Using random forest to predict antimicrobial minimum inhibitory concentrations of nontyphoidal Salmonella in Taiwan.

Antimicrobial resistance (AMR) is a global health issue and surveillance of AMR can be useful for understanding AMR trends and planning intervention strategies. Salmonella, widely distributed in food-producing animals, has been considered the first priority for inclusion in the AMR surveillance program by the World Health Organization (WHO). Recent advances in rapid and affordable whole-genome sequencing (WGS) techniques lead to the emergence of WGS as a one-stop test to predict the antimicrobial susceptibility. Since the variation of sequencing and minimum inhibitory concentration (MIC) measurement methods could result in different results, this study aimed to develop WGS-based random forest models for predicting MIC values of 24 drugs using data generated from the same laboratories in Taiwan. The WGS data have been transformed as a feature vector of 10-mers for machine learning. Based on rigorous validation and independent tests, a good performance was obtained with an average mean absolute error (MAE) less than 1 for both validation and independent test. Feature selection was then applied to identify top-ranked 10-mers that can further improve the prediction performance. For surveillance purposes, the genome sequence-based machine learning methods could be utilized to monitor the difference between predicted and experimental MIC, where a large difference might be worthy of investigation on the emerging genomic determinants.

Differential cytokine profiling and microbial species involved in cecal microbiota modulations in SPF chicks immunized with a dual vaccine against Salmonella Typhimurium infection.

Salmonella Typhimurium (ST) infection in laying hens is a significant threat to public health and food safety. Host resistance against enteric pathogen invasion primarily relies on immunity and gut barrier integrity. This study applied the ST infection model and a dual live vaccine containing Salmonella Enteritidis (SE) strain Sm24/Rif12/Ssq and ST strain Nal2/Rif9/Rtt to investigate the cellular cytokine expression profiles and the differential community structure in the cecal microbiota of specific-pathogen-free (SPF) chicks and field-raised layers. The results showed that ST challenge significantly upregulated expressions of IL-1ß in SPF chicks. Vaccination, on the other hand, led to an elevation in IFNγ expression and restrained IL-1ß levels. In the group where vaccination preceded the ST challenge (S.STvc), heightened expressions of IL-1ß, IL-6, IL-10, and IL-12ß were observed, indicating active involvement of both humoral and cell-mediated immunity in the defense against ST. Regarding the cecal microbiota, the vaccine did not affect alpha diversity nor induce a significant shift in the microbial community. Conversely, ST infection significantly affected the alpha and beta diversity in the cecal microbiota, reducing beneficial commensal genera, such as Blautia and Subdoligranulum. MetagenomeSeq analysis reveals a significant increase in the relative abundance of Faecalibacterium prausnitzii in the groups (S.STvc and STvc) exhibiting protection against ST infection. LEfSe further demonstrated Faecalibacterium prausnitzii as the prominent biomarker within the cecal microbiota of SPF chicks and field layers demonstrating protection. Another biomarker identified in the S.STvc group, Eubacterium coprostanoligenes, displayed an antagonistic relationship with Faecalibacterium prausnitzii, suggesting the limited biological significance of the former in reducing cloacal shedding and tissue invasion. In conclusion, the application of AviPro Salmonella DUO vaccine stimulates host immunity and modulates cecal microbiota to defend against ST infection. Among the microbial modulations observed in SPF chicks and field layers with protection, Faecalibacterium prausnitzii emerges as a significant species in the ceca. Further research is warranted to elucidate its role in protecting layers against ST infection.

Urinary angiotensin-converting enzyme 2 and its activity in cats with chronic kidney disease.

