Astragalus mongholicus Bunge and Panax notoginseng formula (A&P) improves renal mesangial cell damage in diabetic nephropathy by inhibiting the inflammatory response of infiltrated macrophages.

BACKGROUND: Diabetic nephropathy (DN) is one of the main causes of end-stage renal disease with scantly effective treatment. Numerous evidences indicated that macrophages play an important role in the occurrence and pathogenesis of DN by secreting inflammatory cytokines. Mincle is mainly expressed in macrophages and promotes kidney inflammation and damage of acute kidney injury. However, the role of Mincle in DN is unclear. In this study, we aim to investigate the effect of Mincle-related macrophage inflammation on DN, and whether it can be identified as the therapeutic target for Astragalus mongholicus Bunge and Panax notoginseng Formula (A&P), a widely used Chinese herbal decoction for DN treatment. METHODS: In vivo experiments high-fat and high-sugar diet and streptozotocin was used to establish a diabetic nephropathy model, while in vitro experiments inflammation model was induced by high-glucose in mouse Bone Marrow-Derived Macrophages (BMDM) cells and mouse mesangial (MES) cells. Kidney pathological staining is used to detect kidney tissue damage and inflammation, Western blotting, Real-time PCR and ELISA are performed to detect Mincle signaling pathway related proteins and inflammatory cytokines. RESULTS: Mincle was mainly expressed in infiltrated macrophage of DN kidney, and was significant decreased after A&P administration. The in vitro experiments also proved that A&P effectively down-regulated the expression of Mincle in macrophage stimulated by high glucose. Meanwhile, the data demonstrated that A&P can reduce the activation of NFκB, and the expression and secretion of inflammatory cytokines in DN kidney or BMDM cells. Notably, we set up a co-culture system to conform that BMDM cells can aggravate the inflammatory response of mesangial (MES) cells under high glucose stimulation. Furthermore, we found that the anti-injury role of A&P in MES cells was dependent on inhibition of the Mincle in macrophage. CONCLUSION: In summary, our study found that A&P is effective in reducing renal pathological damage and improving renal function and inflammation in diabetic nephropathy by a mechanism mainly related to the inhibition of the Mincle/Card9/NFκB signaling pathway.

Interaction of Host Pattern Recognition Receptors (PRRs) with Mycobacterium Tuberculosis and Ayurvedic Management of Tuberculosis: A Systemic Approach.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb), infects the lungs' alveolar surfaces through aerosol droplets. At this stage, the disease progression may have many consequences, determined primarily by the reactions of the human immune system. However, one approach will be to more actively integrate the immune system, especially the pattern recognition receptor (PRR) systems of the host, which notices pathogen-associated molecular patterns (PAMPs) of Mtb. Several types of PRRs are involved in the detection of Mtb, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), Dendritic cell (DC) -specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), Mannose receptor (MR), and NOD-like receptors (NLRs) related to inflammasome activation. In this study, we focus on reviewing the Mtb pathophysiology and interaction of host PPRs with Mtb as well as adverse drug effects of anti-tuberculosis drugs (ATDs) and systematic TB treatment via Ayurvedic medicine.

The role of Glabridin in antifungal and anti-inflammation effects in Aspergillus fumigatus keratitis.

PURPOSE: To investigate the effect of Glabridin (GLD) in Aspergillus fumigatus keratitis and its associated mechanisms. METHODS: Aspergillus fumigatus (A. fumigatus) conidia was inoculated in 96-well plate, and minimal inhibitory concentration (MIC) and biofilm formation ability were evaluated after GLD treatment. Spore adhesion ability was evaluated in conidia infected human corneal epithelial cells (HCECs). Keratitis mouse model was created by corneal intrastromal injection with A. fumigatus conidia, and GLD treatment started at the day after infection. The number of fungal colonies was calculated by plate count, and degree of corneal inflammation was assessed by clinical score. Flow cytometry, myeloperoxidase (MPO), and immunofluorescence staining (IFS) experiments were used to assess neutrophil infiltrations. PCR, ELISA and Western blot were conducted to determine levels of TLR4, Dectin-1 as well as downstream inflammatory factors. RESULTS: GLD treatment suppressed the proliferation, biofilm formation abilities and adhesive capability of A. fumigatus. In mice upon A. fumigatus infection, treatment of GLD showed significantly decreased severity of corneal inflammation, reduced number of A. fumigatus in cornea, and suppressed neutrophil infiltration in cornea. GLD treatment obviously inhibited mRNA and protein levels of Dectin-1, TLR4 and proinflammatory mediators such as IL-1ß, HMGB1, and TNF-α in mice corneas compared to the control group. CONCLUSION: GLD has antifungal and anti-inflammatory effects in fungal keratitis through suppressing A. fumigatus proliferation and alleviating neutrophil infiltration, and repressing the expression of TLR4, Dectin-1 and proinflammatory mediators.

