Evaluation of Prebiotic Potential of Crude Polysaccharides Extracted from Wild Lentinus polychrous and Lentinus squarrosulus and Their Application for a Formulation of a Novel Lyophilized Synbiotic.

Edible mushrooms, including wild mushrooms, are currently being investigated as natural sources to evaluate their prebiotic potential. This study aimed to evaluate the prebiotic potential of crude polysaccharides (CPSs) extracted from wild Lentinus squarrosulus UBU_LS1 and Lentinus polychrous UBU_LP2 and their application as cryoprotectants in the freeze-drying process to formulate a novel synbiotic product. Based on fruiting body morphology and molecular identification, two wild edible mushrooms named UBU_LS1 and UBU_LP2 were identified as Lentinus squarrosulus and Lentinus polychrous, respectively. L. squarrosulus UBU_LS1 and L. polychrous UBU_LP2 contained high amounts of CPS after hot water extraction. Monosaccharide component analysis showed that CPS_UBU_LS1 and CPS_UBU_LP2 were typical heteropolysaccharides. CPS_UBU_LS1 and CPS_UBU_LP2 showed hydrolysis tolerance to the simulated human gastric acidic pH solution, indicating that these CPSs are capable of reaching the lower gastrointestinal tract. Antioxidant activity determined using the 1,1-diphenyl-2-picrylhydrazyl assay revealed that the CPS_UBU_LS1 and CPS_UBU_LP2 displayed greater antioxidant activity comparable with that of ascorbic acid. It was found that CPS_UBU_LS1 and CPS_UBU_LP2 have a high potential for stimulating growth in all probiotic strains. Moreover, both CPS compounds could possibly be used as cryoprotectants in freeze drying, since the viability of the selected probiotic L. fermentum 47-7 exhibited cell survival of greater than 70% after 90 days of storage at 4 °C. These results highlight that wild edible mushrooms L. squarrosulus UBU_LS1 and L. polychrous UBU_LP2 are potential natural sources of prebiotics and can be applied as cryoprotectants in the freeze-drying process. The crude polysaccharide derived from this study could also be considered as a potent antioxidative compound. Therefore, our study provides evidence to support the application of CPSs from wild edible mushrooms in synbiotic product development and in various functional foods. Finally, further evaluation of these prebiotics, including the determination of the potential rehabilitation of beneficial gut microbes in diseased individuals, is currently being conducted by our research group.

Impaired functions of human monocyte-derived dendritic cells and induction of regulatory T cells by pathogenic Leptospira.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. The disease outcome is influenced by the interplay between innate and adaptive immune responses. Dendritic cells (DCs) play a crucial role in shaping the adaptive immune response. A recent study revealed that pathogenic Leptospira limited the activation of human monocyte-derived dendritic cells (MoDCs) compared to non-pathogenic Leptospira, but their impact on T-cell responses has not been investigated. Our study is the first to explore how viable pathogenic and non-pathogenic Leptospira affect the interaction between human MoDCs and T cells. We found that MoDCs infected with pathogenic leptospires (L. interrogans serovar Pomona and a clinical isolate, MoDCs-P) exhibited lower levels of CD80 and CD83 expression, suggesting partially impaired MoDC maturation, induced regulatory T cells (Tregs) while failing to induce CD4+ T cell proliferation, compared to MoDCs infected with non-pathogenic leptospires (L. biflexa serovar Patoc and L. meyeri serovar Ranarum, MoDCs-NP). In contrast, non-pathogenic leptospires enhanced MoDC maturation and induced higher T cell proliferation including IFN-γ-producing CD4+ T cells, indicative of a Th1-type response. Furthermore, pathogenic leptospires induced higher MoDC apoptosis through a cysteine aspartic acid-specific protease-3 (caspase-3)-dependent pathway and upregulated expression of the prostaglandin-endoperoxide synthase 2 (PTGS2) gene. Notably, prostaglandin E2 (PGE2), a product of the PTGS2 pathway, was found at higher levels in the sera of patients with acute leptospirosis and in the supernatant of MoDCs-P, possibly contributing to Treg induction, compared to those of healthy donors and MoDCs-NP, respectively. In conclusion, this study reveals a novel immunosuppressive strategy employed by pathogenic Leptospira to evade host immunity by partially impairing MoDC maturation and inducing Tregs. These findings deepen our understanding of leptospirosis pathogenesis in humans and may provide a novel strategy to modulate DCs for the prevention and treatment of the disease.