Introduction: Angiotensin-converting enzyme 2 (ACE2) played an important role in the renin-angiotensin-aldosterone system (RAAS) and it was proved to be renoprotective in renal disease. Urinary angiotensin-converting enzyme 2 (uACE2) has been shown to reflect renal injury in human and experimental studies, but its role in feline kidney disease remains unknown. Aims: Our objectives involve comparing uACE2 concentrations and activities in cats across CKD stages with healthy controls, investigating the relationship between uACE2 concentrations, activities, and clinicopathological data in feline CKD patients, and assessing the predictive abilities of both for CKD progression. Methods: A retrospective, case-control study. The concentration and activity of uACE2 were measured by commercial ELISA and fluorometric assay kits, respectively. The concentration was adjusted to give uACE2 concentration-to-creatinine ratios (UACCRs). Results: In total, 67 cats consisting of 24 control and 43 chronic kidney disease (CKD), including 24 early-stage CKD and 19 late-stage CKD, were enrolled in this study. UACCR values were significantly higher in both early-stage (2.100 [1.142-4.242] x 10-6) and late-stage feline CKD (4.343 [2.992-5.0.71] x 10-6) compared to healthy controls (0.894 [0.610-1.076] x 10-6; p < 0.001), and there was also significant difference between-early stage group and late-stage group (p = 0.026). Urinary ACE2 activity (UAA) was significantly lower in CKD cats (1.338 [0.644-2.755] x pmol/min/ml) compared to the healthy cats (7.989 [3.711-15.903] x pmol/min/ml; p < 0.001). UACCR demonstrated an independent, positive correlation with BUN (p < 0.001), and UAA exhibited an independent, negative correlation with plasma creatinine (p < 0.001). Both UACCR and UAA did not yield significant results in predicting CKD progression based on the ROC curve analysis. Conclusion and clinical importance: uACE2 concentration and activity exhibit varying changes as renal function declines, particularly in advanced CKD cats.

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.

Oral supplementation with areca-derived polyphenols attenuates food allergic responses in ovalbumin-sensitized mice.

BACKGROUND: Arecae semen, the dried slice of areca nuts, is a traditional Chinese medicine used to treat intestinal parasitosis, rectal tenesmus and diarrhea. Areca nuts contain a rich amount of polyphenols that have been shown to modulate the functionality of mast cells and T cells. The objective of this study is to investigate the effect of polyphenol-enriched areca nut extracts (PANE) against food allergy, a T cell-mediated immune disorder. METHODS: BALB/c mice were left untreated or administered with PANE (0.05% and 0.1%) via drinking water throughout the entire experiment. The mice were sensitized with ovalbumin (OVA) twice by intraperitoneal injection, and then repeatedly challenged with OVA by gavage to induce food allergic responses. RESULTS: PANE administration attenuated OVA-induced allergic responses, including the occurrence of diarrhea and the infiltration and degranulation of mast cells in the duodenum. The serum level of OVA-specific IgE and the expression of interleukin-4 in the duodenum were suppressed by PANE treatment. In addition, PANE administration induced Gr-1+, IL-10+ and Gr-1+IL-10+ cells in the duodenum. CONCLUSION: These results demonstrate that oral intake of areca-derived polyphenols attenuates food allergic responses accompanied with a decreased Th2 immunity and an enhanced induction of functional myeloid-derived suppressor cells.

Pacific oyster-derived polysaccharides enhance antigen-specific T helper (Th)1 immunity in vitro and in vivo.

Bacterial and fungal polysaccharides are well known as being immunoactive. However, evidence pertaining to the immunomodulatory activity of polysaccharides derived from animal origins is scarce. This study investigated the effects of an extract of oyster (Crassostrea gigas) polysaccharides (OPS) on antigen-specific T-cell immunity. For in vitro studies, ovalbumin (OVA)-primed splenocytes were exposed to OPS and re-stimulated with OVA in culture to induce antigen-specific responses. For in vivo studies, mice were administered with OPS prior to OVA sensitization, and the functionality of their splenocytes was examined. Direct exposure of OVA-primed splenocytes to OPS (10-500 µg/mL) resulted in a marked enhancement of the cell metabolic activity and proliferation. Exposure to OPS also augmented the expression of the T helper (Th)1 cytokine interferon (IFN)-γ, whereas the Th2 cytokine interleukin-4 was suppressed. The enhancement of antigen-specific IFN-γ expression was further demonstrated in splenocytes of mice administered with OPS (100 and 200 mg/kg). Moreover, OPS enhanced the mRNA expression of T-bet, a transcription factor governing the development of Th1 cells, by splenocytes. Taken together, these results demonstrated that OPS modulated antigen-specific T cell responses, including antigen-induced splenocyte proliferation and T-cell cytokine expression. In addition, OPS polarized the Th1/Th2 immunobalance toward the Th1-dominant direction, which may be attributed to the up-regulation of Th1 cell development.

Cytokines and cecal microbiome modulations conferred by a dual vaccine in Salmonella-infected layers.

