Presence and diversity of free-living amoebae and their potential application as water quality indicators.

Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades Ι-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades Ι to VI quality, whereas the Vermamoeba species was present only in Grade Ι water. V. croatica was found exclusively in water with Grade ΙΙ quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.

SCGB1C1 Plays a Critical Role in Suppression of Allergic Airway Inflammation through the Induction of Regulatory T Cell Expansion.

The nanosized vesicles secreted from various cell types into the surrounding extracellular space are called extracellular vesicles (EVs). Although mesenchymal stem cell-derived EVs are known to have immunomodulatory effects in asthmatic mice, the role of identified pulmonary genes in the suppression of allergic airway inflammation remains to be elucidated. Moreover, the major genes responsible for immune regulation in allergic airway diseases have not been well documented. This study aims to evaluate the immunomodulatory effects of secretoglobin family 1C member 1 (SCGB1C1) on asthmatic mouse models. C57BL/6 mice were sensitized to ovalbumin (OVA) using intraperitoneal injection and were intranasally challenged with OVA. To evaluate the effect of SCGB1C1 on allergic airway inflammation, 5 µg/50 µL of SCGB1C1 was administrated intranasally before an OVA challenge. We evaluated airway hyperresponsiveness (AHR), total inflammatory cells, eosinophils in the bronchoalveolar lavage fluid (BALF), lung histology, serum immunoglobulin (Ig), the cytokine profiles of BALF and lung-draining lymph nodes (LLN), and the T cell populations in LLNs. The intranasal administration of SCGB1C1 significantly inhibited AHR, the presence of eosinophils in BALF, eosinophilic inflammation, goblet cell hyperplasia in the lung, and serum total and allergen-specific IgE. SCGB1C1 treatment significantly decreased the expression of interleukin (IL)-5 in the BALF and IL-4 in the LLN, but significantly increased the expression of IL-10 and transforming growth factor (TGF)-ß in the BALF. Furthermore, SCGB1C1 treatment notably increased the populations of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in asthmatic mice. The intranasal administration of SCGB1C1 provides a significant reduction in allergic airway inflammation and improvement of lung function through the induction of Treg expansion. Therefore, SCGB1C1 may be the major regulator responsible for suppressing allergic airway inflammation.

Extract of Torreya nucifera Pericarps Exhibits a Parasiticidal Effect on the Nematode Parasite, Trichinella spiralis.

Benzimidazole derivatives can effectively treat nematode parasitic infections; however, some derivatives demand distinct administrative strategies depending on plasma concentration and patient conditions. Numerous studies have examined the potential of natural extracts to exert parasiticidal activity with minimal side effects. Herein, we examined the potential parasiticidal effects of Torreya nucifera extract. The pericarps of T. nucifera were extracted with methanol, dried, and the pellet was dissolved in hot water (Tn-Phw). We designed four individual mouse experiments to clarify the prophylactic and therapeutic effects of Tn-Phw on Trichinella spiralis infection. Also, 100 L1 larvae were isolated and treated with Tn-Phw (10 mg/mL) in vitro to confirm the killing effect. Furthermore, we microscopically examined the morphology of L1 larvae to confirm the parasite-killing effect and analyzed the morphology using a scanning electron microscope (SEM). The expression of three molting-related genes was confirmed to determine whether Tn-Phw induced morphological changes in L1 larvae. Following treatment with Tn-Phw, L1 larvae death was observed after 16 h. Following SEM examination, the healthy muscle larvae showed striated ridges and wrinkles; this was not observed in extract-treated muscle larvae. Expression levels of the three molting-related genes did not differ between the Tn-Phw-treated and control groups. T. spiralis-infected mice pretreated with Tn-Phw showed significantly reduced muscle larva infection when compared with control mice. In all experiments, treatment with Tn-Phw afforded preventive and therapeutic effects against T. spiralis infection and parasitism. Natural substances against nematode parasites could be developed as therapeutic agents with few side effects and enhanced parasiticidal efficacy.

