Porphyrin-Based Brain Tumor-Targeting Agents: [64Cu]Cu-porphyrin and [64Cu]Cu-TDAP.

The aim of this study is to evaluate a radioactive metal complex platform for brain tumor targeting. Herein, we introduce a new porphyrin derivative, 5,10,15,20-(tetra-N,N-dimethyl-4-aminophenyl)porphyrin (TDAP), in which four N,N-dimethyl-4-p-phenylenediamine (DMPD) moieties are conjugated to the porphyrin labeled with the radiometal 64Cu. DMPD affected the pharmacokinetics of porphyrin in terms of retention time in vivo and tumor-targeting ability relative to those of unmodified porphyrin. [64Cu]Cu-TDAP showed stronger enhancement than [64Cu]Cu-porphyrin in U87MG glioblastoma cells, especially in the cytoplasm and nucleus, indicating its tumor-targeting properties and potential use as a therapeutic agent. In the subcutaneous and orthotopic models of brain-tumor-bearing mice, [64Cu]Cu-TDAP was clearly visualized in the tumor site via positron emission tomography imaging and showed a tumor-to-brain ratio as high as 13. [64Cu]Cu-TDAP deserves attention as a new diagnostic agent that is suitable for the early diagnosis and treatment of brain tumors.

Feasibility of Gd-Based prostate cancer targeted magnetic resonance agents using prostate specific membrane antigen.

The aim of this work was to evaluate Gd-FC705, a prostate-specific membrane antigen (PSMA)-targeted MRI contrast agent. The r1 and r2 relaxivities of Gd-FC705 are 5.94 mM-1s-1 and 17.77 mM-1s-1, respectively, in HSA solution (0.67 mM) at 3 T, which are higher than those of Gd-DOTA. Specific targeting efficacy was found with a 3-fold enhancement between PSMA-negative (PSMA-) and PSMA-positive (PSMA+) cells. The in vivo targeting and bio-distribution of Gd-FC705 were further confirmed using nude mice bearing PC3 human prostate cancer xenografts, which showed a 2-fold increase in the contrast-to-noise ratio (CNR) for PSMA+ tumors compared to PSMA- tumors 1 h post injection and a longer circulation time than Gd-DOTA. These results demonstrate that Gd-FC705 has great potential as a diagnostic agent for prostate cancer.

Protein arginine methyltransferase 1 contributes to the development of allergic rhinitis by promoting the production of epithelial-derived cytokines.

BACKGROUND: Arginine methylation is a posttranslational modification mediated by protein arginine methyltransferases (PRMTs). Although previous studies have shown that PRMT1 contributes to the severity of allergic airway inflammation or asthma, the underlying mechanism is poorly understood. OBJECTIVE: This study aimed to explore the role of PRMT1 and its relevant mechanism in the development of allergic rhinitis (AR). METHODS: The expression levels of PRMTs and cytokines were determined by RT-PCR, and the localization of PRMT1 was determined by immunohistochemistry and confocal microscopy. The levels of house dust mite (HDM)-specific immunoglobulins in serum and of cytokines in nasal lavage fluids were determined by ELISA. PRMT1 inhibition was achieved by siRNA and treatment with the pan PRMT inhibitor arginine N-methyltransferase inhibitor-1. RESULTS: PRMT1 expression was significantly increased in the nasal mucosa of patients and mice with AR. The degree of eosinophilic infiltration in the nasal mucosa was reduced in PRMT1+/- AR mice compared with wild-type mice. PRMT1 haploinsufficiency reduced the levels of HDM-specific immunoglobulins in serum and those of TH2 (IL-4, IL-5, and IL-13) and epithelial (thymic stromal lymphopoietin [TSLP], IL-25, and IL-33) cytokines in the nasal lavage fluids of AR mice. In nasal epithelial cells, HDM and IL-4 cooperate to enhance PRMT1 expression through a mitogen-activated protein kinase-dependent pathway. In addition, PRMT1 was essential for the production of TSLP, IL-25, and IL-33 in response to HDM and IL-4. Arginine N-methyltransferase inhibitor-1 treatment alleviated AR in the mouse model. CONCLUSIONS: PRMT1 plays an important role in AR development by regulating epithelial-derived cytokine production and might be a new therapeutic target for AR.

