Early-life midazolam exposure persistently changes chromatin accessibility to impair adult hippocampal neurogenesis and cognition.

Linkage between early-life exposure to anesthesia and subsequent learning disabilities is of great concern to children and their families. Here we show that early-life exposure to midazolam (MDZ), a widely used drug in pediatric anesthesia, persistently alters chromatin accessibility and the expression of quiescence-associated genes in neural stem cells (NSCs) in the mouse hippocampus. The alterations led to a sustained restriction of NSC proliferation toward adulthood, resulting in a reduction of neurogenesis that was associated with the impairment of hippocampal-dependent memory functions. Moreover, we found that voluntary exercise restored hippocampal neurogenesis, normalized the MDZ-perturbed transcriptome, and ameliorated cognitive ability in MDZ-exposed mice. Our findings thus explain how pediatric anesthesia provokes long-term adverse effects on brain function and provide a possible therapeutic strategy for countering them.

Regulation of Adult Mammalian Neural Stem Cells and Neurogenesis by Cell Extrinsic and Intrinsic Factors.

Tissue-specific stem cells give rise to new functional cells to maintain tissue homeostasis and restore damaged tissue after injury. To ensure proper brain functions in the adult brain, neural stem cells (NSCs) continuously generate newborn neurons that integrate into pre-existing neuronal networks. Proliferation, as well as neurogenesis of NSCs, are exquisitely controlled by extrinsic and intrinsic factors, and their underlying mechanisms have been extensively studied with the goal of enhancing the neurogenic capacity of NSCs for regenerative medicine. However, neurogenesis of endogenous NSCs alone is insufficient to completely repair brains damaged by neurodegenerative diseases and/or injury because neurogenic areas are limited and few neurons are produced in the adult brain. An innovative approach towards replacing damaged neurons is to induce conversion of non-neuronal cells residing in injured sites into neurons by a process referred to as direct reprogramming. This review describes extrinsic and intrinsic factors controlling NSCs and neurogenesis in the adult brain and discusses prospects for their applications. It also describes direct neuronal reprogramming technology holding promise for future clinical applications.

Np95/Uhrf1 regulates tumor suppressor gene expression of neural stem/precursor cells, contributing to neurogenesis in the adult mouse brain.

Adult neurogenesis is a process of generating new neurons from neural stem/precursor cells (NS/PCs) in restricted adult brain regions throughout life. It is now generally known that adult neurogenesis in the hippocampal dentate gyrus (DG) and subventricular zone participates in various higher brain functions, such as learning and memory formation, olfactory discrimination and repair after brain injury. However, the mechanisms underlying adult neurogenesis remain to be fully understood. Here, we show that Nuclear protein 95 KDa (Np95, also known as UHRF1 or ICBP90), which is an essential protein for maintaining DNA methylation during cell division, is involved in multiple processes of adult neurogenesis. Specific ablation of Np95 in adult NS/PCs (aNS/PCs) led to a decrease in their proliferation and an impairment of neuronal differentiation and to suppression of neuronal maturation associated with the impairment of dendritic formation in the hippocampal DG. We also found that deficiency of Np95 in NS/PCs increased the expression of tumor suppressor genes p16 and p53, and confirmed that expression of these genes in NS/PCs recapitulates the phenotype of Np95-deficient NS/PCs. Taken together, our findings suggest that Np95 plays an essential role in proliferation and differentiation of aNS/PCs through the regulation of tumor suppressor gene expression in adult neurogenesis.

Kinetic differentiation mode chromatography using 8-quinolinol and fluorimetric detection for sensitive determination of aluminum adhering to the gastric mucosa.

A highly sensitive method of kinetic differentiation (KD) mode high-performance liquid chromatography (HPLC) with fluorimetric detection was established using 8-quinolinol to measure aluminum adhering to the gastric mucosa. After sucralfate was hydrolyzed by 1 mol/l hydrochloric acid, an 8-quinolinolate-aluminum complex was produced by reacting aluminum with an 8-quinolinol solution. Then contaminants in the gastric mucosa and sucralfate were removed by liquid-liquid extraction with chloroform. Next, the 8-quinolinolate-aluminum complex was separated on a reversed-phase column that was specifically designed to detect aluminum (50 x 4.6-mm I.D.). Separation was done at a flow-rate of 0.8 ml/min, using BES buffer containing sodium dodecyl sulfate (pH 7.0) as the mobile phase. Fluorescence was detected at 370 nm (excitation) and 504 nm (emission). The sensitivity of this method was more than 1000 times greater than that of absorptiometry using 8-quinolinol. The detection and quantitation limits were 1.68 and 5.11 ng/ml, respectively. When tested with aluminum solutions of 10, 30, and 90 ng/ml, the intra-assay and inter-assay coefficients of variation were below 7.1%, with an error of less than 8.3%. Aluminum adhering to the gastric mucosa was determined by HPLC and absorptiometry after administration of sucralfate to rats. The HPLC method showed that aluminum levels were higher at sites of ulceration than in the normal mucosa at all times after sucralfate administration. When the values above zero obtained for absorptiometry were assessed, there was a significant correlation (r=0.993, P<0.0001) between the aluminum concentrations measured by the two methods. This new HPLC method could be applied to the determination of aluminum in small samples, such as human gastric mucosal biopsy specimens.

Anti-tumor immune response induced by the fractions derived from OK-432, a streptococcal preparation, by using a monoclonal antibody TS-2 that neutralizes the interferon-gamma-inducing activity of OK-432: comparison between the TS-2-binding and TS-2-unbinding fraction.

