Induction of Thymus Atrophy and Disruption of Thymocyte Development by Fipronil through Dysregulation of IL-7-Associated Genes.

The susceptibility of the immune system to immunotoxic chemicals is evident, particularly in the thymus, a vital primary immune organ prone to atrophy due to exposure to toxicants. Fipronil (FPN), a widely used insecticide, is of concern due to its potential neurotoxicity, hepatotoxicity, and immunotoxicity. Our previous study showed that FPN disturbed the antigen-specific T-cell functionality in vivo. As T-cell lineage commitment and thymopoiesis are closely interconnected with the normal function of the T-cell-mediated immune responses, this study aims to further examine the toxic effects of FPN on thymocyte development. In this study, 4-week-old BALB/c mice received seven doses of FPN (1, 5, 10 mg/kg) by gavage. Thymus size, medulla/cortex ratio, total thymocyte counts, double-positive thymocyte population, and IL-7-positive cells decreased dose-dependently. IL-7 aids the differentiation of early T-cell precursors into mature T cells, and several essential genes contribute to the maturation of T cells in the thymus. Foxn1 ensures that the thymic microenvironment is suitable for the maturation of T-cell precursors. Lyl1 is involved in specifying lymphoid cells and maintaining T-cell development in the thymus. The c-Kit/SCF collaboration fosters a supportive thymic milieu to promote the formation of functional T cells. The expression of IL-7, IL-7R, c-Kit, SCF, Foxn1, and Lyl1 genes in the thymus was significantly diminished in FPN-treated groups with the concordance with the reduction of IL-7 signaling proteins (IL-7, IL-7R, c-KIT, SCF, LYL1, FOXO3A, and GABPA), suggesting that the dysregulation of T-cell lineage-related genes may contribute to the thymic atrophy induced by FPN. In addition, FPN disturbed the functionality of thymocytes with an increase of IL-4 and IFN-γ production and a decrease of IL-2 secretion after T-cell mitogen stimulation ex vivo. Collectively, FPN significantly deregulated genes related to T-cell progenitor differentiation, survival, and expansion, potentially leading to impaired thymopoiesis.

Asymmetric Synthesis of Nabscessin A from Inositol and d-Camphor.

An enantiomer of nabscessin A (1), an aminocyclitol amide with antimicrobial activity, was synthesized from myo-inositol and dimethyl d-camphor acetal in 14 steps. Formal synthesis of natural nabscessin A was also achieved through the new approach to access both enantiomers of 4,5-di-O-benzyl-myo-inositol, derived from the same set of starting materials. This synthesis features utilizations of the existing framework of myo-inositol and a regioselective esterification.

vHDvDB 2.0: Database and Group Comparison Server for Hepatitis Delta Virus.

The hepatitis delta virus (HDV) is a unique pathogen with significant global health implications, affecting individuals who are coinfected with the hepatitis B virus (HBV). HDV infection has profound clinical consequences, manifesting either as coinfection with HBV, resulting in acute hepatitis and potential liver failure, or as superinfection in chronic HBV cases, substantially increasing the risk of cirrhosis and hepatocellular carcinoma. Given the complex dynamics of HDV infection and the urgent need for advanced research tools, this article introduces vHDvDB 2.0, a comprehensive HDV full-length sequence database. This innovative platform integrates data preprocessing, secondary structure prediction, and epidemiological research tools. The primary goal of vHDvDB 2.0 is to consolidate HDV sequence data into a user-friendly repository, thereby facilitating access for researchers and enhancing the broader scientific understanding of HDV. The significance of this database lies in its potential to streamline HDV research by providing a centralized resource for analyzing viral sequences and exploring genotype-specific characteristics. It will also enable more in-depth research within the HDV sequence domains.

Iron oxide nanoparticles suppress the production of IL-1beta via the secretory lysosomal pathway in murine microglial cells.