Zoonotic Salmonella infection is a critical and challenging issue for public health. Since human infections are mainly associated with consuming contaminated chicken products, strategies to reduce Salmonella carriage and shedding are essential. Here we investigate the mechanisms of the live attenuated Salmonella vaccine (AviPro Salmonella Duo) against Salmonella Enteritidis (SE) infection. We focused on inflammatory-related cytokine expressions and cecal microbiota modulations in specific-pathogen-free (SPF) and field layers. Forty-eight 2-day-old SPF layers were randomly allotted into S.SEvc, S.SEc, S.Vc, and S.Ct groups in trial 1. The equal number of filed layers at 25 wk were allocated into SEvc, SEc, Vc, and Ct groups in trial 2. Each group contained 12 layers. Groups were further assigned for vaccination (S.Vc and Vc groups), SE challenge (S.SEc and SEc groups), vaccination and the following SE challenge (S.SEvc and SEvc groups), or the placebo treatment (S.Ct and Ct groups). Cecal tissues and contents of layers on day 14 post-SE-challenges were collected for cytokine mRNA expression and 16S rRNA metagenomic analyses. We found that SE challenges significantly upregulated expressions of IFNγ, IL-1ß, IL-12ß, and NFκB1A in SPF layers. The vaccine notably counteracted the levels of IFNα, IFNγ, and NFκB1A activated by SE attacks. The vaccination, SE challenge, and their combination did not significantly affect alpha diversities but promoted dissimilarities in microbial communities between groups. Eubacterium_coprostanoligenes and Faecalibacterium_prausnitzii were identified as contributory taxa in the cecal microbiota of SE-challenged and vaccinated SPF layers. A significantly higher abundance of Faecalibacterium_prausnitzii in the ceca further correlated with the vaccination conferred protection against SE infection. In contrast, Oscillibacter_valericigenes and Mediterraneibacter_glycyrrhizinilyticus were featured taxa in Salmonella-infected field layers. Megamonas_hypermegale and Megamonas_rupellensis were identified as featured taxa in vaccinated field layers compared to SE-infected layers. To conclude, applying a dual Salmonella vaccine in this study modulated expressions of inflammatory-related cytokines and the cecal microbiome in layers, contributing to protection against SE infection. The feature microbes are promising for developing predictive indices and as antibiotic alternatives added to feed to reduce the risk of Salmonella shedding and contamination.

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.

Investigation of a potential scintigraphic tracer for imaging apoptosis: radioiodinated annexin V-Kunitz protease inhibitor fusion protein.

Radiolabeled annexin V (ANV) has been widely used for imaging cell apoptosis. Recently, a novel ANV-Kunitz-type protease inhibitor fusion protein, ANV-6L15, was found to be a promising probe for improved apoptosis detection based on its higher affinity to phosphatidylserine (PS) compared to native ANV. The present paper investigates the feasibility of apoptosis detection using radioiodinated ANV-6L15. Native ANV and ANV-6L15 were labeled with iodine-123 and iodine-125 using Iodogen method. The binding between the radioiodinated proteins and erythrocyte ghosts or chemical-induced apoptotic cells was examined. ANV-6L15 can be radioiodinated with high yield (40%-60%) and excellent radiochemical purity (>95%). (123)I-ANV-6L15 exhibited a higher binding ratio to erythrocyte ghosts and apoptotic cells compared to (123)I-ANV. The biodistribution of (123)I-ANV-6L15 in mice was also characterized. (123)I-ANV-6L15 was rapidly cleared from the blood. High uptake in the liver and the kidneys may limit the evaluation of apoptosis in abdominal regions. Our data suggest that radiolabeled ANV-6L15 may be a better scintigraphic tracer than native ANV for apoptosis detection.

Areca nut extracts enhance the development of CD11b(+) Gr-1(+) cells with the characteristics of myeloid-derived suppressor cells in antigen-stimulated mice.

BACKGROUND: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre-cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid-derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen-stimulated mice, we hypothesized that ANE might enhance the development of MDSC. METHODS: Ovalbumin (OVA)-sensitized BALB/c mice were daily administered with ANE (5-50 mg/kg), polyphenol-enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. RESULTS: ANE and PANE treatment significantly increased the spleen index and the population of CD11b(+) Gr-1(+) cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL-10, arginase-I and iNOS in splenic CD11b(+) cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr-1(+) IL-10(+) cells in the footpads challenged with OVA. CONCLUSIONS: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid-derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca-related oral diseases.