Expression of Transcripts in Marmoset Oocytes Retrieved during Follicle Isolation Without Gonadotropin Induction.

The in vitro maturation of oocytes is frequently used as an assisted reproductive technique (ART), and has been successfully established in humans and rodents. To overcome the limitations of ART, novel procedures for the in vitro maturation of early follicles are emerging. During the follicle isolation procedure, the unintended rupture of each follicle leads to a release of extra oocytes. Such oocytes, which are obtained during follicle isolation from marmosets, can be used for early maturation studies. Marmoset (Callithrix jacchus), which is classified as a new-world monkey, is a novel model that has been employed in reproductive biomedical research, as its reproductive physiology is similar to that of humans in several aspects. The ovaries of female marmosets were collected, and the excess oocytes present during follicle isolation were retrieved without pre-gonadotropin induction. Each oocyte was matured in vitro for 48 h in the presence of various concentrations of human chorionic gonadotropin (hCG) and epidermal growth factor (EGF), and the maturity of oocytes and optimal maturation conditions were evaluated. Each oocyte was individually reverse-transcribed, and the expression of mRNAs and microRNAs (miRs) were analyzed. Concentrations of hCG significantly affected the maturation rate of oocytes [the number of metaphase II (MII) oocytes]. The expression of BMP15 and ZP1 was highest when the oocytes were matured using 100 IU/L of hCG without pre-treatment with gonadotropins, and that of Cja-mir-27a was highest when cultured with follicle stimulating hormone. These results suggest that these up-regulated miRs affect the maturation of oocytes. Interactions with other protein networks were analyzed, and a strong association of BMP15 and ZP1 with sperm binding receptor (ACR), anti-Müllerian hormone (AMH), and AMH receptor was demonstrated, which is related to the proliferation of granulosa cells. Collectively, on the basis of these results, the authors propose optimal maturation conditions of excess oocytes of marmoset without in vivo gonadotropin treatment, and demonstrated the roles of miRs in early oocyte maturation at the single-cell level in marmosets.

A comprehensive study on in vitro and in vivo toxicological evaluation of Artemisia capillaris.

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.

Absence of subchronic oral toxicity and genotoxicity of rice koji with Aspergillus terreus.

Koji products have been considered as an effective fermented food consumed in East Asia with many health benefits. Particularly, rice koji with Aspergillus terreus (RAT) has been reported to be able to prevent hyperlipidemia and hepatic steatosis through regulating cholesterol synthesis. Despite its biological activities, there is a lack of comprehensive information to give an assurance of its safety. Therefore, the objective of this study was to perform a series of toxicological studies (repeated dose oral toxicity and genotoxicity) according to test guidelines published by the Organization for Economic Cooperation and Development. Along with acute toxicity study using rats and beagle dogs, a 13-week toxicity study revealed no clear RAT-related toxic changes, including body weight, mortality, hematology, serum biochemistry, organ weight, and histopathology after oral administration at doses of 500, 1000, and 2000 mg/kg BW. The no-observed-adverse-effect level of RAT was considered to be more than 2000 mg/kg BW/day in rats of both genders. In addition, potential genotoxicity was evaluated using a standard battery of tests (Ames test, chromosome aberration assay, and micronucleus assay) which revealed that RAT showed no genotoxicity. Accordingly, these results suggest that RAT is a safe and non-toxic functional food for human consumption at proper dose.

Human umbilical cord-derived mesenchymal stem cells in acute liver injury: Hepatoprotective efficacy, subchronic toxicity, tumorigenicity, and biodistribution.