Anti-obesity effects by parasitic nematode (Trichinella spiralis) total lysates.

Background: Obesity is an inducible factor for the cause of chronic diseases and is described by an increase in the size and number of adipocytes that differentiate from precursor cells (preadipocytes). Parasitic helminths are the strongest natural trigger of type 2 immune system, and several studies have showed that helminth infections are inversely correlated with metabolic syndromes. Methodology/Principal findings: To investigate whether helminth-derived molecules have therapeutic effects on high-fat diet (HFD)-induced obesity, we isolated total lysates from Trichinella spiralis muscle larvae. We then checked the anti-obesity effect after intraperitoneal administration and intraoral administration of total lysate from T. spiralis muscle larvae in a diet-induced obesity model. T. spiralis total lysates protect against obesity by inhibiting the proinflammatory response and/or enhancing M2 macrophages. In addition, we determined the effects of total lysates from T. spiralis muscle larvae on anti-obesity activities in 3T3-L1 preadipocytes by investigating the expression levels of key adipogenic regulators, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding protein alpha (C/EBPα) and adipocyte protein 2 (aP2). Oil Red O staining showed that the total lysates from T. spiralis muscle larvae decreased the differentiation of 3T3-L1 preadipocytes by decreasing the number of lipid droplets. In addition, the production levels of proinflammatory cytokines IL-1ß, IL-6, IFN-γ and TNF-α were examined by enzyme-linked immunosorbent assay (ELISA). T. spiralis total lysates decreased intracellular lipid accumulation and suppressed the expression levels of PPARγ, C/EBPα and aP2. Conclusion/Significance: These results show that T. spiralis total lysate significantly suppresses the symptoms of obesity in a diet- induced obesity model and 3T3-L1 cell differentiation and suggest that it has potential for novel anti-obesity therapeutics.

Free-Living Amoeba Vermamoeba vermiformis Induces Allergic Airway Inflammation.

The high percentage of Vermamoeba was found in tap water in Korea. This study investigated whether Vermamoeba induced allergic airway inflammation in mice. We selected 2 free-living amoebas (FLAs) isolated from tap water, which included Korean FLA 5 (KFA5; Vermamoeba vermiformis) and 21 (an homolog of Acanthamoeba lugdunensis KA/ E2). We axenically cultured KFA5 and KFA21. We applied approximately 1 × 106 to mice's nasal passages 6 times and investigated their pathogenicity. The airway resistance value was significantly increased after KFA5 and KFA21 treatments. The eosinophil recruitment and goblet cell hyperplasia were concomitantly observed in bronchial alveolar lavage (BAL) fluid and lung tissue in mice infected with KFA5 and KFA21. These infections also activated the Th2-related interleukin 25, thymic stromal lymphopoietin, and thymus and activation-regulated chemokines gene expression in mouse lung epithelial cells. The CD4+ interleukin 4+ cell population was increased in the lung, and the secretion of Th2-, Th17-, and Th1-associated cytokines were upregulated during KFA5 and KFA21 infection in the spleen, lung-draining lymph nodes, and BAL fluid. The pathogenicity (allergenicity) of KFA5 and KFA21 might not have drastically changed during the long-term in vitro culture. Our results suggested that Vermamoeba could elicit allergic airway inflammation and may be an airway allergen.

Trichinella spiralis nurse cell formation is regulated via CCR7+ dendritic cells.