BACKGROUND: Superparamagnetic iron oxide nanoparticles (IONPs) have been used as magnetic resonance imaging contrast agents for various research and diagnostic purposes, such as the detection of neuroinflammation and blood-brain-barrier integrity. As the central resident macrophage-like cells, microglia are responsible for managing foreign agents invading the CNS. The present study investigated the direct effect of IONPs on the production of pro-inflammatory cytokines by murine microglia stimulated with lipopolysaccharide (LPS). METHODS: Primary murine microglial cells were pretreated with IONPs (1-50 µg Fe/mL) for 30 min and then stimulated with LPS (100 ng/mL) for 24 h. Confocal microscopy is used to visualize the intracellular IONP distribution and secretory lysosomes after staining with LysoTracker and Rab27a, respectively. The production of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α was quantified by ELISA. The activity of IL-1ß converting enzyme (ICE) and TNF-α converting enzyme (TACE) was measured by fluorescent microplate assay using specific substrates. The lysosomal number, alkalinity, permeability and cathepsin B activity were determined by flow cytometry with ectodermal dysplasia-1, lysosensor and acridine orange staining, and using cathepsin B specific substrate, respectively. RESULTS: Confocal imaging revealed that IONPs were markedly engulfed by microglia. Exposure to IONPs attenuated the production of IL-1ß, but not TNF-α. Concordantly, the activity of ICE, but not the TACE, was suppressed in IONP-treated cells. Mechanistic studies showed that IONPs accumulated in lysosomes and the number of lysosomes was increased in IONP-treated cells. In addition, exposure to IONPs increased lysosomal permeability and alkalinity, but decreased the activity of cathepsin B, a secretory lysosomal enzyme involved in the activation of ICE. CONCLUSIONS: Our results demonstrated a contrasting effect of IONPs on the production of IL-1ß and TNF-α by LPS-stimulated microglia, in which the attenuation of IL-1ß by IONPs was mediated by inhibiting the secretory lysosomal pathway of cytokine processing.

Cannabidiol-induced apoptosis in murine microglial cells through lipid raft.

Cannabidiol (CBD), the major nonpsychotropic phytocannabinoid, induces apoptosis in both immortalized and primary lymphocytes and monocytes. However, contrasting effects of CBD on the apoptosis between normal and immortalized glial cells have been reported. This study investigated the proapoptotic effect of CBD on primary microglial cells. Treatment of murine primary microglial cultures with CBD resulted in a time- and concentration-dependent induction of apoptosis, as shown by increase in hypodiploid cells and DNA strand breaks, and marked activation of both caspase-8 and -9. Mechanistic studies revealed that antioxidants, including N-acetyl-L-cysteine and glutathione, the G protein-coupled receptor 55 agonist abnormal-CBD and specific antagonists for vanilloid, and CB1 and CB2 cannabinoid receptors did not counteract the apoptosis induced by CBD. In contrast, methyl-ß-cyclodextrin (MCD), a lipid raft disruptor, potently attenuated CBD-induced microglial apoptosis and caspase activation. Furthermore, CBD induced lipid raft coalescence and augmented the expression of GM1 ganglioside and caveolin-1, all of which were attenuated by MCD. Taken together, these results suggest that CBD induces a marked proapoptotic effect in primary microglia through lipid raft coalescence and elevated expression of GM1 ganglioside and caveolin-1.

Cannabidiol induced a contrasting pro-apoptotic effect between freshly isolated and precultured human monocytes.

It has been documented that cannabidiol (CBD) induced apoptosis in a variety of transformed cells, including lymphocytic and monocytic leukemias. In contrast, a differential sensitivity between normal lymphocytes and monocytes to CBD-mediated apoptosis has been reported. The present study investigated the pro-apoptotic effect of CBD on human peripheral monocytes that were either freshly isolated or precultured for 72h. CBD markedly enhanced apoptosis of freshly isolated monocytes in a time- and concentration-dependent manner, whereas precultured monocytes were insensitive. By comparison, both cells were sensitive to doxorubicin-induced apoptosis. CBD significantly diminished the cellular thiols and glutathione in freshly isolated monocytes. The apoptosis induced by CBD was abrogated in the presence of N-acetyl-L-cysteine, a precursor of glutathione. In addition, precultured monocytes contained a significantly greater level of glutathione and heme oxygenase-1 (HO-1) compared to the freshly isolated cells. The HO-1 competitive inhibitor zinc protoporphyrin partially but significantly restored the sensitivity of precultured monocytes to CBD-mediated apoptosis. Collectively, our results demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the cellular level of glutathione and the antioxidative capability of the cells.