Areca-nut extract modulates antigen-specific immunity and augments inflammation in ovalbumin-sensitized mice.

Areca-nut chewing has been linked to oral cancer and many other diseases, in which immune deterioration and tissue inflammation are plausibly involved. Recent studies reported that areca-nut extract (ANE) affected the functionality of lymphocytes and neutrophils in vitro. In the present study, we investigated the immunomodulatory effect of ANE in vivo. Ovalbumin (OVA)-sensitized mice were daily administered with ANE (5-50 mg/kg) for 10 doses by intraperitoneal injection from days 1 to 5 and from 8 to 12. The mice were systemically sensitized with OVA on day 3, and their footpads were challenged with OVA to induce delayed-type hypersensitivity (DTH) reactions on day 13. The serum level of OVA-specific IgM and IgG(1) was significantly attenuated by 5 and 25 mg/kg of ANE, whereas OVA-specific IgG(2a) was markedly enhanced by 50 mg/kg of ANE. The production of interferon (IFN)-γ by splenocytes reexposed to OVA in culture was markedly augmented by ANE (25 and 50 mg/kg). In addition, ANE (25 and 50 mg/kg) demonstrated an enhancing effect on DTH reactions, including the tissue swelling, the infiltration of CD3(+) and F4/80(+) cells, and the expression of IFN-γ and tumor necrosis factor (TNF)-α in the footpads challenged with OVA. The phagocytic activity and TNF-α production by the splenic CD11b(+) cells were also enhanced in ANE-treated groups. Taken together, these results demonstrated that ANE modulated antigen-specific immune responses and promoted inflammatory reactions in vivo, which may contribute to immune deregulation associated with areca-related diseases.

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.

Diosgenin, a plant-derived sapogenin, enhances regulatory T-cell immunity in the intestine of mice with food allergy.

It was hypothesized that the suppressive effect of diosgenin (1) on the intestinal T helper (Th)2 responses is associated with an enhancement of the regulatory T-cell immunity. Ovalbumin (OVA)-sensitized BALB/c mice were gavaged daily with 1 and received repeatedly oral OVA challenges to induce intestinal allergic responses. The expression of Th2- and Treg-related cytokines and transcription factors was examined by immunohistochemical staining and RT-PCR. Administration of 1 markedly attenuated the intestinal expression of interleukin (IL)-4 and GATA3. In addition, administration of 1 reversed the diminished density of intestinal Foxp3(+) cells induced by OVA oral challenges and enhanced the expression of IL-10 by Foxp3(+) cells markedly. These results suggest that the suppressive effect of 1 on allergen-induced intestinal Th2 responses is closely associated with an up-regulation of the regulatory T-cell immunity in the inflammatory site.

Cannabidiol attenuates delayed-type hypersensitivity reactions via suppressing T-cell and macrophage reactivity.

AIM: to investigate the effects cannabidiol (CBD) on delayed-type hypersensitivity (DTH) reactions and antigen-induced T-cell cytokine expression. METHODS: DTH was induced by subcutaneous ovalbumin (OVA) challenge to the footpads of mice sensitized with OVA. Inflammatory reactions were measured by footpad swelling and histological analysis. Antigen-induced cytokine expression by OVA-primed splenocytes was measured using ELISA and RT-PCR. RESULTS: CBD (1-10 mg/kg) administration, in a dose-dependent fashion, significantly attenuated inflammatory reactions associated with DTH in the footpads of mice sensitized and challenged with OVA. Histological examination revealed that CBD suppressed the infiltration of T cells and macrophages, and the expression of interferon (IFN)-γ and tumor necrosis factor-α, two pro-inflammatory cytokines implicated in DTH in the inflammatory site. In contrast, the expression of interleukin (IL)-10 in the footpads was enhanced by CBD administration. In addition, CBD at concentrations devoid of cytotoxic effects (1-4 micromol/L) attenuated OVA-induced IFN-γ production by OVA-primed splenocytes, whereas IL-4 was unaffected. CONCLUSION: CBD curbs DTH reactions via suppressing the infiltration and functional activity of T cells and macrophages in the inflammatory site, suggesting a therapeutic potential for CBD for the treatment of type IV hypersensitivity.