The chemokine receptor CCR7 is a well-established homing receptor for dendritic cells (DCs) and T-cells. Interaction with the CCL19 and CCL21 ligands promotes priming of immune responses in lymphoid tissues; however, the mechanism underlying CCR7-induced immune responses against helminth parasite infection remains unknown. Thus, we examined the role of CCR7 in generating protective immune responses against intracellular Trichinella spiralis infection. The results showed significantly increased CCR7, CCL19 and CCL21 expression in the muscle tissue compared to that in the intestinal tissue in T. spiralis-infected mice. The CCR7-expressing DC population increased in the mesenteric and peripheral lymph nodes (PLNs) during T. spiralis infection. Notably, the number of CCR7-expressing cells in PLNs increased by more than 30% at 28 days post-infection; however, this increase was significantly inhibited in CCR7-blocked mice treated with CCR7-specific antibodies. T helper 2 (Th2)-and regulatory T (Treg )-related cytokine levels were also reduced by CCR7-specific antibody treatment. CCR7-blocked mice lost their resistance to T. spiralis infection in the muscle phase but not in the intestinal phase. Furthermore, fewer eosinophils around the nurse cells and reduced total and T. spiralis-specific IgE in the serum were observed in CCR7-blocked mice compared to those infected with only T. spiralis. CCR7 blockade led to the T. spiralis infection-induced suppression of Th2- and Treg -related cytokine production in vitro. These results suggest that CCR7 in DCs might play an essential role in host defence mechanisms against T. spiralis infection, particularly in the muscle stage of the infection, by accelerating Th2 and Treg cell responses.

Bioorthogonal Catalysis for Treatment of Solid Tumors Using Thermostable, Self-Assembling, Single Enzyme Nanoparticles and Natural Product Conversion with Indole-3-acetic Acid.

Bioorthogonal catalysis (BC) generates chemical reactions not present in normal physiology for the purpose of disease treatment. Because BC catalytically produces the desired therapy only at the site of disease, it holds the promise of site-specific treatment with little or no systemic exposure or side effects. Transition metals are typically used as catalytic centers in BC; however, solubility and substrate specificity typically necessitate a coordinating enzyme and/or stabilizing superstructure for in vivo application. We report the use of self-assembling, porous exoshells (tESs) to encapsulate and deliver an iron-containing reaction center for the treatment of breast cancer. The catalytic center is paired with indole-3-acetic acid (IAA), a natural product found in edible plants, which undergoes oxidative decarboxylation, via reduction of iron(III) to iron(II), to produce free radicals and bioactive metabolites. The tES encapsulation is critical for endocytic uptake of BC reaction centers and, when followed by administration of IAA, results in apoptosis of MDA-MB-231 triple negative cancer cells and complete regression of in vivo orthotopic xenograft tumors (p < 0.001, n = 8 per group). When Renilla luciferase (rLuc) is substituted for horseradish peroxidase (HRP), whole animal luminometry can be used to monitor in vivo activity.

Intranasally Administered Extracellular Vesicles from Adipose Stem Cells Have Immunomodulatory Effects in a Mouse Model of Asthma.

Asthma is a chronic eosinophilic airway disease characterized by type 2 helper T cell-driven inflammation. Adipose stem cells (ASCs) and the ASC culture supernatant are known to improve allergic airway inflammation; however, the immunomodulatory effects of ASC-derived extracellular vesicles (EVs) on allergic airway diseases remain unclear. Thus, we assessed the effects of ASC-derived EVs on allergic airway inflammation in a mouse model of asthma. EVs were isolated from the culture supernatant of murine ASCs and characterized. Six-week-old female C57BL/6 mice were sensitized to ovalbumin (OVA) by intraperitoneal injection and challenged intranasally with OVA. Before the OVA challenge, 10 µg/50 µl of ASC-derived EVs was administered intranasally to the experimental group. ASC-derived EVs significantly attenuated airway hyperresponsiveness (AHR) in asthmatic mice (p = 0.023). ASC-derived EVs resulted in a remarkable reduction of the total number of inflammatory cells (p = 0.005) and eosinophils (p = 0.023) in the bronchoalveolar lavage fluid (BALF), the degree of eosinophilic lung inflammation (p < 0.001), and the serum total and OVA-specific immunoglobulin (Ig)E (p = 0.048 and p = 0.001) and total IgG1 (p < 0.001). Interleukin- (IL-) 4 was significantly inhibited with ASC-derived EV pretreatment in the BALF and lung draining lymph nodes (LLNs) (p = 0.040 and p = 0.011). Furthermore, ASC-derived EV administration resulted in a significant increase of the regulatory T cell (Treg) populations in LLNs. ASC-derived EVs alleviated AHR and allergic airway inflammation caused by the induction of Treg expansion in a mouse model of asthma. There seems to be a role for ASC-derived EVs as a modifier in allergic airway disease.