Host Cell and SARS-CoV-2-Associated Molecular Structures and Factors as Potential Therapeutic Targets.

Coronavirus disease 19 (COVID-19) is caused by an enveloped, positive-sense, single-stranded RNA virus, referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the realm Riboviria, order Nidovirales, family Coronaviridae, genus Betacoronavirus and the species Severe acute respiratory syndrome-related coronavirus. This viral disease is characterized by a myriad of varying symptoms, such as pyrexia, cough, hemoptysis, dyspnoea, diarrhea, muscle soreness, dysosmia, lymphopenia and dysgeusia amongst others. The virus mainly infects humans, various other mammals, avian species and some other companion livestock. SARS-CoV-2 cellular entry is primarily accomplished by molecular interaction between the virus's spike (S) protein and the host cell surface receptor, angiotensin-converting enzyme 2 (ACE2), although other host cell-associated receptors/factors, such as neuropilin 1 (NRP-1) and neuropilin 2 (NRP-2), C-type lectin receptors (CLRs), as well as proteases such as TMPRSS2 (transmembrane serine protease 2) and furin, might also play a crucial role in infection, tropism, pathogenesis and clinical outcome. Furthermore, several structural and non-structural proteins of the virus themselves are very critical in determining the clinical outcome following infection. Considering such critical role(s) of the abovementioned host cell receptors, associated proteases/factors and virus structural/non-structural proteins (NSPs), it may be quite prudent to therapeutically target them through a multipronged clinical regimen to combat the disease.

Ganoderma lucidum polysaccharides ameliorated depression-like behaviors in the chronic social defeat stress depression model via modulation of Dectin-1 and the innate immune system.

Major depressive disorder (MDD) is a prevalent, chronic, and recurrent disease. At least one-third of patients have treatment-resistant depression; therefore, there is an urgent need for novel drug development. Cumulative studies have suggested an inflammatory mechanism for the pathophysiology of MDD. Ganoderma lucidum polysaccharides (GLP) is an anti-inflammatory and immunomodulatory agent. Here, we found that an injection of GLP led to a rapid and robust antidepressant effect after 60 min in the tail suspension test. This antidepressant effect remained after 5 days of treatment with GLP in the forced swim test. Unlike psychostimulants, GLP did not show a hyperactive effect in the open field test. After 60 min or 5 days of treatment, GLP exhibited an antidepressant effect in a chronic social defeat stress (CSDS) depression animal model. Moreover, after 5 days of treatment, GLP attenuated the expression of the proinflammatory cytokines IL-1ß and TNF-α, enhanced the expression of the anti-inflammatory cytokine IL-10 and the neurotrophic factor BDNF, and inhibited the activation of microglia and proliferation of astrocytes in the hippocampus of CSDS mice. In addition, after 5 days of treatment, GLP significantly enhanced GluA1 S845 phosphorylation as well as GluA1 and GluA2 expression levels in the hippocampus of CSDS mice. To determine whether the antidepressant effect was mediated by Dectin-1, we found that GLP treatment enhanced Dectin-1 expression in the hippocampus in CSDS mice, and the Dectin-1-specific inhibitor laminarin almost completely blocked the antidepressant effect of GLP. This study identified GLP, an agonist of Dectin-1, as a novel and rapid antidepressant with clinical potential and multiple beneficial mechanisms, particularly in regulating the neuroimmune system and, subsequently, AMPA receptor function.

Possibility of Japanese Cedar Pollen Causing False Positives in the Deep Mycosis Test.