Development of Nanosome-Encapsulated Honokiol for Intravenous Therapy Against Experimental Autoimmune Encephalomyelitis.

BACKGROUND: Honokiol has been reported to possess anti-inflammatory and neuroprotective activities. However, the poor aqueous solubility of honokiol limits its clinical application for systemic administration. PURPOSE: This study aims to develop a novel formulation of nanosome-encapsulated honokiol (NHNK) for intravenous therapy against mouse experimental autoimmune encephalomyelitis (EAE) that mimics human multiple sclerosis. METHODS: Nanosomes and NHNK were prepared by using an ultra-high pressure homogenization (UHPH) method. Mice were treated with NHNK or empty nanosomes during the peak phase of EAE symptoms. Symptoms of EAE were monitored and samples of the spinal cord were obtained for histopathological examinations. RESULTS: The stock of NHNK containing honokiol in the nanosome formulation, which showed the structure of single phospholipid bilayer membranes, was well formulated with the particle size of 48.0 ± 0.1 nm and the encapsulation efficiency 58.1 ± 4.2%. Intravenous administration of NHNK ameliorated the severity of EAE accompanied by a significant reduction of demyelination and inflammation in the spinal cord. Furthermore, NHNK decreased the number of IL-6+, Iba-1+TNF +, Iba-1+IL-12 p40+, and CD3+IFN-γ+ cells infiltrating the spinal cord. CONCLUSION: The UHPH method simplified the preparation of NHNK with uniformly distributed nanosize and high encapsulation efficiency. Intravenous administration of NHNK ameliorated the severity of EAE by suppressing the infiltration of activated microglia and Th1 cells into the spinal cord. Collectively, these results suggest that the formulation of NHNK is a prospective therapeutic approach for inflammatory CNS diseases, such as multiple sclerosis.

CD5-low expression lymphocytes in canine peripheral blood show characteristics of natural killer cells.

NK cell markers and receptors have been discovered in many mammalian species, such as humans, mice, rats, pigs, and cows. However, there is still a lack of information concerning NK cell markers or receptors in canines. We have discovered that canine CD5-low density (CD5lo) cells in PBL are closely associated with NK cell characteristics. CD5lo cells comprised 14.9 +/- 6.68% of the total PBL. A high proportion of the CD5lo cell population expressed CD3 (96.6%), CD8alpha (77.7%), CD8beta (53%), alpha/beta TCR (83%), and CD11/18 (80%), but the expression of gamma/delta TCR (6.5%), CD4 (10.6%), and CD21 (2.4%) was low. CD5lo cells were larger than CD5-high density (CD5hi) cells. Light and electron microscopy revealed numerous large cytoplasmic granules in CD5lo cells, especially after IL-2 stimulation, which was in contrast to CD5hi, in which intracytoplasmic granules were not frequently seen. After IL-2 stimulation, CD5lo cells had significantly stronger NK cytotoxicity than CD5hi cells. CD5lo cells had much higher mRNA levels for NKG2D, CD16, CD94, CD160, perforin, and granzyme than CD5hi. Following IL-2 stimulation, CD5lo cells had significantly higher mRNA levels of NKp30, NKp44, CD16, and CD94 than CD5hi cells. In addition, IL-2-stimulated, CD5lo-depleted PBL showed a loss of NK cytotoxicity. CD5lo cells also showed significantly lower antigen-specific cytotoxic T cell activity as compared with CD5hi cells. Taken together, the CD5lo subset in canine PBL is closely related to canine NK cells, and CD5lo can be used as a phenotypic marker for an IL-2-dependent canine NK cell enrichment.

Diosgenin attenuates allergen-induced intestinal inflammation and IgE production in a murine model of food allergy.

Diosgenin, the major sapogenin contained in the Chinese yam, has recently been shown to promote systemic T helper 1-type immunity in a murine model of airway hypersensitivity. In this study, we hypothesized that diosgenin might be effective in modulating food allergy. BALB/c mice were either left untreated (naïve; NA) or administered daily with vehicle (VH; olive oil) and/or diosgenin (100 or 200 mg/kg) by gavage throughout the experiment. Except for the NA group, the mice were sensitized with ovalbumin (OVA) and repeatedly challenged with intragastric OVA to induce intestinal allergic responses. Diosgenin demonstrated a suppressive effect on the intestinal inflammation, including the occurrence of diarrhea, the infiltration and degranulation of mast cells, and the presence of mucin-containing goblet cells in the duodenum. A protective effect by diosgenin on reducing the crypt depth of the intestine was also observed in OVA-sensitized and challenged mice. Furthermore, the serum production of OVA-specific IgE, and the total IgE was suppressed. In contrast, OVA-specific IgG (2a) was enhanced by diosgenin treatment in OVA-sensitized mice. These results demonstrated the IN VIVO anti-allergic activity of diosgenin, which is associated with the suppression of IgE production and mast cell infiltration and degranulation.