Extracellular vesicles of Echinococcus granulosus have therapeutic effects in allergic airway inflammation.

BACKGROUND: Previous studies have shown that Echinococcus granulosus cystic fluid can alleviate Th2 allergic airway inflammatory responses by increasing the number of CD4+ CD25+ Foxp3+ T (regulatory T; Treg) cells. Parasite-derived extracellular vesicles (EV) are known to not only promote parasite infection by communicating between parasites but also regulate the inflammatory response by acting as an immunomodulatory agent in the host. METHODS: To evaluate the effect of EV extracted from the cystic fluid of E. granulosus on allergic airway inflammation, gene expression was investigated after administering EV to mouse lung epithelial cells (MLE-12) following 2 h of pretreatment with Aspergillus proteins. An allergic airway inflammation animal model was used to investigate the regulation of the inflammatory response by EV and induced with ovalbumin. RESULTS: EV treatment significantly reduced airway resistance and the number of eosinophils and other immune cells in the bronchoalveolar lavage fluid and Th2- and Th17-related cytokine levels. EV pretreatment decreased the number of IL-4+ CD4+ T cells and increased the number of Treg cells in the lung-draining lymph nodes and spleen. CONCLUSIONS: Echinococcus granulosus cystic fluid derived EV ameliorated Th2 allergic airway inflammatory through Treg cells, similar to whole cystic fluid treatment. Thus, EV may be important immunomodulatory molecules in cystic fluid.

Acceleration of Trichinella spiralis worm expulsion by leukotriene B4 receptor binding inhibition.

AIMS: Helminth infection typically induces a Th2 inflammatory response that is characterized by eosinophilia, high levels of IgE and mast cells. LTB4 is generated from innate immune cells, such as neutrophils, macrophages and mast cells, in response to a range of stimuli. It mainly acts on myeloid leukocytes, inducing the activation of integrins, adhesion to endothelium walls, and chemotaxis. METHODS AND RESULTS: The objective of the present study was to determine the role of the LTB4 receptor in Trichinella spiralis expulsion. We treated mice with the LTB4 receptor antagonist before infection with T. spiralis. We observed that the number of mast cells and worm infection decreased following treatment with the BLT antagonist during the intestinal phase. We also demonstrated that blocking the LTB4 receptor inhibited neutrophil and eosinophil infiltration. CONCLUSIONS: Further studies are required to investigate the specific mechanism of mast cell number decrease and worm infection and the in vitro interactions between LTB4 and worm expulsion.

Trichinella spiralis infection ameliorated diet-induced obesity model in mice.

Obesity is an increasingly prevalent disease worldwide, and genetic and environmental factors are known to regulate the development of obesity and associated metabolic diseases. Emerging studies indicate that innate and adaptive immune cell responses in adipose tissue play critical roles in the regulation of metabolic homeostasis. Parasitic helminths are the strongest natural inducers of type 2 inflammatory responses, and several studies have revealed that helminth infections inversely correlate with metabolic syndrome. Hence, this study investigated whether helminth infections could have preventative effects on high fat diet-induced obesity. Female C57BL/6 mice were maintained on either a low fat diet (LFD, 10% fat) or a high fat diet (HFD, 60% fat) for 6 weeks after Trichinella spiralis infection. The mice were randomly divided into four groups and were fed a normal diet, LFD, LFD after T. spiralis infection (Inf + LFD), a high fat diet (HFD), or HFD after T. spiralis infection (HFD + inf). All groups were assayed for body weight, food efficiency ratio (FER), total body weight gain (g)/total food intake amount (g) fat weight, and blood biochemical parameters. Our data indicate that the HFD + inf group significantly reduced body weight gain, fat mass, total cholesterol, and FER. Analysis of immune cell composition by flow cytometry revealed that T. spiralis promoted strong decreases in proinflammatory adipose macrophages (F4/80+CD11c+) and T cells. The alterations in microbiota from fecal samples of mice were analyzed, which showed that T. spiralis infection decreased the ratio of Firmicutes to Bacteriodetes, thereby restoring the previously increased ratio of Firmicutes to Bacteriodetes in HFD-fed mice. Moreover, elimination of T. spiralis retained the protective effects in the HFD-fed obese mice whereas flubendazole (FLBZ) treatment increased levels of the families Lachnospiraceae and Ruminococcaceae. In summary, we provided novel data suggesting that helminth infection protects against obesity and the protection was closely related to M2 macrophage proliferation, an inhibiting proinflammatory response. In addition, it alters the microbiota in the gut.