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive factor. However, given that the LAL-assay-positive substances in the JCP extract were not distinguishable by the three BG-specific quantitative methods, we conclude that they may cause the background to rise. Therefore, in Japan false positives arising from JCP contamination should be considered when testing patients for deep mycosis.

Induction of IL-10-producing type 2 innate lymphoid cells by allergen immunotherapy is associated with clinical response.

The role of innate immune cells in allergen immunotherapy that confers immune tolerance to the sensitizing allergen is unclear. Here, we report a role of interleukin-10-producing type 2 innate lymphoid cells (IL-10+ ILC2s) in modulating grass-pollen allergy. We demonstrate that KLRG1+ but not KLRG1- ILC2 produced IL-10 upon activation with IL-33 and retinoic acid. These cells attenuated Th responses and maintained epithelial cell integrity. IL-10+ KLRG1+ ILC2s were lower in patients with grass-pollen allergy when compared to healthy subjects. In a prospective, double-blind, placebo-controlled trial, we demonstrated that the competence of ILC2 to produce IL-10 was restored in patients who received grass-pollen sublingual immunotherapy. The underpinning mechanisms were associated with the modification of retinol metabolic pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathways in the ILCs. Altogether, our findings underscore the contribution of IL-10+ ILC2s in the disease-modifying effect by allergen immunotherapy.

Chenopodium Quinoa and Salvia Hispanica Provide Immunonutritional Agonists to Ameliorate Hepatocarcinoma Severity under a High-Fat Diet.

Complex interactions between immunonutritional agonist and high fat intake (HFD), the immune system and finally gut microbiota are important determinants of hepatocarcinoma (HCC) severity. The ability of immunonutritional agonists to modulate major aspects such as liver innate immunity and inflammation and alterations in major lipids profile as well as gut microbiota during HCC development is poorly understood. 1H NMR has been employed to assess imbalances in saturated fatty acids, MUFA and PUFA, which were associated to variations in iron homeostasis. These effects were dependent on the botanical nature (Chenopodium quinoa vs. Salvia hispanica L.) of the compounds. The results showed that immunonutritional agonists' promoted resistance to hepatocarcinogenesis under pro-tumorigenic inflammation reflected, at a different extent, in increased proportions of F4/80+ cells in injured livers as well as positive trends of accumulated immune mediators (CD68/CD206 ratio) in intestinal tissue. Administration of all immunonutritional agonists caused similar variations of fecal microbiota, towards a lower obesity-inducing potential than animals only fed a HFD. Modulation of Firmicutes to Bacteroidetes contents restored the induction of microbial metabolites to improve epithelial barrier function, showing an association with liver saturated fatty acids and the MUFA and PUFA fractions. Collectively, these data provide novel findings supporting beneficial immunometabolic effects targeting hepatocarcinogenesis, influencing innate immunity within the gut-liver axis, and providing novel insights into their immunomodulatory activity.

Endo-1,3(4)-ß-Glucanase-Treatment of Oat ß-Glucan Enhances Fermentability by Infant Fecal Microbiota, Stimulates Dectin-1 Activation and Attenuates Inflammatory Responses in Immature Dendritic Cells.

Background: Non-digestible carbohydrates are added to infant formula to mimic the effects of human milk oligosaccharide by acting as prebiotics and stimulating the immune system. Although not yet used in infant formulas, ß-glucans are known to have beneficial health effects, and are therefore of potential interest for supplementation. Methods and results: We investigated the in vitro fermentation of native and endo-1,3(4)-ß-glucanase-treated oat ß-glucan using pooled fecal inocula of 2- and 8-week-old infants. While native oat ß-glucan was not utilized, both inocula specifically utilized oat ß-glucan oligomers containing ß(1→4)-linkages formed upon enzyme treatment. The fermentation rate was highest in the fecal microbiota of 2-week-old infants, and correlated with a high lactate production. Fermentation of media supplemented with native and enzyme-treated oat ß-glucans increased the relative abundance of Enterococcus and attenuated pro-inflammatory cytokine production (IL-1ß, IL-6, TNFα) in immature dendritic cells. This attenuating effect was more pronounced after enzyme treatment. This attenuation might result from the enhanced ability of fermented oat ß-glucan to stimulate Dectin-1 receptors. Conclusion: Our findings demonstrate that endo-1,3(4)-ß-glucanase treatment enhances the fermentability of oat ß-glucan and attenuates pro-inflammatory responses. Hence, this study shows that especially enzyme-treated oat ß-glucans have a high potential for supplementation of infant formula.