A comparative study on cannabidiol-induced apoptosis in murine thymocytes and EL-4 thymoma cells.

It has been shown that leukemia and glioma cells are sensitive to cannabidiol (CBD)-induced apoptosis, whereas primary monocytes and glia cells are relatively insensitive. In the current study, the cellular events and sensitivity to CBD-induced apoptosis between murine thymocytes and EL-4 thymoma cells were compared. Cannabidiol markedly induced apoptosis in a time- and concentration-related manner in both cells. The efficacy of CBD to induce apoptosis was comparable between the 2 types of T cells, whereas CBD induced apoptosis in thymocytes with a slightly greater potency than in EL4 cells. Time-course analyses revealed CBD-mediated apoptosis occurred earlier in EL-4 cells than that in thymocytes. An increased level of cellular reactive oxygen species (ROS) was detected in both cells with the peak response at 2 h post CBD treatment. Concordantly, CBD triggered a gradual diminishment in the cellular thiols. The presence of N-acetyl-L-cysteine (NAC), a precursor of glutathione, markedly attenuated the induction of apoptosis, and restored the diminished levels of cellular thiols. The results demonstrated that both thymocytes and EL-4 thymoma cells were susceptible to CBD-induced apoptosis and that ROS played a critical role in the apoptosis induction.

Methamphetamine and diazepam suppress antigen-specific cytokine expression and antibody production in ovalbumin-sensitized BALB/c mice.

Methamphetamine is a widely abused psychostimulant. Abusing methamphetamine causes various adverse effects, such as immune dysfunction. The present study investigated the effect of diazepam, a central depressant, on methamphetamine-induced immunosuppression. BALB/c mice were daily administered with diazepam and methamphetamine (5mg/kg of each), either alone or in combination, for 5 consecutive days followed by sensitization with ovalbumin (OVA). Two days later the same dosing and sensitization regimen was repeated once. The production of serum anti-OVA antibodies, and the cellularity and functional activities of splenocytes were measured 7 days post the 2nd OVA sensitization. The results demonstrated that methamphetamine and/or diazepam significantly attenuated the production of OVA-specific IgM, IgG(1) and IgG(2a). Concordantly, splenocytes of mice administered with diazepam and/or methamphetamine produced less IL-4 and IFN-gamma upon ex vivo re-stimulation with OVA, as compared to the vehicle-treated control. In contrast, the cellularity and metabolic activity of splenocytes were not altered by the drug treatment. These results indicated that the central depressant diazepam did not affect methamphetamine-mediated immunosuppression. Rather, both drugs markedly suppressed antigen-specific antibody production and T-cell reactivity.

Cannabidiol induced apoptosis in human monocytes through mitochondrial permeability transition pore-mediated ROS production.

Cannabidiol (CBD) has been reported to induce apoptosis in immune cells through oxidative stress-related mechanisms. The objective of the present study was to investigate the cellular mechanisms for CBD-induced apoptosis and oxidative stress in human monocytes. Exposure of freshly isolated human monocytes to CBD induced apoptosis in a time- and concentration-dependent manner. Time-course analyses revealed the induction of intracellular reactive oxygen species (ROS) at 1-2 h post CBD (16 µM) exposure. By comparison, the CBD treatment rapidly elicited the depolarization of mitochondrial membrane potential (MMP) within 5 min, and the oxidation of cardiolipin, a major lipid component of the mitochondrial inner membrane, within 15 min. Moreover, CBD induced the release of cytochrome c (Cyt c) from mitochondria. Mechanistic studies revealed that CBD-induced ROS production and apoptosis were not associated with the alteration of mitochondrial superoxide dismutase activity, the electron leakage through mitochondrial respiratory chain, and Fe2+- and Ca2+-mediated mechanisms. In contrast, CBD-induced apoptosis and MMP depolarization were markedly attenuated by the mitochondrial permeability transition pore (MPTP) inhibitor cyclosporin A (CsA), but not the calcineurin inhibitor FK506. Furthermore, CsA prevented cardiolipin oxidation and the MPTP opening induced by CBD. The present study suggests that CBD acts on the mitochondria to elicit ROS generation and apoptosis through MPTP opening and provides critical insights into the cellular mechanisms for CBD-induced oxidative stress in apoptotic monocytes.