Effects of lifelong spontaneous exercise on the M1/M2 macrophage polarization ratio and gene expression in adipose tissue of super-aged mice.

In the adipose tissue (AT), an increase in the M1 macrophage (M1Ø)/M2 macrophage (M2Ø) polarization ratio can be a risk factor enhancing the inflammatory response during aging, as well as increasing the risk of chronic disease, thereby reducing lifespan, or at least reducing "healthy" lifespan. The purpose of this study was to analyze and compare the AT M1Ø/M2Ø polarization ratio at the final lifespan stage in aged and control animals performing lifelong spontaneous wheel running. Based on flow cytometric analysis, the AT ratio of macrophages revealed M2Ø polarization following lifelong spontaneous exercise (LSE) regardless of age. However, for Icam1 and Tnf, the qPCR analysis showed no difference in gene expressions in young mice; Arg1 expression was higher in Young-EXE (exercising) than in Young-CON (control) mice (p < .0001). In Old-EXE, Icam1 (p < .0001) and Tnf (p < .0001) expression were lower than in Old-CON; for Arg1, gene expression in Old-EXE was higher than in Old-CON (p < .0001). LSE prevents deterioration of physical fitness owing to aging, maintaining high M2Ø polarization levels in the AT. Additionally, LSE does not downregulate Icam1 and Tnf in the AT but appears to suppress the increased M1Ø polarization ratio attributed to aging by upregulating Arg1.

Hippo/MST blocks breast cancer by downregulating WBP2 oncogene expression via miRNA processor Dicer.

WBP2 transcription coactivator is an emerging oncoprotein and a key node of convergence between EGF and Wnt signaling pathways. Understanding how WBP2 is regulated has important implications for cancer therapy. WBP2 is tightly controlled by post-translational modifications, including phosphorylation and ubiquitination, leading to changes in subcellular localization, protein-protein interactions, and protein turnover. As the function of WBP2 is intricately linked to YAP and TAZ, we hypothesize that WBP2 is negatively regulated by the Hippo tumor suppressor pathway. Indeed, MST is demonstrated to negatively regulate WBP2 expression in a kinase-dependent but LATS-independent manner. This was observed in the majority of the breast cancer cell lines tested. The effect of MST was enhanced by SAV and concomitant with the inhibition of the transcription co-activation, in vitro and in vivo tumorigenesis activities of WBP2, resulting in good prognosis in xenografts. Downregulation of WBP2 by MST involved miRNA but not proteasomal or lysosomal degradation. Our data support the existence of a novel MST-Dicer signaling axis, which in turn regulates both WBP2 CDS- and UTR-targeting miRNAs expression, including miR-23a. MiR-23a targets the 3'UTR of WBP2 mRNA directly. Significant inverse relationships between WBP2 and MST or miR23a expression levels in clinical specimens were observed. In conclusion, WBP2 is a target of the Hippo/MST kinase; MST is identified as yet another rheostat in the regulation of WBP2 and its oncogenic function. The findings have implications in targeted therapeutics and precision medicine for breast cancer.

Prevalence of scabies in long-term care hospitals in South Korea.