Multi-functional nanocomplex codelivery of Trp2 and R837 to activate melanoma-specific immunity.

Antigen-adjuvant combination could induce a protective and long-lasting anti-tumor immune response. However, exploiting system which could co-deliver melanoma antigen peptide Trp2 (Tyrosinase-related protein 2) and Toll-like-receptor-7 (TLR7) agonists imiquimod (R837) both are poor aqueous solubility is still challenging. Our new nanocomplex was explored for specific delivery of Trp2 and R837 into antigen-presenting cells (APCs). R837 was loaded into mannosylated-ß-cyclodextrin (Man-CD) to target dendritic cells (DCs) by binding mannose receptors (MR) on DCs. A fusion peptide (WT) was constructed by incorporating the amino acid region of TAT (cell-penetrating peptide) into Trp2 to improve the TAT-mediated intracellular efficiency. Negatively charged sodium alginate (SA), a biocompatible polymer, which can induce adjuvant responses by affecting the functions of DCs, was complexed with Man-CD/R837 and WT via physical adsorption. The optimized nanocomplex promoted the cellular uptake and showed remarkable efficacy to enhance the secreting of Th1-cytokines. This multi-functional nanocomplex system may allow effective targeting-codelivery of multi-hydrophobic drugs and be a promising subunit vaccine candidate as a potential prevention action of tumor.

Synthetic mycobacterial molecular patterns partially complete Freund's adjuvant.

Complete Freund's adjuvant (CFA) has historically been one of the most useful tools of immunologists. Essentially comprised of dead mycobacteria and mineral oil, we asked ourselves what is special about the mycobacterial part of this adjuvant, and could it be recapitulated synthetically? Here, we demonstrate the essentiality of N-glycolylated peptidoglycan plus trehalose dimycolate (both unique in mycobacteria) for the complete adjuvant effect using knockouts and chemical complementation. A combination of synthetic N-glycolyl muramyl dipeptide and minimal trehalose dimycolate motif GlcC14C18 was able to upregulate dendritic cell effectors, plus induce experimental autoimmunity qualitatively similar but quantitatively milder compared to CFA. This research outlines how to substitute CFA with a consistent, molecularly-defined adjuvant which may inform the design of immunotherapeutic agents and vaccines benefitting from cell-mediated immunity. We also anticipate using synthetic microbe-associated molecular patterns (MAMPs) to study mycobacterial immunity and immunopathogenesis.

Identification of a pro-elongation effect of diallyl disulfide, a major organosulfur compound in garlic oil, on microglial process.

Microglia are the innate immune cells in the nervous system. In the resting state, they display a ramified morphology, while upon disease stimulation their processes would be retracted, along with pro-inflammatory cytokine overproduction. Reversing microglial process retraction may help reduce pro-inflammatory cytokine production and restore microglia's ability to scan surrounding environments, rendering brain function regulation to be more effective. We found that diallyl disulfide (DADS), a major organosulfur compound in garlic oil, administered at different doses and time points, promoted microglial process elongation in both cultured systems and prefrontal cortexes in mice in a reversible manner. Lipopolysaccharide (LPS), a classical activator of microglia, did not affect this pro-elongation effect of DADS at conditions in vitro and in vivo. Functional studies revealed that DADS pre-treatment attenuated LPS-induced decreases in levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) mRNA as well as LPS-induced increases in levels of IL-10 and CD206 mRNA in both cultured microglia and prefrontal cortexes in mice. Protein kinase B (Akt) inhibition attenuated the pro-elongation effect of DADS on microglial process and blocked the regulatory effects of DADS on LPS-induced inflammatory responses in both cultured microglia and prefrontal cortexes in mice. In an in vivo model of neuroinflammation, DADS pre-treatment prevented LPS-induced retraction of microglial process in the prefrontal cortex in mice and attenuated LPS-induced increase in immobility time in the tail suspension test and forced swim test. These results indicate that DADS induces an Akt-dependent elongation of microglia process, along with the induction of an anti-inflammatory phenotype.