Systemic exposure to a single dose of ferucarbotran aggravates neuroinflammation in a murine model of experimental autoimmune encephalomyelitis.

BACKGROUND: Medicinal preparations of iron oxide nanoparticles (IONPs) have been used as MRI contrast agents for the diagnosis of hepatic tumors and the assessment of neuroinflammation and blood-brain barrier integrity. However, it remains mostly unclear whether exposure to IONPs affects neuroinflammation under disease conditions. The present study aims to investigate the impact of IONPs on autoimmune-mediated neuroinflammation using a murine model of experimental autoimmune encephalomyelitis (EAE) that mimics human multiple sclerosis. METHODS: Mice were either left untreated or immunized with myelin oligodendrocyte glyco-protein on day 0 followed by two injections of pertussis toxin for EAE induction. The EAE mice were intravenously administered with a single dose of the carboxydextran-coated IONPs, ferucarbotran (20 mg Fe/kg) and/or saline (as vehicle) on day 18. Symptoms of EAE were daily monitored until the mice were killed on day 30. Tissue sections of the brain and spinal cord were prepared for histopathological examinations. Iron deposition, neuron demyelination and inflammatory cell infiltration were examined using histochemical staining. The infiltration of microglial and T cells, and cytokine expression were examined by immunohistochemical staining and/or reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Iron deposition was detected in both the brain and spinal cord of EAE mice 3 days post-ferucarbotran treatment. The clinical and pathological scores of EAE, percentage of myelin loss and infiltration of inflammatory cells into the spinal cord were significantly deteriorated in EAE mice treated with ferucarbotran. Furthermore, ferucarbotran treatment increased the number of CD3+, Iba-1+, IL-6+, Iba-1+TNF-α+ and CD3+IFN-γ+ cells in the spinal cord of EAE mice. CONCLUSION: A single exposure to ferucarbotran exacerbated neuroinflammation and disease severity of EAE, which might be attributed to the enhanced activation of microglia and T cells. These results demonstrated that the pro-inflammatory effect of ferucarbotran on the central nervous system is closely associated with the deterioration of autoimmunity.

Neural anti-inflammatory action mediated by two types of acetylcholine receptors in the small intestine.

Gastrointestinal prokinetic agents function as serotonin-4 receptor (5-HT4R) agonists to activate myenteric plexus neurons to release acetylcholine (ACh), which then induce anti-inflammatory action. Details of this pathway, however, remain unknown. The aim of this study is to clarify the anti-inflammatory mechanism underlying the 5-HT4R agonist, mosapride citrate (MOS)-induced anti-inflammatory action on postoperative ileus (POI). POI models were generated from wild-type C57BL6/J (WT), 5-HT4R knock-out (S4R KO), α7 nicotinic AChR KO (α7 R KO), and M2 muscarinic ACh receptor KO (M2R KO) mice. MOS attenuated leukocyte infiltration in WT. MOS-induced anti-inflammatory action was completely abolished in both S4R KO and S4R KO mice upon wild-type bone marrow transplantation. MOS-induced anti-inflammatory action against macrophage infiltration, but not neutrophil infiltration, was attenuated in α7 R KO mice. Selective α7nAChR agonists (PNU-282987 and AR-R17779) also inhibited only macrophage infiltration in POI. MOS-mediated inhibition of neutrophil infiltration was diminished by atropine, M2AChR antagonist, methoctramine, and in M2R KO mice. Stimulation with 5-HT4R inhibits leukocyte infiltration in POI, possibly through myenteric plexus activation. Released ACh inhibited macrophage and neutrophil infiltration likely by activation of α7nAChR on macrophages and M2AChR. Thus, macrophage and neutrophil recruitment into inflamed sites is regulated by different types of AChR in the small intestine.

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.