BACKGROUND: Scabies is a common contagious skin disease. With the economic growth in South Korea, the incidence of scabies has decreased. However, with the recent advancements in medical facilities, mainly the establishment of long-term care hospitals (LTCHs), scabies is now considered an emerging public health problem. METHODOLOGY/PRINCIPAL FINDINGS: To examine the prevalence and management of scabies in LTCHs in South Korea, we contacted all 1,336 LTCHs registered at the Health Insurance Review and Assessment Service in South Korea in 2018. A total of 110 LTCHs completed a questionnaire, and we analyzed their responses. In the last 5 years, 71.8% (79/110) of LTCHs had a high incidence of scabies (suspected/confirmed cases). Usually, patients aged older than 80 years (45.5%) were diagnosed with the disease, with more women being affected than men. Only 30.0% of the patients were transferred to scabies-restricted rooms, and very few LTCHs (7.0%) had special departments for scabies. Fifty-five (61.1%) of 90 LTCHs reported contact between scabies patients and nurses, nurse aides, caregivers, and other employees (hereinafter, referred to as primary exposure), with 29 (32.2%) LTCHs reporting infections due to primary exposure. The most common challenges in managing scabies were patient isolation (47.8%), diagnosis (31.1%), management of individuals exposed to an individual with scabies (17.8%), lack of staff for managing the patients (16.7%), and treatment (11.1%). CONCLUSIONS: The incidence rate of scabies in LTCHs in South Korea has increased. Regular and enhanced staff training is needed, considering that most hospitals rarely focused on the handling of equipment and furniture used by scabies patients and on educating their healthcare staff. These findings can be used to develop various strategies to reduce the prevalence of scabies.

Screening and Functional Pathway Analysis of Pulmonary Genes Associated with Suppression of Allergic Airway Inflammation by Adipose Stem Cell-Derived Extracellular Vesicles.

BACKGROUND: Although mesenchymal stem cell- (MSC-) derived extracellular vesicles (EVs) are as effective as MSCs in the suppression of allergic airway inflammation, few studies have explored the molecular mechanisms of MSC-derived EVs in allergic airway diseases. The objective of this study was to evaluate differentially expressed genes (DEGs) in the lung associated with the suppression of allergic airway inflammation using adipose stem cell- (ASC-) derived EVs. METHODS: C57BL/6 mice were sensitized to ovalbumin (OVA) by intraperitoneal injection and challenged intranasally with OVA. To evaluate the effect of ASC-derived EVs on allergic airway inflammation, 10 µg/50 µL of EVs were administered intranasally prior to OVA challenge. Lung tissues were removed and DEGs were compared pairwise among the three groups. DEG profiles and hierarchical clustering of the identified genes were analyzed to evaluate changes in gene expression. Real-time PCR was performed to determine the expression levels of genes upregulated after treatment with ASC-derived EVs. Enrichment analysis based on the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were also performed to further identify the function of DEGs. RESULTS: Expression of paraoxonase 1 (PON1), brain-expressed X-linked 2 (Bex2), insulin-like growth factor binding protein 6 (Igfbp6), formyl peptide receptor 1 (Fpr1), and secretoglobin family 1C member 1 (Scgb1c1) was significantly increased in asthmatic mice following treatment with ASC-derived EVs. GO enrichment and KEGG pathway analysis showed that these genes were strongly associated with immune system processes and their regulation, cellular processes, single-organism processes, and biological regulation. CONCLUSION: These results suggest that the DEGs identified in this study (PON1, Bex2, Igfbp6, Fpr1, and Scgb1c1) may be involved in the amelioration of allergic airway inflammation by ASC-derived EVs.

Differences in macrophage polarization in the adipose tissue of obese mice under various levels of exercise intensity.