Poor clinical outcomes of intratumoral dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin-positive macrophages associated with immune evasion in gastric cancer.

AIM: Tumour-associated macrophages (TAMs) are prominent immune cells infiltrating in solid tumours with phenotypic and functional heterogeneity. However, the clinical significance of heterogeneous subtypes of TAMs in gastric cancer still remains obscure. Here, we aimed to explore the clinical significance of TAMs expressing dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) and its relevance with immune contexture in gastric cancer. METHODS: We selected 453 formalin-fixed and paraffin-embedded samples and 51 fresh tissue specimens of patients with gastric cancer from Zhongshan Hospital. The association of DC-SIGN+ macrophages with clinicopathological parameters, overall survival (OS) and responsiveness to fluorouracil-based adjuvant chemotherapy (ACT) was inspected. Immunohistochemistry (IHC) and flow cytometry (FCM) were applied to characterize immune cells in gastric cancer. RESULTS: We demonstrated that high intratumoral DC-SIGN+ macrophages infiltration predicted poor OS and inferior therapeutic responsiveness to fluorouracil-based ACT in patients with gastric cancer. Furthermore, higher infiltration of DC-SIGN+ macrophages indicated an increased number of Foxp3+ regulatory T cells (Tregs), CD8+ T cells and a higher ratio of Foxp3+/CD8+ within the tumour microenvironment (TME). In addition, CD8+ T cells in DC-SIGN+ macrophages high subgroup were functionally impaired, showing decreased interferon-γ (IFN-γ), granzyme B (GZMB) and perforin production yet elevated programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression. CONCLUSIONS: DC-SIGN+ macrophages were associated with immunoinvasive TME and indicated poor prognosis and inferior therapeutic responsiveness to fluorouracil-based ACT. DC-SIGN+ macrophages might be an independent prognosticator and a potential immunotherapeutic target for gastric cancer.

Astragalus propinquus Schischkin and Panax notoginseng (A&P) compound relieved cisplatin-induced acute kidney injury through inhibiting the mincle maintained macrophage inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute kidney injury (AKI) is a common disease in hospitalized patients, especially in critically ill patients. It is characterised with high morbidity and mortality, and is also an important cause of chronic kidney disease and chronic renal failure. Astragalus propinquus Schischkin and Panax notoginseng (A&P) compound, a famous traditional Chinese medicine, consists of Astragalus propinquus Schischkin, Panax notoginseng, Angelica sinensis, Achyranthes bidentata, and Ecklonia kurome, has been widely used for the treatment of various kidney diseases in the southwest of China. However, the effects of A&P on treatment of AKI and its underlying mechanism are needed to be uncovered. AIM OF THE STUDY: Recent researches reported that Mincle (Macrophage-inducible C-type lectin) plays a key role in renal injury of AKI by regulating the expression and secretion of inflammatory cytokines on macrophage through modulating NF-κB signaling pathway. Here, we aimed to investigate the renoprotective effect of A&P on AKI and whether by inhibiting Mincle. MATERIALS AND METHODS: We established a lipopolysaccharide (LPS)-induced Bone Marrow-Derived Macrophage (BMDM) inflammatory cell model and a cisplatin-induced mouse AKI model in vitro and in vivo. Renal histopathology staining was performed to observe kidney morphology. The expression and secretion of inflammatory cytokines were detected by real-time PCR and Enzyme-linked immunosorbent assay. Western blotting was used to detect the protein levels and Flow cytometry performed to detect polarization of macrophage. RESULTS: The results showed that A&P significantly reduced the mRNA expression of IL-1ß, IL-6, TNFα and MCP-1 in LPS-stimulated BMDM cells, and secretion of IL-1ß and IL-6 in supernatant. The same results were found in Cisplatin-induced AKI kidney and serum after treatment with A&P. The data also showed that A&P strongly reduced the mRNA and protein levels of Mincle in vitro and vivo, and also inhibited the activation of Syk and NF-κB. Notably, A&P down-regulated the M1 macrophage marker iNOS, which may relate to the inhibition of Mincle. Interestingly, both overexpression of Mincle by transfection of pcDNA3.1-Mincle plasmid and administration of TDB (a ligand of Mincle) can significantly abolished the A&P-inhibited inflammation in BMDM, suggesting Mincle pathway play a key role in macrophage inflammation in AKI. CONCLUSION: Our findings indicated that A&P protected kidney from inhibiting inflammation through down-regulating of Mincle pathway in macrophage in AKI. It provides a potential medicine compound for the treatment of AKI.