Immunosuppressive Polymeric Nanoparticles Targeting Dendritic Cells Alleviate Lupus Disease in Fcgr2b-/- Mice by Mediating Antigen-Specific Immune Tolerance.

Dendritic cells (DCs) are the most potent antigen-presenting cells that have multifaceted functions in the control of immune activation and tolerance. Hyperresponsiveness and altered tolerogenicity of DCs contribute to the development and pathogenesis of system lupus erythematosus (SLE); therefore, DC-targeted therapies aimed at inducing specific immune tolerance have become of great importance for the treatment of SLE. This study developed a new nanoparticle (NP) containing a biodegradable PDMAEMA-PLGA copolymer for target-oriented delivery to DCs in situ. PDMAEMA-PLGA NPs provided sustained drug release and exhibited immunosuppressive activity in FLT3L and GM-CSF-derived bone marrow in conventional DCs (BM-cDCs). PDMAEMA-PLGA NPs improved dexamethasone capability to convert wild-type and Fcgr2b-/- BM-cDCs from an immunogenic to tolerogenic state, and BM-cDCs treated with dexamethasone-incorporated PDMAEMA-PLGA NPs (Dex-NPs) efficiently mediated regulatory T cell (Treg) expansion in vitro. Dex-NP therapy potentially alleviated lupus disease in Fcgr2b-/- mice by mediating Foxp3+ Treg expansion in an antigen-specific manner. Our findings substantiate the superior efficacy of DC-targeted therapy using the PDMAEMA-PLGA NP delivery system and provide further support for clinical development as a potential therapy for SLE. Furthermore, PDMAEMA-PLGA NP may be a versatile platform for DC-targeted therapy to induce antigen-specific immune tolerance to unwanted immune responses that occur in autoimmune disease, allergy, and transplant rejection.

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-ß secretions.

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4+ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-ß) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.

Dendritic cells as key players in systemic lupus erythematosus.

System lupus erythematosus (SLE) is a chronic autoimmune disorder affecting multiple organs, and persistent disease activity is associated with increased morbidity and mortality. Impairment of immune cell function and loss of immune tolerance to self-antigens are significant determinants that trigger inflammation and drive SLE pathogenesis. Dendritic cells (DCs) are the most potent antigen-presenting cells that serve as a critical link between innate and adaptive immune system. SLE development and pathogenesis are associated with aberrant regulation in homeostasis and function of DCs, therefore, DC-targeted therapies have become of importance for treatment of SLE and autoimmune diseases. This review focus on the significance of DCs in promoting of SLE pathogenesis, and further discuss the clinical potential of DCs in SLE therapy. The insights on the roles of DCs in SLE will provide the improvement of treatment strategy for SLE patients.

Current Paradigms of Tolerogenic Dendritic Cells and Clinical Implications for Systemic Lupus Erythematosus.

Tolerogenic dendritic cells (tolDCs) are central players in the initiation and maintenance of immune tolerance and subsequent prevention of autoimmunity. Recent advances in treatment of autoimmune diseases including systemic lupus erythematosus (SLE) have focused on inducing specific tolerance to avoid long-term use of immunosuppressive drugs. Therefore, DC-targeted therapies to either suppress DC immunogenicity or to promote DC tolerogenicity are of high interest. This review describes details of the typical characteristics of in vivo and ex vivo tolDC, which will help to select a protocol that can generate tolDC with high functional quality for clinical treatment of autoimmune disease in individual patients. In addition, we discuss the recent studies uncovering metabolic pathways and their interrelation intertwined with DC tolerogenicity. This review also highlights the clinical implications of tolDC-based therapy for SLE treatment, examines the current clinical therapeutics in patients with SLE, which can generate tolDC in vivo, and further discusses on possibility and limitation on each strategy. This synthesis provides new perspectives on development of novel therapeutic approaches for SLE and other autoimmune diseases.