Animal studies have demonstrated that the ratio of M1 (M1Φ) to M2 (M2Φ) macrophage-specific gene expression in adipose tissue (AT) may be altered by chronic exercise; however, whether macrophage polarization is induced under these conditions has not yet been reported. Therefore, this study aimed to investigate the effect of chronic exercise on M1Φ/M2Φ polarization in the AT of high-fat diet (HFD)-induced obese mice. Exercise-induced differences in M1Φ/M2Φ polarization were verified via an exercise intensity study (EIS) in which different levels of exercise intensity were evaluated. Obesity was induced in male C57BL/6 J mice by feeding them with an HFD for 6 weeks. The study consisted of four groups: control group (CON), HFD-fed group (HFD), HFD-fed with exercise group (HFD + EXE), dietary conversion from HFD to normal diet (ND) group (DC), and dietary conversion from HFD to ND group (DC + EXE). For EIS, the HFD + EXE group was divided into three subgroups: low- (LI), mid- (MI), and high- (HI) intensity exercise. The total intervention period was 8 weeks. M1Φ/M2Φ polarization was confirmed by flow cytometry. M2Φ polarization in the AT of obese mice was significantly higher in HFD + EXE mice than in HFD mice, despite the HFD intake. In the EIS, M2Φ polarization was most pronounced in HFD + EXE-HI mice than in HFD mice. It can be proposed that the enhanced insulin resistance and inflammation by obesity can be improved by the increase of M2Φ polarization which is achieved by relatively high-intensity exercise.

Dendritic cells and M2 macrophage play an important role in suppression of Th2-mediated inflammation by adipose stem cells-derived extracellular vesicles.

Although stem cell-derived extracellular vesicles (EVs) have been shown to facilitate regeneration of injured tissue, there is no report that evaluates the immune-modulating effect of stem cell-derived EVs on Th2-mediated inflammation. In this study, we evaluated the immunomodulatory effects of adipose stem cells (ASCs)-derived EVs on Th2-mediated inflammation induced by Aspergillus protease antigen in lung epithelial cells. The EVs were isolated from supernatant of ASCs and the diameters of EVs were measured by using dynamic light scattering. The mice primary lung epithelial cells and mouse lung epithelial cell line (MLE12) were pre-treated with 200 ng/ml of Aspergillus protease and then treated with 1 µg/ml of ASC-derived EVs. Real time PCR was performed to determine the expression levels of eotaxin, IL-25, TGF-ß, and IL-10 mRNAs after EV treatment. To evaluate the role of EVs in macrophage polarization and dendritic cells (DCs) differentiation, in vitro bone marrow-derived macrophage and DCs stimulation assay was performed. EV treatment significantly decreased the expression of eotaxin and IL-25 and increased TGF-ß and IL-10 in both lung epithelial cells. EV treatment significantly increased the expression of co-stimulatory molecules such as CD40, CD80, and CD 86 in immature DCs. Furthermore, EV treatment significantly enhanced the gene expression of M2 macrophage marker such as Arg1, CCL22, IL-10, and TGF-ß. In conclusion, EVs of ASCs ameliorated Th2-mediated inflammation induced by Aspergillus protease antigen through the activation of dendritic cells and M2 macrophage, accompanied by down-regulation of eotaxin and IL-25, and up-regulation of TGF-ß and IL-10 in mouse lung epithelial cells.

Adoptive transfer of Trichinella spiralis-activated macrophages can ameliorate both Th1- and Th2-activated inflammation in murine models.