A ß-glucan from Grifola frondosa effectively delivers therapeutic oligonucleotide into cells via dectin-1 receptor and attenuates TNFα gene expression.

Grifola frondosa is an edible and medicinal mushroom with great nutritional values and bioactivities. In the present study, a soluble homogeneous ß-glucan, GFPS, with high molecular mass of 5.42 × 106 Da was purified from the fruit bodies of Grifola frondosa using 5% cold NaOH. The structure of GFPS was determined with FT-IR, NMR, and monosaccharide composition analysis, and was identified to be a ß-D-(1-3)-linked glucan backbone with a single ß-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. Our results indicated that GFPS had a triple helical structure and could form complex with polydeoxyadenylic acid (poly[A]). Further studies demonstrated that GFPS could interact with poly[A] moiety of a designed antisense oligonucleotide (ASO) targeting the primary transcript of proinflammatory cytokine TNFα (TNFα-A60). This GFPS-based complex could incorporate TNFα-A60 into the macrophage cells via dectin-1 receptor and attenuate lipopolysaccharide-induced secretion of TNFα. Our results suggested that GFPS could be applied to deliver therapeutic oligonucleotides for the treatment of diseases such as inflammation and cancers.

Topical Therapy with Antisense Tumor Necrosis Factor Alpha Using Novel ß-Glucan-Based Drug Delivery System Ameliorates Intestinal Inflammation.

Anti-tumor necrosis factor alpha (TNF-α) antibodies are effective in patients with inflammatory bowel disease (IBD). However, the effect is not optimal because a sufficient concentration of antibodies cannot be maintained at the site of inflammation. Thus, a macromolecular complex was developed with schizophyllan (SPG) and antisense oligonucleotides. In the present study, an SPG-antisense TNF-α complex was prepared, and its therapeutic efficacy was examined using a dextran sodium sulfate (DSS)-induced colitis model. The TNF-α production in CD11b+ macrophages significantly increased in the colon of DSS-treated mice. Dectin-1, a receptor of SPG, binds with SPG and is subsequently taken into the cells via phagocytosis. The expression of dectin-1 by CD11b+ macrophages significantly increased in DSS-treated mice. Flow cytometry revealed that the uptake of SPG-antisense TNF-α in the macrophages was efficient. TNF-α production was suppressed significantly by SPG-antisense TNF-α in vitro, which was administered via enema to evaluate its efficacy. The intrarectal administration of SPG-antisense TNF-α ameliorated the intestinal inflammation. In this study, we showed that the delivery system that conjugates SPG and antisense can have higher therapeutic efficacy. Thus, the new therapeutic approach presented in this study may be used in the management of IBD.

The mycelium of the Trametes versicolor (Turkey tail) mushroom and its fermented substrate each show potent and complementary immune activating properties in vitro.

BACKGROUND: The medicinal mushroom Trametes versicolor (Tv, Turkey Tail) is often prepared for consumption as a powder from the fungal mycelium and the fermented substrate on which it grew. The goal for this study was to evaluate the immune-modulating properties of the mycelium versus the fermented substrate, to document whether an important part of the immune-activating effects resides in the metabolically fermented substrate. METHODS: Tv mycelium was cultured on rice flour. The mycelium and the fermented substrate were mechanically separated, dried, and milled. The initial substrate served as a control. Aqueous fractions were extracted and passed through 0.22-µm filters. The remaining solids were passed through homogenization spin columns without filtration. The aqueous and solid fractions of the initial substrate (IS), the fermented substrate (FS), and the Trametes versicolor mycelium (TvM) were tested for immune-activating and modulating activities on human peripheral blood mononuclear cell cultures, to examine expression of the CD69 activation marker on lymphocytes versus monocytes, and on the T, NKT, and NK lymphocyte subsets. Culture supernatants were tested for cytokines using Luminex arrays. RESULTS: Both aqueous and solid fractions of TvM triggered robust induction of CD69 on lymphocytes and monocytes, whereas FS only triggered minor induction of CD69, and IS had no activating effect. The aqueous extract of TvM had stronger activating effects than the solid fraction. In contrast, the solid fraction of IS triggered a reduction in CD69, below levels on untreated cells. Both aqueous and solid fractions of FS triggered large and dose-dependent increases in immune-activating pro-inflammatory cytokines (IL-2, IL-6), anti-inflammatory cytokines Interleukin-1 receptor antagonist (IL-1ra) and Interleukin-10 (IL-10), anti-viral cytokines interferon-gamma (IFN-γ) and Macrophage Inflammatory Protein-alpha (MIP-1α), as well as Granulocyte-Colony Stimulating Factor (G-CSF) and Interleukin-8 (IL-8). TvM triggered more modest cytokine increases. The aqueous extract of IS showed no effects, whereas the solid fraction showed modest effects on induction of cytokines and growth factors. CONCLUSION: The results demonstrated that the immune-activating bioactivity of a mycelial-based medicinal mushroom preparation is a combination of the mycelium itself (including insoluble beta-glucans, and also water-soluble components), and the highly bioactive, metabolically fermented substrate, not present in the initial substrate.