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) therapy might be an alternative to liver transplantation for acute or chronic liver injury. The aim of this study was to evaluate the efficacy of human UC-MSCs on carbon tetrachloride (CCl4)-induced acute liver injury. In addition, its toxicity, tumorigenicity, and biodistribution were determined. Significant hepatoprotective effects of hUC-MSCs with decreased levels of hepatocellular necrosis and lobular neutrophilic infiltration were found. Regarding the safety of hUC-MSCs, no serious hUC-MSCs-related changes (body weight, food/water consumption, clinical symptom, urinalysis, hematology, clinical chemistry, organ weight, and histopathology) were observed in a 13-week subchronic toxicity study. In a 26-week tumorigenicity study, no mice developed tumor related to hUC-MSCs transplantation up to 1 × 108 cells/kg. In particular, human mitochondrial sequence detection revealed that most hUC-MSCs were cleared from the major organs of the mice at 13 weeks after transplantation. There was no systemic toxicity or neoplastic finding either. Taken together, these results suggested that hUC-MSCs have great potential for future clinical treatment of acute liver disease.

Impact of IL-1ß on lung pathology caused by Mycobacterium abscessus infection and its association with IL-17 production.

Mycobacterium abscessus (MAB), a non-tuberculous mycobacterium (NTM), causes chronic pulmonary inflammation in humans. The NLRP3 inflammasome is a multi-protein complex that triggers IL-1ß maturation and pyroptosis through the cleavage of caspase-1. In this study, we investigated the roles of NLRP3 and IL-1ß in the host's defense against MAB. The IL-1ß production by MAB was completely abolished in NLRP3, but not NLRC4, deficient macrophages. The NLRP3 inflammasome components, which are ASC and caspase-1 were also found to be essential for IL-1ß production in response to MAB. NLRP3 and IL-1ß deficiency did not affect the intracellular growth of MAB in macrophages, and the bacterial burden in lungs of NLRP3- and IL-1ß-deficient mice was also comparable to the burden observed in WT mice. In contrast, IL-1ß deficiency ameliorated lung pathology in MAB-infected mice. Notably, the lung homogenates of IL-1ß-deficient mice had reduced levels of IL-17, but not IFN-γ and IL-4 when compared with WT counterparts. Furthermore, in vitro co-culture analysis showed that IL-1ß signaling was essential for IL-17 production in response to MAB. Finally, we observed that the anti-IL-17 antibody administration moderately mitigated MAB-induced lung pathology. These findings indicated that IL-1ß production contribute to MAB-induced lung pathology via the elevation of IL-17 production.

Preclinical evaluation of [18F]FP-CIT, the radiotracer targeting dopamine transporter for diagnosing Parkinson's disease: pharmacokinetic and efficacy analysis.

BACKGROUND: N-(3-fluoropropyl)-2ß-carboxymethoxy-3ß-(4-iodophenyl) nortropane (FP-CIT), the representative cocaine derivative used in dopamine transporter imaging, is a promising biomarker, as it reflects the severity of Parkinson's disease (PD). 123I- and 18F-labeled FP-CIT has been used for PD diagnosis. However, preclinical studies evaluating [18F]FP-CIT as a potential diagnostic biomarker are scarce. Among translational research advancements from bench to bedside, translating preclinical findings into clinical practice is one-directional. The aim of this study is to employ a circular approach, beginning back from the preclinical stage, progressing to the supplementation of [18F]FP-CIT, and subsequently returning to clinical application. We investigated the pharmacokinetic properties of [18F]FP-CIT and its efficacy for PD diagnosis using murine models. RESULTS: Biodistribution, metabolite and excretion analyses were performed in mice and PD models were induced in rats using 6-hydroxydopamine (6-OHDA). The targeting efficiency of [18F]FP-CIT for the dopamine receptor was assessed through animal PET/CT imaging. Subsequently, correlation analysis was conducted between animal PET/CT imaging results and immunohistochemistry (IHC) targeting tyrosine hydroxylase. Rapid circulation was confirmed after [18F]FP-CIT injection. [18F]FP-CIT reached the highest uptake of 23.50 ± 12.46%ID/g in the striatum 1 min after injection, and it was rapidly excreted within 60 min. The major metabolic organs of [18F]FP-CIT were confirmed to be the intestines, liver, and kidneys. Its uptake in the intestine was approximately 5% ID/g. The uptake in the liver gradually increased, with excretion beginning after reaching a maximum after 60 min. The kidneys exhibited rapid elimination after 10 min. In the excretion study, rapid elimination was verified, with 21.46 ± 9.53% of the compound excreted within a 6 h period. Additionally, the efficacy of [18F]FP-CIT PET was demonstrated in the PD model, with a high correlation with IHC for both the absolute value (R = 0.803, p = 0.0017) and the ratio value (R = 0.973, p = 0.0011). CONCLUSIONS: This study fills the gap regarding insufficient preclinical studies on [18F]FP-CIT, including its ADME, metabolites, and efficiency. The pharmacological results, including accurate diagnosis, rapid circulation, and [18F]FP-CIT excretion, provide complementary evidence that [18F]FP-CIT can be used safely and efficiently to diagnose PD in clinics, although it is already used in clinics.