Suppressive effects of cannabidiol on antigen-specific antibody production and functional activity of splenocytes in ovalbumin-sensitized BALB/c mice.

Cannabidiol (CBD) and cannabis-based medicines are potential therapeutic agents. Because the immune system has been widely demonstrated to be affected by psychoactive cannabinoids, such as Delta(9)-tetrahydrocannabinol, the objective of the present studies is to investigate the immunomodulatory effect of CBD, the major non-psychoactive cannabinoid in marijuana. BALB/c mice were intraperitoneally administered with a single dose of CBD (5-20 mg/kg) prior to ovalbumin (OVA) sensitization, and the serum production of antigen-specific antibodies was measured 7 days post OVA sensitization. The serum level of OVA-specific IgM was significantly attenuated by a high dose of CBD (20 mg/kg), and OVA-specific IgG(1) and IgG(2a) by all 3 doses of CBD. Concordantly, splenocytes of mice administered with CBD (5 or 20 mg/kg) produced less IL-2, IL-4 and IFN-gamma than those of vehicle-treated controls, upon ex vivo stimulation with phorbol ester plus calcium ionophore. Likewise, T-cell mitogen (concanavalin A)-induced proliferation of splenocytes was also markedly suppressed in mice administered with CBD. Furthermore, the observed ex vivo effects of CBD on cytokine production and T-cell proliferation were confirmed in splenocytes directly exposed to CBD (1-8 microM) in vitro, indicating a direct effect by CBD. Taken together, the results demonstrated that CBD markedly suppressed antigen-specific antibody production in OVA-sensitized mice, and suggest that CBD-mediated suppression of humoral immunity could be mediated by the impaired functions of splenocytes.

Pacific oyster-derived polysaccharides attenuate allergen-induced intestinal inflammation in a murine model of food allergy.

Oyster-derived polysaccharides (OPS) have been shown to modulate the T helper (Th)1/Th2 immunobalance toward the Th1-dominant direction in antigen-primed splenocytes. In the present study, we hypothesized that OPS might attenuate intestinal inflammation associated with food allergy, a Th2-dominant immune disorder. BALB/c mice were sensitized twice with ovalbumin (OVA) absorbed to alum and then repeatedly challenged with intragastric OVA to induce intestinal allergic responses. The mice were administered by gavage with OPS and/or vehicle (distilled water) once/d during the two sensitization phases, and once every other day during the challenge phase. Administration with OPS attenuated OVA challenge-elicited diarrhea, and the infiltration of mast cells in the intestine. OPS demonstrated a protective effect on the reduced ratio of villus length over crypt depth of the intestine in allergic mice. Furthermore, OPS administration markedly attenuated the intestinal expression of the Th2 signature cytokine interleukin-4 (IL-4). Collectively, these results demonstrated the in vivo antiallergic activity of OPS, which is associated with the suppression of allergen-induced intestinal Th2 responses and mast cell activation.

Polyethylene glycol-coated graphene oxide attenuates antigen-specific IgE production and enhanced antigen-induced T-cell reactivity in ovalbumin-sensitized BALB/c mice.