We have previously isolated a lipoteichoic acid (LTA)-related molecule (OK-PSA) from OK-432, a streptococcal agent, by affinity chromatography on a CNBr-activated Sepharose 4B bound TS-2 monoclonal antibody (mAb) that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. In the current study, we compared the cytokine-inducing and anti-tumor activities of OK-PSA, a TS-2-binding fraction, with those of OK-PTF, a TS-2-unbinding fraction, in order to determine the efficacy of OK-PSA for clinical use in affinity chromatography using TS-2. In the in vitro experiments using human peripheral blood mononuclear cells (PBMCs), OK-PSA markedly induced Th1-type cytokines, while interleukin (IL)-6 and IL-10, Th2-type cytokines, were induced by OK-PTF. Th1-cytokine induction by OK-PTF was not dose-dependent and was suppressed when PBMCs were treated with a high concentration of OK-PTF. In a mouse model, Th1 cytokines were also induced by OK-PSA and Th2 cytokines were induced by OK-PTF. Th2 cytokine-inducing activity of OK-PTF was accelerated in tumor-bearing mice relative to that in healthy mice. Although the anti-tumor effect of OK-PTF was statistically significant, it was much weaker than that of OK-PSA. A significant difference between the anti-tumor effect of OK-PSA and that of OK-PTF was observed (P<0.05). Finally, OK-PSA elicited its cytokine-inducing effect via Toll-like receptor (TLR) 4, whereas OK-PTF-induced signaling was mediated by both TLR2 and TLR4. These findings strongly suggested that the affinity chromatography using TS-2 is a useful strategy to separate the effective component for cancer therapy (OK-PSA) from other components.

Involvement of Toll-like receptor 4 signaling in interferon-gamma production and antitumor effect by streptococcal agent OK-432.

BACKGROUND: The streptococcal agent OK-432 has been used for immunotherapy of head and neck cancer, among other malignancies, but its mechanism of action is unknown. Because the Toll-like receptor 4 (TLR4)/MD-2 complex is important in enabling the mammalian immune system to recognize bacterial components, we investigated whether expression of the TLR4 and MD-2 genes is associated with OK-432-induced anticancer immunity. METHODS: Peripheral blood mononuclear cells (PBMCs) from 28 patients with head and neck cancer were analyzed for TLR4 and MD-2 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis. PBMCs were treated in vitro with OK-432 or with OK-PSA (a lipoteichoic-acid-related molecule that is an active component of OK-432), and interferon-gamma (IFN-gamma) mRNA expression, an immune response measure, was analyzed by RT-PCR. Patient sera collected 24 hours after OK-432 administration were examined for IFN-gamma protein using an enzyme-linked immunosorbent assay. Lewis lung carcinoma-bearing wild-type C57BL/6 and TLR4-deficient mice (four mice per group) received intraperitoneal injections of OK-432, and tumor volumes and sera IFN-gamma levels were measured over time. All statistical tests were two-sided. RESULTS: Twenty patients expressed both TLR4 and MD-2. Expression of TLR4 and MD-2 genes was associated with the in vivo IFN-gamma induction in 19 patients administered OK-432 (Fisher's exact test P<.001). Although both OK-432 and OK-PSA induced IFN-gamma expression from PBMCs in vitro, expression of TLR4 and MD-2 was associated only with IFN-gamma expression induced by OK-PSA (P<.001). In vivo intraperitoneal administration of OK-432 resulted in an increase of IFN-gamma in sera from wild-type mice but not in sera from TLR4-deficient mice. Tumors in wild-type mice treated with OK-432 were statistically significantly smaller than those in mice treated with saline (P =.007). By contrast, in TLR4-deficient mice, there was no difference in tumor volume between the two treatment groups. CONCLUSIONS: TLR4 and MD-2 may mediate OK-432-induced anticancer immunity.

Enhancement of anti-tumor immunity by lipoteichoic acid-related molecule isolated from OK-432, a streptococcal agent, in athymic nude mice bearing human salivary adenocarcinoma: role of natural killer cells.

BACKGROUND: OK-PSA, a lipoteichoic acid (LTA)-related molecule isolated from a streptococcal agent OK-432, enhances anti-tumor immunity as a potent inducer of Th1-type cytokines. Recently, we obtained the data suggesting that natural killer (NK) cells may play a significant role for OK-PSA-induced cytokine production in vitro. MATERIALS AND METHODS: We conducted the animal experiments using athymic nude mice bearing human salivary adenocarcinoma to examine the role of NK cells in OK-PSA-induced anti-tumor immunity. OK-PSA was peritumorally injected into the mice. Cytokines in the sera were analyzed by ELISA. mRNAs for cytokines were detected by RT-PCR. 51Cr release test was performed to measure killer cell activities. RESULTS: OK-PSA markedly increased the amounts of IFN-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18 that are generally called "Th1-type cytokines" in the sera derived from tumor-bearing nude mice, and also accelerated the killing activities of tumor-infiltrating lymphocytes as well as of draining lymph node cells. Furthermore, OK-PSA administration resulted in significant inhibition of tumor growth, but the effect of OK-PSA was almost completely inhibited by the deletion of NK cells using anti-asialo GM1 antibody. CONCLUSION: These findings strongly suggested that NK cells are closely involved in OK-PSA-mediated anti-tumor immunity.