Comparison of the Effects of DOTA and NOTA Chelators on 64Cu-Cudotadipep and 64Cu-Cunotadipep for Prostate Cancer.

BACKGROUND: This study compared the effects of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as 64Cu-chelating agents in newly developed prostate-specific membrane antigen (PSMA) target compounds, 64Cu-cudotadipep and 64Cu-cunotadipep, on pharmacokinetics. METHODS: The in vitro stability of the chelators was evaluated using human and mouse serum. In vitro PSMA-binding affinity and cell uptake were compared using human 22Rv1 cells. To evaluate specific PSMA-expressing tumor-targeting efficiency, micro-positron emission tomography (mcroPET)/computed tomography (CT) and biodistribution analysis were performed using PSMA+ PC3-PIP and PSMA- PC3-flu tumor xenografts. RESULTS: The serum stability of DOTA- or NOTA-conjugated 64Cu-cudotadipep and 64Cu-cunotadipep was >97%. The Ki value of the NOTA derivative, cunotadipep, in the in vitro affinity binding analysis was higher (2.17 ± 0.25 nM) than that of the DOTA derivative, cudotadipep (6.75 ± 0.42 nM). The cunotadipep exhibited a higher cellular uptake (6.02 ± 0.05%/1 × 106 cells) compared with the cudotadipep (2.93 ± 0.06%/1 × 106 cells). In the biodistribution analysis and microPET/CT imaging, the 64Cu-labeled NOTA derivative, 64Cu-cunotadipep, demonstrated a greater tumor uptake and lower liver uptake than the DOTA derivative. CONCLUSIONS: This study indicates that the PSMA-targeted 64Cu-cunotadipep can be applied in clinical practice owing to its high diagnostic power for prostate cancer.

Oral Administration of Lactobacillus sakei CVL-001 Improves Recovery from Dextran Sulfate Sodium-Induced Colitis in Mice by Microbiota Modulation.

Inflammatory bowel disease (IBD) is an intestinal chronic inflammatory disease, and its incidence is steadily increasing. IBD is closely related to the intestinal microbiota, and probiotics are known to be a potential therapeutic agent for IBD. In our study, we evaluated the protective effect of Lactobacillus sakei CVL-001, isolated from Baechu kimchi, on dextran sulfated sodium (DSS)-induced colitis in mice. The oral administration of L. sakei CVL-001 according to the experimental schedule alleviated weight loss and disease activity in the mice with colitis. Furthermore, the length and histopathology of the colon improved. The expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß genes decreased in the colons of mice that were administered L. sakei CVL-001, whereas that of IL-10 increased. The expressions of genes coding for E-cadherin, claudin3, occludin, and mucin were also restored. In co-housed conditions, L. sakei CVL-001 administration did not improve disease activity, colon length, and histopathology. Microbiota analysis revealed that L. sakei CVL-001 administration increased the abundance of microbiota and altered Firmicutes/Bacteroidetes ratio, and decreased Proteobacteria. In conclusion, L. sakei CVL-001 administration protects mice from DSS-induced colitis by regulating immune response and intestinal integrity via gut microbiota modulation.