BACKGROUND: Graphene oxide (GO) is a promising nanomaterial for potential application in the versatile field of biomedicine. Graphene-based nanomaterials have been reported to modulate the functionality of immune cells in culture and to induce pulmonary inflammation in mice. Evidence pertaining to the interaction between graphene-based nanomaterials and the immune system in vivo remains scarce. The present study investigated the effect of polyethylene glycol-coated GO (PEG-GO) on antigen-specific immunity in vivo. METHODS: BALB/c mice were intravenously administered with a single dose of PEG-GO (0.5 or 1 mg/kg) 1 hour before ovalbumin (OVA) sensitization, and antigen-specific antibody production and splenocyte reactivity were measured 7 days later. RESULTS: Exposure to PEG-GO significantly attenuated the serum level of OVA-specific immunoglobulin E. The production of interferon-γ and interleukin-4 by splenocytes restimulated with OVA in culture was enhanced by treatment with PEG-GO. In addition, PEG-GO augmented the metabolic activity of splenocytes restimulated with OVA but not with the T-cell mitogen concanavalin A. CONCLUSION: Collectively, these results demonstrate that systemic exposure to PEG-GO modulates several aspects of antigen-specific immune responses, including the serum production of immunoglobulin E and T-cell functionality.

Cannabidiol hydroxyquinone-induced apoptosis of splenocytes is mediated predominantly by thiol depletion.

Cannabidiol, the major nonpsychotropic phytocannabinoid, has been recently demonstrated to induce apoptosis in primary lymphocytes via an oxidative stress-dependent mechanism. Cannabidiol can be converted by microsomal enzymes to the hydroxyquinone metabolite HU-331 that forms adducts with glutathione. The present study tested the hypothesis that HU-331 could cause apoptosis via the depletion of thiols in splenocytes. Our results showed that HU-331 treatment significantly enhanced splenocyte apoptosis in a time- and concentration-dependent manner. Concordantly, a gradual diminishment in the cellular thiols and glutathione was detected in HU-331-treated splenocytes. The apoptosis and thiol diminishment induced by HU-331 were abrogated in the presence of thiol antioxidants, including N-acetyl-(L)-cysteine and N-(2-mercaptopropionyl) glycine, whereas the non-thiol antioxidants catalase and pyruvate were ineffective. In comparison, both thiol and non-thiol antioxidants were capable of attenuating H(2)O(2)-induced thiol diminishment and reactive oxygen species generation in splenocytes. Collectively, these results suggest that HU-331 might be an active metabolite of cannabidiol potentially contributing to the induction of apoptosis in splenocytes, and that the apoptosis is primarily mediated by the loss of cellular thiols.

Cannabidiol-induced apoptosis in primary lymphocytes is associated with oxidative stress-dependent activation of caspase-8.

We recently reported that cannabidiol (CBD) exhibited a generalized suppressive effect on T-cell functional activities in splenocytes directly exposed to CBD in vitro or isolated from CBD-administered mice. To investigate the potential mechanisms of CBD effects on T cells, we characterized the pro-apoptotic effect of CBD on primary lymphocytes. The apoptosis of splenocytes was markedly enhanced following CBD exposure in a time- and concentration-dependent manner, as evidenced by nuclear hypodiploidity and DNA strand breaks. Exposure of splenocytes to CBD elicited an early production of reactive oxygen species (ROS) with the peak response at 1 h post CBD treatment. In parallel with the ROS production, a gradual diminishment in the cellular glutathione (GSH) content was detected in CBD-treated splenocytes. Both CBD-mediated ROS production and GSH diminishment were remarkably attenuated by the presence of N-acetyl-L-cysteine (NAC), a thiol antioxidant. In addition, CBD treatment significantly stimulated the activation of caspase-8, which was abrogated in the presence of NAC or GSH. Pretreatment of splenocytes with a cell-permeable inhibitor for caspase-8 significantly attenuated, in a concentration-dependent manner, CBD-mediated apoptosis, but not ROS production. Collectively, the present study demonstrated that the apoptotic effect of CBD in primary lymphocytes is closely associated with oxidative stress-dependent activation of caspase-8.