A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction.

Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.

Effect of Dehydrocostus Lactone Isolated from the Roots of Aucklandia lappa on the Apoptosis of Endometriotic Cells and the Alternative Activation of Endometriosis-Associated Macrophages.

The roots of Aucklandia lappa have been used in traditional medicine in Asia to treat inflammation and diseases associated with pain, including endometriosis. The aim of this study was to investigate the anti-endometriotic effect of dehydrocostus lactone, an active compound in A. lappa roots, using human endometriotic cells and macrophages stimulated by these cells. Dehydrocostus lactone induced apoptotic cell death in 12Z human endometriotic cells. Dehydrocostus lactone stimulated the activation of caspase-3, -8, and -9, while caspase inhibitors significantly reversed the dehydrocostus lactone-induced cell death in 12Z cells. In addition, dehydrocostus lactone decreased the production of PGE2 and neurotrophins (BDNF, NGF, NT3, and NT4/5), which are regarded as endometriosis-associated pain factors in human endometriotic cells. Moreover, dehydrocostus lactone inhibited the expression of M2 markers (CD206, and Trem-2), IL-10, VEGF, and MMP-2/-9 in endometriosis-associated macrophages (EAMs). Furthermore, dehydrocostus lactone inhibited the Akt and NFκB pathways in both endometriotic cells and EAMs. Taken together, our findings suggest that dehydrocostus lactone, an active compound of A, lappa, has anti-endometriotic activities via induction of apoptosis and downregulation of pain factors in endometriotic cells and inhibition of the alternative activation of EAMs.

Development and Characterization of a Preclinical Model for the Evaluation of CD205-Mediated Antigen Delivery Therapeutics in Type 1 Diabetes.

Dendritic cells (DCs) are crucial for the production of adaptive immune responses to disease-causing microbes. However, in the steady state (i.e., in the absence of an infection or when Ags are experimentally delivered without a DC-activating adjuvant), DCs present Ags to T cells in a tolerogenic manner and are important for the establishment of peripheral tolerance. Delivery of islet Ags to DCs using Ag-linked Abs to the DC endocytic receptor CD205 has shown promise in the NOD mouse model of type 1 diabetes (T1D). It is important to note, however, that all myeloid DCs express CD205 in humans, whereas in mice, only one of the classical DC subsets does (classical DC1; CD8α+ in spleen). Thus, the evaluation of CD205-targeted treatments in mice will likely not accurately predict the results observed in humans. To overcome this challenge, we have developed and characterized a novel NOD mouse model in which all myeloid DCs transgenically express human CD205 (hCD205). This NOD.hCD205 strain displays a similar T1D incidence profile to standard NOD mice. The presence of the transgene does not alter DC development, phenotype, or function. Importantly, the DCs are able to process and present Ags delivered via hCD205. Because Ags taken up via hCD205 can be presented on both class I and class II MHC, both CD4+ and CD8+ T cells can be modulated. As both T cell subsets are important for T1D pathogenesis, NOD.hCD205 mice represent a unique, patient-relevant tool for the development and optimization of DC-directed T1D therapies.