Water Extract of Desalted Salicornia europaea Inhibits RANKL-Induced Osteoclast Differentiation and Prevents Bone Loss in Ovariectomized Mice.

Osteoporosis, which is often associated with increased osteoclast activity due to menopause or aging, was the main focus of this study. We investigated the inhibitory effects of water extract of desalted Salicornia europaea L. (WSE) on osteoclast differentiation and bone loss in ovariectomized mice. Our findings revealed that WSE effectively inhibited RANKL-induced osteoclast differentiation, as demonstrated by TRAP staining, and also suppressed bone resorption and F-actin ring formation in a dose-dependent manner. The expression levels of genes related to osteoclast differentiation, including NFATc1, ACP5, Ctsk, and DCSTAMP, were downregulated by WSE. Oral administration of WSE improved bone density and structural parameters in ovariectomized mice. Dicaffeoylquinic acids (DCQAs) and saponins were detected in WSE, with 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA being isolated and identified. All tested DCQAs, including the aforementioned types, inhibited osteoclast differentiation, bone resorption, and the expression of osteoclast-related genes. Furthermore, WSE and DCQAs reduced ROS production mediated by RANKL. These results indicate the potential of WSE and its components, DCQAs, as preventive or therapeutic agents against osteoporosis and related conditions.

The sesquiterpene lactone estafiatin exerts anti-inflammatory effects on macrophages and protects mice from sepsis induced by LPS and cecal ligation puncture.

BACKGROUND: Previously, we found that the water extract of Artermisia scoparia Waldst. & Kit suppressed the cytokine production of lipopolysaccharide (LPS)-stimulated macrophages and alleviated carrageenan-induced acute inflammation in mice. Artemisia contains various sesquiterpene lactones and most of them exert immunomodulatory activity. PURPOSE: In the present study, we investigated the immunomodulatory effect of estafiatin (EST), a sesquiterpene lactone derived from A. scoparia, on LPS-induced inflammation in macrophages and mouse sepsis model. STUDY DESIGN AND METHODS: Murine bone marrow-derived macrophages (BMDMs) and THP-1 cells, a human monocytic leukemia cell line, were pretreated with different doses of EST for 2 h, followed by LPS treatment. The gene and protein expression of pro-inflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS) were measured by quantitative real-time polymerase chain reaction (qPCR) and Western blot analysis. The activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) was also evaluated at the level of phosphorylation. The effect of EST on inflammatory cytokine production, lung histopathology, and survival rate was assessed in an LPS-induced mice model of septic shock. The effect of EST on the production of cytokines in LPS-stimulated peritoneal macrophages was evaluated by in vitro and ex vivo experiments and protective effect of EST on cecal ligation and puncture (CLP) mice was also assessed. RESULTS: The LPS-induced expression of IL-6, TNF-α, and iNOS was suppressed at the mRNA and protein levels in BMDMs and THP-1 cells, respectively, by pretreatment with EST. The half-maximal inhibitory concentration (IC50) of EST on IL-6 and TNF-α production were determined as 3.2 µM and 3.1 µM in BMDMs, 3 µM and 3.4 µM in THP1 cells, respectively. In addition, pretreatment with EST significantly reduced the LPS-induced phosphorylation p65, p38, JNK, and ERK in both cell types. In the LPS-induced mice model of septic shock, serum levels of IL-6, TNF-α, IL-1ß, CXCL1, and CXCL2 were lower in EST-treated mice than in the control animals. Histopathology analysis revealed that EST treatment ameliorated LPS-induced lung damage. Moreover, while 1 of 7 control mice given lethal dose of LPS survived, 3 of 7 EST-treated (1.25 mg/kg) mice and 5 of 7 EST-treated (2.5 mg/kg) mice were survived. Pretreatment of EST dose-dependently suppressed the LPS-induced production of IL-6, TNF-α and CXCL1 in peritoneal macrophages. In CLP-induced mice sepsis model, while all 6 control mice was dead at 48 h, 1 of 6 EST-treated (1.25 mg/kg) mice and 3 of 6 EST-treated (2.5 mg/kg) mice survived for 96 h. CONCLUSION: These results demonstrated that EST exerts anti-inflammatory effects on LPS-stimulated macrophages and protects mice from sepsis. Our study suggests that EST could be developed as a new therapeutic agent for sepsis and various inflammatory diseases.

D-galactose Intake Alleviates Atopic Dermatitis in Mice by Modulating Intestinal Microbiota.

Atopic dermatitis (AD) is one of the most prevalent, chronic and persistent inflammatory skin diseases closely associated with intestinal microbiota. To evaluate the effect of D-galactose intake on AD, we orally administered D-galactose to BALB/c mice whose ears and skin were treated with 2,4-dinitrochlorobenzene (DNCB). D-galactose alleviated DNCB-induced AD-like phenotypes such as redness, scaling/dryness and excoriation. Ear thickness was also decreased by D-galactose administration. Histopathological analysis revealed decreased epidermal thickening, infiltration of immune cells, especially mast cells, in the dermis. Total levels of serum IgE representing the immunological response of AD were decreased by D-galactose administration. Microbiota analysis showed that D-galactose administration restored gut microbiota profiles, which were altered in AD mice, characterized by increased abundance of Bacteroidetes and decreased abundance of Firmicutes. The increased abundance of Bacteroides and the decreased abundance of Prevotella and Ruminococcus were reversed by D-galactose treatment, following improvement of AD. Our results suggest the possible use of D-galactose as a prebiotic to alleviate AD by altering gut microbiota.

Porphyrin-Based Tumor-Targeting Theranostic Agent: Gd-TDAP.

The aim of this work was to evaluate a tumor-targeting porphyrin-based gadolinium complex (Gd-TDAP) for use as an MR/optical imaging agent and potential therapeutic agent. Gd-TDAP had higher longitudinal relaxivity (11.8 mM-1 s-1) than a commercial MRI contrast agent (Omniscan; 3.7 mM-1 s-1) in HSA solution (0.67 mM) at 3 T. The tumor-targeting characteristics were confirmed by T1-weighted MR imaging and optical imaging using an orthotopic brain tumor mouse model, which showed 1.3-fold higher uptake in tumor compared to normal brain tissues. The cell fraction data using U87MG glioblastoma cells indicated the potential for gadolinium neutron capture therapy (Gd-NCT), which requires gadolinium to be inside the cell nucleus. In addition, porphyrin derivatives can be used for photodynamic therapy (PDT), and the results demonstrated that Gd-TDAP has great potential not only as a bimodal imaging agent but also for treatment.

Combination of vegetable soup and glucan demonstrates synergistic effects on macrophage-mediated immune responses.

Vegetable soup (VS), a plant-based functional food, has been used as a traditional folk medicine and is attracting attention for its ability to enhance the immune response. ß-Glucan, a well-established and effective immunomodulator, has synergistic effects when used in combination with some bioactive compounds. In the present study, we aimed to evaluate the synergistic immunomodulatory effects of the combination of VS and ß-glucan on macrophage-mediated immune responses. ß-Glucan was demonstrated to synergistically enhance the VS-stimulated immune response, including the production of interleukin-6, tumor necrosis factor-α, and nitric oxide, mainly through the mitogen-activated protein kinase pathway in macrophages. In addition, this combination has the potential for further development in functional foods with immune-enhancing activity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00888-x.

Water extract of Artemisia scoparia Waldst. & Kitam suppresses LPS-induced cytokine production and NLRP3 inflammasome activation in macrophages and alleviates carrageenan-induced acute inflammation in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia scoparia Waldst. & Kitam (A. scoparia) is a perennial herbal plant that is widely used as a folk remedy in Asian countries. Several studies have demonstrated that A. scoparia has various physiological effects, including anti-inflammation, anti-hypertension, anti-obesity, anti-hepatotoxicity, and anti-oxidant effects. AIM OF THE STUDY: The objective of the present study was to examine the anti-inflammatory effects of water extract of A. scoparia (WAS). MATERIALS AND METHODS: Murine bone marrow-derived macrophages (BMDMs), human monocyte THP-1 and murine fibroblast 3T3-L1 cells were used for the in vitro experiments. Cell viability and cytokine production were determined by the MTT assay and ELISA, respectively. RT-PCR was performed to determine iNOS gene expression and the Griess reaction was used to measure nitrite levels. iNOS protein expression, activation of NF-κB and MAPKs, and cleavage of caspase-1 and IL-1ß were determined by Western blot analysis. A carrageenan-induced mouse model of acute inflammation was used in the in vivo experiments. RESULTS: Pretreatment with WAS concentration-dependently suppressed gene expression and IL-6, TNF-α, CXCL1 and iNOS protein levels in BMDMs stimulated with LPS. In addition, pretreatment with WAS inhibited LPS-induced production of IL-6 and TNF-α in THP-1 cells and CXCL1 in 3T3-L1. Furthermore, LPS induced phosphorylation of p65 in BMDMs, and this induction was dramatically suppressed by WAS pretreatment. We further investigated whether WAS regulates activation of the NLRP3 inflammasome, which is known to be essential for IL-1ß processing. WAS inhibited the production of IL-1ß, but not IL-6, in response to adenosine triphosphate (ATP) and monosodium uric acid (MSU) crystals in LPS-primed BMDMs. Cleavage of caspase-1 and IL-1ß was also reduced by WAS. We finally evaluated the in vivo anti-inflammatory effects of WAS in a mouse model of carrageenan-induced acute inflammation. Subcutaneous administration of WAS reduced production of the inflammatory cytokines IL-6, TNF-α, CXCL1, and IL-1ß. Recruitment of immune cells, mostly neutrophils, was also reduced by administration of WAS. Infiltration of inflammatory cells and edema in the submucosa of air pouch tissues were markedly improved in the WAS-treated groups. CONCLUSIONS: Our results indicate that WAS possesses potent anti-inflammatory properties. These findings suggest that A. scoparia is a candidate functional food targeting several inflammatory diseases.

Type I Interferons Are Involved in the Intracellular Growth Control of Mycobacterium abscessus by Mediating NOD2-Induced Production of Nitric Oxide in Macrophages.

Mycobacterium abscessus (MAB) is one of the rapidly growing, multidrug-resistant non-tuberculous mycobacteria (NTM) causing various diseases including pulmonary disorder. Although it has been known that type I interferons (IFNs) contribute to host defense against bacterial infections, the role of type I IFNs against MAB infection is still unclear. In the present study, we show that rIFN-ß treatment reduced the intracellular growth of MAB in macrophages. Deficiency of IFN-α/ß receptor (IFNAR) led to the reduction of nitric oxide (NO) production in MAB-infected macrophages. Consistently, rIFN-ß treatment enhanced the expression of iNOS gene and protein, and NO production in response to MAB. We also found that NO is essential for the intracellular growth control of MAB within macrophages in an inhibitor assay using iNOS-deficient cells. In addition, pretreatment of rIFN-ß before MAB infection in mice increased production of NO in the lungs at day 1 after infection and promoted the bacterial clearance at day 5. However, when alveolar macrophages were depleted by treatment of clodronate liposome, rIFN-ß did not promote the bacterial clearance in the lungs. Moreover, we found that a cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2) is required for MAB-induced TANK binding kinase 1 (TBK1) phosphorylation and IFN-ß gene expression in macrophages. Finally, increase in the bacterial loads caused by reduction of NO levels was reversed by rIFN-ß treatment in the lungs of NOD2-deficient mice. Collectively, our findings suggest that type I IFNs act as an intermediator of NOD2-induced NO production in macrophages and thus contribute to host defense against MAB infection.

Receptor-interacting protein kinase 2 contributes to host innate immune responses against Fusobacterium nucleatum in macrophages and decidual stromal cells.

PROBLEM: Chorioamnionitis is caused by a bacterial infection that ascends from the vagina and can cause adverse pregnancy outcomes (APOs). Fusobacterium nucleatum (F. nucleatum) is a periodontal pathogen associated with the occurrence of APOs. In this study, we evaluated whether receptor-interacting protein kinase 2 (Ripk2), an adaptor protein of the cytosolic receptors nucleotide-binding oligomerization domain (NOD)1 and NOD2, in macrophages and human decidual stromal cells (hDSCs) contributes to immune responses against F. nucleatum. METHOD OF STUDY: Bone marrow-derived macrophages (BMDMs) isolated from wild-type (WT) and Ripk2-deficient mice and hDSCs were cultured with F. nucleatum (MOI 1, 10, 100). BMDMs and hDSCs were assessed using enzyme-linked immunosorbent assay, Western blot analysis, real-time PCR, and nitrite assay. RESULTS: Fusobacterium nucleatum-induced production of IL-6, but not of TNF-α and IL-10, was lower in Ripk2-deficient BMDMs than in WT cells. Western blotting revealed a decrease in F. nucleatum-induced p65 phosphorylation in Ripk2-deficient macrophages, whereas mitogen-activated protein kinases activation was comparable between WT and Ripk2-deficient cells. The production of nitric oxide (NO) in response to F. nucleatum and the gene and protein expression of inducible NO synthase was impaired in Ripk2-deficient BMDMs. In hDSCs, F. nucleatum upregulated the gene and protein expression of NOD1, NOD2, and Ripk2 in a time-dependent manner. F. nucleatum also increased the production of IL-6, CXCL8, and CCL2, whereas this production was decreased by the Ripk2 inhibitors SB203580 and PP2. CONCLUSIONS: In conclusion, Ripk2 signaling appears to contribute to the F. nucleatum-induced immune response and can be a preventive and therapeutic target against APOs.

Cathelicidin-related Antimicrobial Peptide Contributes to Host Immune Responses Against Pulmonary Infection with Acinetobacter baumannii in Mice.

Acinetobacter baumannii is known for its multidrug antibiotic resistance. New approaches to treating drug-resistant bacterial infections are urgently required. Cathelicidin-related antimicrobial peptide (CRAMP) is a murine antimicrobial peptide that exerts diverse immune functions, including both direct bacterial cell killing and immunomodulatory effects. In this study, we sought to identify the role of CRAMP in the host immune response to multidrug-resistant Acinetobacter baumannii. Wild-type (WT) and CRAMP knockout mice were infected intranasally with the bacteria. CRAMP-/- mice exhibited increased bacterial colony-forming units (CFUs) in bronchoalveolar lavage (BAL) fluid after A. baumannii infection compared to WT mice. The loss of CRAMP expression resulted in a significant decrease in the recruitment of immune cells, primarily neutrophils. The levels of IL-6 and CXCL1 were lower, whereas the levels of IL-10 were significantly higher in the BAL fluid of CRAMP-/- mice compared to WT mice 1 day after infection. In an in vitro assay using thioglycollate-induced peritoneal neutrophils, the ability of bacterial phagocytosis and killing was impaired in CRAMP-/- neutrophils compared to the WT cells. CRAMP was also essential for the production of cytokines and chemokines in response to A. baumannii in neutrophils. In addition, the A. baumannii-induced inhibitor of κB-α degradation and phosphorylation of p38 MAPK were impaired in CRAMP-/- neutrophils, whereas ERK and JNK phosphorylation was upregulated. Our results indicate that CRAMP plays an important role in the host defense against pulmonary infection with A. baumannii by promoting the antibacterial activity of neutrophils and regulating the innate immune responses.