Heterologous protection elicited by a live, attenuated, Leptospira vaccine.

A previously-described live, attenuated vaccine (M1352, serovar Manilae, serogroup Pyrogenes) was tested in the hamster model of infection for cross-protective immunity. The vaccine elicited strong, significant cross-protection against lethal infection by strains representing four serologically distinct leptospiral serovars (Grippotyphosa, Australis, Canicola, and Autumnalis). Combined with our previously reported protection against serovars Pomona and Manilae, this work demonstrates unequivocal proof of concept for cross-protective immunity in leptospirosis.

CpG oligodeoxynucleotides with crude parasite antigens reduce worm recovery in Opisthorchis viverrini infected hamsters.

Opisthorchis viverrini, a human liver fluke, is still an endemic parasitic infection in Thailand and nearly all countries in Southeast Asia. O. viverrini induces a chronic stage of infection in hamsters. During the first 2 weeks of infection, Th1 inducing cytokine, IL-12, increased but was down regulated in chronic infection. In this study it was found that unmethylated-CpG ODN (oligodeoxynucleotides) 1826 increased hamster mononuclear cell proliferation and stimulated IFN-γ production in vitro. The IFN-γ levels in hamster sera were significantly increased in hamsters injected with CpG ODN 1826 alone or plus crude somatic antigens (CSAg). Further investigation using the flow cytometer found that CD4+T cells and IFN-γ+ CD4+T cells (Th1-like cells) in the hamster blood were significantly increased. The role of these cells in the protective responses in hamsters was evaluated by challenging with 25 metacercaria and observation for 3 months. The number of worms recovered was significantly reduced in the hamsters injected with CpG ODN 1826 with CSAg, but not in CpG ODN 1826 alone groups when compared to PBS control. The percent of reduction in hamsters against this parasite were 32.95% and 21.49% in the CpG ODN 1826 with CSAg and CpG ODN 1826 alone. This study indicates that CpG ODN 1826 plus parasite antigens elicit a Th1-like response that leads to the enhancement of worm reduction.

Protective immunization of hamsters against Opisthorchis viverrini infection is associated with the reduction of TGF-ß expression.

Opisthorchis viverrini infection is a significant health problem in Thailand and other countries in Southeast Asia. There is little known about the mechanisms of the immune response to O. viverrini in immunoprotection. However, it has been reported that this parasite can suppress both cell and antibody mediated immune responses. The TGF-ß and IL-10 are immunosuppressive cytokines that play an important role in inhibition of host immune response leading to worm survival. In this study, we immunized hamsters to protect against O. viverrini infection and the IL-4, IL-10, TGF-ß and IFN-γ expression in spleen was investigated by real time PCR analysis. An O. viverrini-crude somatic antigen preparation (CSAg) administered with complete Freund's adjuvant (CFA) or with alum was used to stimulate immune responses in O. viverrini-primed hamsters. The greatest percent protection (48.4%) was seen following immunization with CSAg plus alum. The mean number±SD of worms recovered in the PBS control, CFA alone, CSAg plus CFA, alum alone and CSAg plus alum was 17.4±2.3, 17.1±3.3, 14.5±3.8, 14.5±2.3 and 9±2.7, respectively. Significant protection correlated with the reduction of TGF-ß and IL-10, but not IL-4, IFN-γ expressions. Since TGF-ß expression is significantly increased in the spleens of hamsters with opisthorchiasis, stimulation of this cytokine by parasite antigens was confirmed by using CSAg and primary hamster spleen cells. Antigen fractions with molecular masses of 81-92, 64-72 and 19-21.4kDa were found to significantly induce TGF-ß production. Our results suggested that TGF-ß induction by O. viverrini may have an important role in parasite survival.