding affinity of the D2101 minibody by chelate conjugation and radiolabeling procedures was observed. The biodistribution of Cu-D2101 minibody showed high uptake in AGS tumors and low uptake in MKN74. The blood half-life of Cu-D2101 minibody was 6.5 h. Improved blood clearance of Cu-D2101 minibody provided high tumor-to-blood ratios compared with the previous results of parental IgG in AGS xenograft mice. PET studies showed consistent results with biodistribution studies. CONCLUSIONS: Cu-D2101 minibody exhibited higher tumor-to-blood ratios at earlier time points than those of the radiolabeled parental IgG. Cu-D2101 minibody has potential as an immunoimaging agent for CDH17-positive tumors.

Human Influenza Virus Hemagglutinins Contain Conserved Oligomannose N-Linked Glycans Allowing Potent Neutralization by Lectins.

Hemagglutinins (HAs) from human influenza viruses adapt to bind α2-6-linked sialosides, overcoming a receptor-defined species barrier distinct from the α2-3 specificity of avian virus progenitors. Additionally, human-adapted HAs gain glycosylation sites over time, although their biological function is poorly defined. Using quantitative glycomic analysis, we show that HAs from human pandemic viruses exhibit significant proportions of high-mannose type N-linked glycans throughout the head domain. By contrast, poorly adapted avian-origin HAs contain predominately complex-type glycans, which have greater structural diversity. Although oligomannose levels vary, they are present in all tested recombinant HAs and whole viruses and can be specifically targeted for universal detection. The positions of high-mannose glycosites on the HA of human H1N1 and H3N2 strains are conserved. Additionally, high-mannose-binding lectins possess a broad capacity to neutralize and prevent infection with contemporary H3N2 strains. These findings reveal the biological significance of HA glycosylation and therapeutic potential of targeting these structures.

Synergistic Cytotoxic Effect on Gastric Cancer Cells of an Immunotoxin Cocktail in Which Antibodies Recognize Different Epitopes on CDH17.

Cadherin-17 (CDH17) is highly expressed in gastric cancer and is thus considered to be a good target for antibody therapy. CDH17 is classified as a nonclassical cadherin, in that it is composed of seven extracellular cadherin domains. We generated anti-CDH17 monoclonal antibodies (mAbs) which recognize the extracellular domain of CDH17. Competitive assay using AGS, a gastric cancer cell line, cells revealed that five selected anti-CDH17 mAbs recognize different epitopes on CDH17. As AGS cells were shown to exhibit broad expression pattern of CDH17 by flow cytometry, we separated three clones with a low (10,000/cell), medium (50,000/cell), and high (200,000/cell) expression level, designating them as AGSlow, AGSmed, and AGShigh, respectively. The mAbs, coupled with saporin, exhibited effective cytotoxicity to AGShigh, but poor cytotoxicity to AGSlow. By contrast, the immunotoxin cocktail using the three clones D2101, D2005, and D2008, which recognize different epitopes, exhibited efficient cytotoxicity, even to the AGSlow group. The effect of the immunotoxin cocktail is synergistic, as the combination index was demonstrated to be below 1.0, as calculated by the method of Chou and Talalay using CalcuSyn software. These results suggest that the immunotoxin cocktail targeted to multiple epitopes has synergistic effects on low expression level cells, which expand the applicable range of immunotoxin therapy for cancer.

Propofol Induces Apoptosis of Neurons but Not Astrocytes, Oligodendrocytes, or Neural Stem Cells in the Neonatal Mouse Hippocampus.

It has been shown that propofol can induce widespread apoptosis in neonatal mouse brains followed by long-term cognitive dysfunction. However, selective brain area and cell vulnerability to propofol remains unknown. This study was aimed to dissect toxic effect of propofol on multiple brain cells, including neurons, astrocytes, oligodendrocytes, and neural stem cells (NSCs). Seven-day-old mice were intraperitoneally administrated propofol or intralipid as a vehicle control for 6 hours. To identify vulnerable cells undergoing apoptosis following propofol exposure, brain sagittal sections were co-stained with antibodies against an apoptosis marker along with neuron, astrocyte, oligodendrocyte, or NSC markers using immunofluorescence staining. The results showed widespread apoptosis in propofol-treated brains (apoptotic cells: 1.55 ± 0.04% and 0.06 ± 0.01% in propofol group and intralipid-treated control group, respectively). Apoptotic cell distribution exhibits region- and cell-specific patterns. Several brain regions (e.g., cerebral cortex and hippocampus) were more vulnerable to propofol than other brain regions. Most apoptotic cells in the hippocampus were located in the cornus ammonis 1 (CA1) subfield. These apoptotic cells were only detected in neurons and not astrocytes, oligodendrocytes, or NSCs. These data demonstrate that different brain regions, subfields, and different types of neuronal cells in mice exhibit various vulnerabilities to propofol. Understanding region- and cell-specific susceptibility to propofol will help to better understand cellular contribution to developmental neurotoxicity and further develop novel therapeutic targets.

Effects of remifentanil on the sphincter of Oddi in a 3-year-old child: a case report.