Trichinella spiralis is a zoonotic nematode and food borne parasite and infection with T. spiralis leads to suppression of the host immune response and other immunopathologies. Alternative activated macrophages (M2) as well as Treg cells, a target for immunomodulation by the helminth parasite, play a critical role in initiating and modulating the host immune response to parasite. The precise mechanism by which helminths modulate host immune response is not fully understood. To determine the functions of parasite-induced M2 macrophages, we compared the effects of M1 and M2 macrophages obtained from Trichinella spiralis-infected mice with those of T. spiralis excretory/secretory (ES) protein-treated macrophages on experimental intestinal inflammation and allergic airway inflammation. T. spiralis infection induced M2 macrophage polarization by increasing the expression of CD206, ARG1, and Fizz2. In a single application, we introduced macrophages obtained from T. spiralis-infected mice and T. spiralis ES protein-treated macrophages into mice tail veins before the induction of dextran sulfate sodium (DSS)-induced colitis, ovalbumin (OVA)-alum sensitization, and OVA challenge. Colitis severity was assessed by determining the severity of colitis symptoms, colon length, histopathologic parameters, and Th1-related inflammatory cytokine levels. Compared with the DSS-colitis group, T. spiralis-infected mice and T. spiralis ES protein-treated macrophages showed significantly lower disease activity index (DAI) at sacrifice and smaller reductions of body weight and proinflammatory cytokine level. The severity of allergic airway inflammation was assessed by determining the severity of symptoms of inflammation, airway hyperresponsiveness (AHR), differential cell counts, histopathologic parameters, and levels of Th2-related inflammatory cytokines. Severe allergic airway inflammation was induced after OVA-alum sensitization and OVA challenge, which significantly increased Th2-related cytokine levels, eosinophil infiltration, and goblet cell hyperplasia in the lung. However, these severe allergic symptoms were significantly decreased in T. spiralis-infected mice and T. spiralis ES protein-treated macrophages. Helminth infection and helminth ES proteins induce M2 macrophages. Adoptive transfer of macrophages obtained from helminth-infected mice and helminth ES protein-activated macrophages is an effective treatment for preventing and treating airway allergy in mice and is promising as a therapeutic for treating inflammatory diseases.

Therapeutic effects of Echinococcus granulosus cystic fluid on allergic airway inflammation.

Previous studies showed that Echinococcus granulosus infection reduces allergic airway inflammation in experimentally infected hosts and the cystic fluid of E. granulosus is known to activate regulatory T (CD4+CD25+Foxp3+T, Treg) cells. To evaluate the effects of cystic fluid of E. granulosus on allergic airway inflammation, we investigated the regulation of the inflammatory reaction by cystic fluid using an allergic airway inflammation animal model. Cystic fluid was administered to C57BL/6 mice seven times every other day, after which allergic airway inflammation was induced using ovalbumin and aluminum. The airway resistance, number of eosinophils and other immune cells in the bronchoalveolar lavage fluid, and levels of Th2 and Th17-related cytokines were significantly reduced by cystic fluid pre-treatment in allergic airway inflammation-induced mice. The number IL-4+CD4+ T cells decreased, the number of Treg cells increased in the lung-draining lymph nodes and spleen of cystic fluid pre-treated mice. In conclusion, E. granulosus-derived cystic fluid may alleviate the Th2 allergic airway inflammatory response via Treg cells. Further studies of the immune regulation of cystic fluid may lead to the development of therapeutic agents for immune disorders.

An advanced protocol for the purification of whipworm eggs from feces for use as therapeutic agents.

Recent studies have attempted to treat autoimmune diseases using Trichuris suis. whipworm) eggs. Large quantities of eggs can be obtained efficiently by collecting from the feces of the porcine hosts rather than by extracting from the female worm uterus. However, it is difficult to process large amounts of feces using the current methods. In the present study, we propose a method to collect the eggs from bulk feces more efficiently. Collecting the eggs using washing meshes (25 µm sieve) yields 65.7% (56.0-70.7) of eggs (median, min-max) from 100 g feces. Our method, which uses ethyl acetate and simulated gastric fluid, yielded 91.4% (91.4-94.0) of the eggs from 100 g feces into the separated aqueous solution. Egg collection using simulated gastric fluid (SGF) method was also 60 min faster than that using the sieve method. As the SGF used in the experiment is a strongly acidic reagent with a pH of 1-2, embryonation of the eggs was induced by the rapid pH change. As a result, 37.1% (8.0-77.8) of the eggs had embryonated two months after SGF stimulation. Using the developed method, we could process the feces quickly and efficiently. Furthermore, after purification, egg embryonation could be induced without any harmful reagent treatment. This method is expected to be helpful for further research using Trichuris suis eggs.