Opioids cause spasm of the sphincter of Oddi. Remifentanil is metabolized enzymatically throughout the body. Its context-sensitive half-time is 3 to 4minutes. The effect of remifentanil on the sphincter of Oddi is unknown, especially in children. We recently encountered a patient in whom the administration of remifentanil caused spasm of the sphincter of Oddi, which resolved rapidly after discontinuation of remifentanil. A 3-year-old girl weighing 11.3kg was scheduled to undergo common bile duct excision with ductoplasty. Her diagnosis was congenital biliary dilatation. In the operating room, after achieving the initial induction through sevoflurane (5%) and intravenous rocuronium (10mg), she was intubated and administered a continuous paravertebral block by levobupivacaine (25mg/10mL +2.5mg/h). General anesthesia was maintained with sevoflurane (2%), remifentanil (0.5 µg kg(-1) min(-1)), and oxygen (fractional inspired oxygen tension, 0.33). The first intraoperative cholangiogram obtained via the cystic duct tube showed obstruction at the terminal end of the common bile duct. We injected scopolamine butylbromide (5mg, intravenous) to relax the sphincter of Oddi. However, the next cholangiogram obtained 3minutes later still showed an obstruction. We speculated that the obstruction may have been caused by remifentanil-induced spasm of the sphincter of Oddi. Therefore, we stopped administering remifentanil; 2minutes later, we achieved satisfactory passage of the contrast material to the duodenum. The predicted plasma concentrations of remifentanil at the time of stopping its administration and at the time of disobliteration were 6.38and 2.55ng/mL, respectively. The patient's postoperative course was uneventful. In patients who have spasms of the sphincter of Oddi during the administration of remifentanil, the resultant obstruction can be treated effectively by reducing the infusion rate of remifentanil.

Comparison of Cardiomyocyte Differentiation Potential Between Type 1 Diabetic Donor- and Nondiabetic Donor-Derived Induced Pluripotent Stem Cells.

Type 1 diabetes mellitus (T1DM) is the most common type of diabetes in children and adolescents. Diabetic subjects are more likely to experience a myocardial infarction compared to nondiabetic subjects. In recent years, induced pluripotent stem cells (iPSCs) have received increasing attention from basic scientists and clinicians and hold promise for myocardial regeneration due to their unlimited proliferation potential and differentiation capacity. However, cardiomyogenesis of type 1 diabetic donor-derived iPSCs (T1DM-iPSCs) has not been investigated yet. The aim of the study was to comparatively analyze cardiomyocyte (CM) differentiation capacity of nondiabetic donor-derived iPSCs (N-iPSCs) and T1DM-iPSCs. The differentiated CMs were confirmed by both expression of cardiac-specific markers and presence of cardiac action potential. Since mitochondrial bioenergetics is vital to every aspect of CM function, extracellular acidification rates and oxygen consumption rates were measured using Seahorse extracellular flux analyzer. The results showed that N-iPSCs and T1DM-iPSCs demonstrated similar capacity of differentiation into spontaneously contracting CMs exhibiting nodal-, atrial-, or ventricular-like action potentials. Differentiation efficiency was up to 90%. In addition, the CMs differentiated from N-iPSCs and T1DM-iPSCs (N-iPSC-CMs and T1DM-iPSC-CMs, respectively) showed 1) well-regulated glucose utilization at the level of glycolysis and mitochondrial oxidative phosphorylation and 2) the ability to switch metabolic pathways independent of extracellular glucose concentration. Collectively, we demonstrate for the first time that T1DM-iPSCs can differentiate into functional CMs with well-regulated glucose utilization as shown in N-iPSCs, suggesting that T1DM-iPSC-CMs might be a promising autologous cell source for myocardial regeneration in type 1 diabetes patients.

2a-[4-(Tetrahydropyridoindol-2-yl)butyl]tetrahydrobenzindole derivatives: new selective antagonists of the 5-hydroxytryptamine7 receptor.

A series of tetrahydrobenzindoles was prepared, and the affinity of these compounds for the 5-hydroxytryptamine7 (5-HT7) receptor and other receptors was evaluated. Most of the compounds showed high affinity for the 5-HT7 receptor, and 2a-[4-(tetrahydropyridoindol-2-yl)butyl]tetrahydrobenzindole derivatives (26a-j) exhibited high selectivity for this receptor. The nature of the substituent at the 9-position of the tetrahydropyridindole ring affected the affinity for the 5-HT7 receptor, and the 9-carbamoyl moiety afforded increased selectivity. Compound 26j exhibited high affinity for the 5-HT7 receptor, with at least 280-fold selectivity over the 5-HT2 receptor. In a functional model of 5-HT7 receptor activation, this compound was confirmed to have 5-HT7 receptor antagonist activity. It should be a useful tool for clarifying the biological role of the 5-HT7 receptor.

Ketamine induces toxicity in human neurons differentiated from embryonic stem cells via mitochondrial apoptosis pathway.

Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨm), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose- and time-dependently caused cell death. In addition, ketamine decreased ΔΨm and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨm changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time- and dose-dependently induces human neurotoxicity at supraclinical concentrations via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies.