Polysaccharide-Containing Macromolecules in a Kampo (Traditional Japanese Herbal) Medicine, Hochuekkito: Dual Active Ingredients for Modulation of Immune Functions on Intestinal Peyer's Patches and Epithelial cells.
ABSTRACT: A traditional Japanese herbal (Kampo) medicine, Hochuekkito (Bu-Zhong-Yi-Qi-Tang in Chinese, TJ-41) is a well-known Kampo formula, and has been found to enhance antigen-specific antibody response in not only local mucosal immune system in upper respiratory tract, but also systemic immune system through upper respiratory mucosal immune system. Although this immunopharmacological effect has been proposed to express by modulation of intestinal immune system including Peyer's patches and intestinal epithelial cells, active ingredients are not known. TJ-41 directly affected the production of bone marrow cell-proliferative growth factors from murine Peyer's patch immunocompetent cells in vitro. Among low molecular, intermediate size and macromolecular weight fractions prepared from TJ-41, only fraction containing macromolecular weight ingredients showed Peyer's patch-mediated bone marrow cell-proliferation enhancing activity. Anion-exchange chromatography and gel filtration gave 17 subfractions comprising polysaccharides and lignins from the macromolecular weight fraction of TJ-41, and some of the subfractions showed significant enhancing activities having different degrees. Some of the subfractions also expressed stimulating activity on G-CSF-production from colonic epithelial cells, and statistically significant positive correlation was observed among enhancing activities of the subfractions against Peyer's patch immunocompetent cells and epithelial cells. Among the fractions from TJ-41 oral administration of macromolecular weight ingredient fraction to mice succeeded to enhance antigen-specific antibody response in systemic immune system through upper respiratory mucosal immune system, but all the separated fractions failed to enhance the in vivo antibody response in upper respiratory tract.
Project description:Previously, we revealed that several kampo medicines used for patients with excess and/or medium patterns (kakkonto (TJ-1), shosaikoto (TJ-9), hangeshashinto (TJ-14), and orento (TJ-120)) reduced prostaglandin (PG)E2 levels using LPS-treated human gingival fibroblasts (HGFs). Recently, we examined other kampo medicines used for patients with the deficiency pattern [bakumondoto (TJ-29), shinbuto (TJ-30), ninjinto (TJ-32), and hochuekkito (TJ-41)] and the herbs comprising shinbuto and ninjinto using the same experimental model. Shinbuto and ninjinto concentration-dependently reduced LPS-induced PGE2 production by HGFs, whereas hochuekkito weakly reduced and bakumondoto did not reduce PGE2 production. Shinbuto and ninjinto did not alter cyclooxygenase (COX) activity or the expression of molecules involved in the arachidonic acid cascade. Therefore, we next examined which herbs compromising shinbuto and ninjinto reduce LPS-induced PGE2 production. Among these herbs, shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) strongly and concentration-dependently decreased LPS-induced PGE2 production. However, both shokyo and kankyo increased the expression of cytosolic phospholipase (cPL)A2 but did not affect annexin1 or COX-2 expression. These results suggest that shokyo and kankyo suppress cPLA2 activity. We demonstrated that kampo medicines suppress inflammatory responses in patients with the deficiency pattern, and in those with excess or medium patterns. Moreover, kampo medicines that contain shokyo or kankyo are considered to be effective for the treatment of inflammatory diseases.
Project description:Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.
Project description:Daiokanzoto (TJ-84) is a traditional Japanese herbal medicine (Kampo formulation). While many Kampo formulations have been reported to regulate inflammation and immune responses in oral mucosa, there is no evidence to show that TJ-84 has beneficial effects on oral mucositis, a disease resulting from increased cell death induced by chemotherapeutic agents such as 5-fluorouracil (5-FU). In order to develop effective new therapeutic strategies for treating oral mucositis, we investigated (i) the mechanisms by which 5-FU induces the death of human gingival cells and (ii) the effects of TJ-84 on biological events induced by 5-FU. 5-FU-induced lactate dehydrogenase (LDH) release and pore formation in gingival cells (Sa3 cell line) resulted in cell death. Incubating the cells with 5-FU increased the expression of nucleotide-binding domain and leucine-rich repeat containing PYD-3 (NLRP3) and caspase-1. The cleavage of caspase-1 was observed in 5-FU-treated cells, which was followed by an increased secretion of interleukin (IL)-1β. The inhibition of the NLRP3 pathway slightly decreased the effects of 5-FU on cell viability and LDH release, suggesting that NLRP3 may be in part involved in 5-FU-induced cell death. TJ-84 decreased 5-FU-induced LDH release and cell death and also significantly inhibited the depolarization of mitochondria and the up-regulation of 5-FU-induced reactive oxygen species (ROS) and nitric oxide (NO) production. The transcriptional factor, nuclear factor-κB (NF-κB) was not involved in the 5-FU-induced cell death in Sa3 cells. In conclusion, we provide evidence suggesting that the increase of ROS production in mitochondria, rather than NLRP3 activation, was considered to be associated with the cell death induced by 5-FU. The results also suggested that TJ-84 may attenuate 5-FU-induced cell death through the inhibition of mitochondrial ROS production.
Project description:OBJECTIVE:Exposure to lethal doses of radiation has severe effects on normal tissues. Exposed individuals experience a plethora of symptoms in different organ systems including the gastrointestinal (GI) tract, summarized as Acute Radiation Syndrome (ARS). There are currently no approved drugs for mitigating GI-ARS. A recent high-throughput screen performed at the UCLA Center for Medical Countermeasures against Radiation identified compounds containing sulfonylpiperazine groups with radiation mitigation properties to the hematopoietic system and the gut. Among these 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound #5) efficiently mitigated gastrointestinal ARS. However, the mechanism of action and target cells of this drug is still unknown. In this study we examined if Compound #5 affects gut-associated lymphoid tissue (GALT) with its subepithelial domes called Peyer's patches. METHODS:C3H mice were irradiated with 0 or 12?Gy total body irradiation (TBI). A single dose of Compound #5 or solvent was administered subcutaneously 24?h later. 48?h after irradiation the mice were sacrificed, and the guts examined for changes in the number of visible Peyer's patches. In some experiments the mice received 4 daily injections of treatment and were sacrificed 96?h after TBI. For immune histochemistry gut tissues were fixed in formalin and embedded in paraffin blocks. Sections were stained with H&E, anti-Ki67 or a TUNEL assay to assess the number of regenerating crypts, mitotic and apoptotic indices. Cells isolated from Peyer's patches were subjected to immune profiling using flow cytometry. RESULTS:Compound #5 significantly increased the number of visible Peyer's patches when compared to its control in non-irradiated and irradiated mice. Additionally, assessment of total cells per Peyer's patch isolated from these mice demonstrated an overall increase in the total number of Peyer's patch cells per mouse in Compound #5-treated mice. In non-irradiated animals the number of CD11bhigh in Peyer's patches increased significantly. These Compound #5-driven increases did not coincide with a decrease in apoptosis or an increase in proliferation in the germinal centers inside Peyer's patches 24?h after drug treatment. A single dose of Compound #5 significantly increased the number of CD45+ cells after 12?Gy TBI. Importantly, 96?h after 12?Gy TBI Compound #5 induced a significant rise in the number of visible Peyer's patches and the number of Peyer's patch-associated regenerating crypts. CONCLUSION:In summary, our study provides evidence that Compound #5 leads to an influx of immune cells into GALT, thereby supporting crypt regeneration preferentially in the proximity of Peyer's patches.
Project description:Lymphocytes express integrin receptors, termed lymphocyte Peyer's patch high endothelial venule (HEV) adhesion molecules (LPAMs), that mediate their organ-specific adhesion to specialized HEVs found in mucosal lymphoid organs (Peyer's patches). LPAM-1 consists of a murine integrin alpha 4 noncovalently associated with integrin beta p. Here, we describe the cloning and expression of a mouse cDNA encoding beta p, which is an 806-amino acid transmembrane glycoprotein. The genomic Southern blot analysis indicates that beta p is the murine homologue of human beta 7. The function of alpha 4 beta 7 as a Peyer's patch-specific adhesion molecule was tested directly by expression of the murine beta 7 cDNA in an alpha 4+ beta 7-B-cell line or coexpression of the alpha 4 and beta 7 cDNAs in an alpha 4-beta 7-T-cell line. The transfected cells exhibited a new Peyer's patch-specific adhesive phenotype that could be specifically blocked by monoclonal antibodies against alpha 4 and beta 7. Moreover, an anti-beta 7 monoclonal antibody specifically blocked binding of normal lymphocytes to Peyer's patch HEV but did not inhibit their binding to peripheral lymph node HEVs, indicating that beta 7 is a unique component of the Peyer's patch-specific homing receptor.
Project description:The initiation of the mucosal immune response in Peyer’s patch (PP) relies on the sampling, processing and efficient presentation of foreign antigens by dendritic cells (DC). PP DC encompass five subsets, among which CD11b+ conventional DC (cDC) and LysoDC have distinct progenitors and functions but share many cell surface markers. This has previously led to confusion between these two subsets. In addition, another PP DC subset, termed double-negative (DN), remains poorly characterized. Here, we have studied the genetic relatedness of the different subsets of PP cDC at steady state and under TLR7 ligand stimulation. We also provide the transcriptional profiles of subepithelial TIM-4- and interfollicular TIM-4+ macrophages. Overall design: Three independent replicates of TIM-4- and TIM-4+ LysoMac, CD8a+ and DN dome cDC and five independent replicates of CD11b+ dome cDC were sorted from Peyer's patch of C57BL/6 mice at steady state. In addition, three and five independant replicates of CD11b+ and CD8a+ dome cDC, respectively were sorted from Peyer's patch of C57BL/6 mice 9 hours after R848 gavage. Total RNA of Peyer's patch sorted cells was extracted with a Qiagen RNeasy Plus Micro Kit. Quantity, quality and absence of genomic DNA contamination were assessed with a Bioanalyser (Agilent). Microarray experiments were performed by the Plateforme Biopuces of Strasbourg (France) using the Affymetrix GeneChip® Mouse Gene 1.0 ST Array.
Project description:Streptococcus pneumoniae may colonize the nasopharynx, and as pneumococcal colonization causes invasive diseases and the subsequent transmission, reducing bacterial burden in the nasal cavity is critical. Hochu-ekki-to (TJ-41) is a traditional Japanese herbal medicine that exerts immunomodulatory effects in host cells. In this study, we investigated the potency of TJ-41 in modulating pneumococcal colonization clearance by activating host immunity. Mice, intranasally inoculated with pneumococci, were treated orally with TJ-41. During colonization, TJ-41 treatment significantly reduced pneumococcal burden and increased macrophage population in the nasopharynx. Furthermore, interleukin 17A production was significantly enhanced after TJ-41 treatment. In vitro experiment using nasal-derived cells revealed that pneumococcal antigen exposure upregulated the transcription of interleukin 17A in the TJ-41-treated group compared with that in the control group. Macrophages activated by killed bacteria were significantly increased in the presence of TJ-41 in an interleukin 17A-dependent manner. Moreover, TJ-41 enhanced phagocytosis, inhibited bacterial growth, and improved the antigen-presenting capacity of macrophages. Our results demonstrate that TJ-41 accelerates the clearance of pneumococcal nasopharyngeal colonization via macrophage activation. Subsequent production of interleukin 17A provides an additional benefit to effector cells.
Project description:OBJECTIVES:To investigate the cause of an acute respiratory tract infection (ARTI) outbreak. METHODS:Thirty-eight clinical samples were collected from 19 patients in an ARTI outbreak that occurred in a physical training facility in January 2013; patient demographic information was also collected. In addition, 60 influenza virus-negative samples from febrile respiratory patients were collected from the same community at the same time to determine whether these were the same infections. Multiplex PCR (multi-PCR) was used to detect the possible pathogen in these samples. All human adenovirus (HAdV)-positive samples were inoculated onto Hep-2 cells for isolation. HAdV isolates were typed by hexon gene, fiber gene, and whole genome sequencing using primers designed in-house and compared to different type/serotype HAdVs downloaded from GenBank. Phylogenetic analysis was used to determine the type of the HAdV. RESULTS:Of the 38 samples, 34 from 17 cases were HAdV-positive; two of them were co-infected, one with respiratory syncytial virus A and the other with human rhinovirus. The hexon gene open reading frame (ORF; 2841 nucleotides (nt)) and fiber gene ORF (978 nt) were obtained from four HAdV strains (TJ-2013-92, TJ-2013-94, TJ-2013-100, TJ-2013-122) from three upper respiratory infection cases and one pneumonia case. They were all completely identical. One HAdV isolate, TJ-2013-90, was selected for whole genome sequencing; 34238 nt were obtained. Phylogenetic analysis showed the whole genome of TJ-2013-90 to be clustered together with HAdV-B55/HAdV-B11a. Three of 60 influenza virus-negative specimens were HAdV-positive, but hexon and fiber gene analysis showed that they were grouped in different branches to the HAdV isolates from this outbreak. CONCLUSIONS:The cause of this ARTI outbreak was HAdV-B55. This was another outbreak caused by this re-emerging virus. Continuous surveillance of respiratory adenovirus is necessary for disease control.
Project description:Salmonella enterica serovar Choleraesuis is an enteric pathogen of swine, producing septicemia, enterocolitis, pneumonia, and hepatitis. The initial molecular events at the site of Salmonella infection are hypothesized to be critical in the initiation of innate and adaptive immune responses; however, the acute immune response elicited by porcine intestinal tissues is not well understood. To address this need, we employed explants of jejunal Peyer's patch (JPP) mucosa from pigs to examine Salmonella-induced immune responses under controlled conditions as well as to overcome limitations of whole animal approaches. JPP explants mounted in Ussing chambers maintained normal histological structure for 2 h and stable short-circuit current and electrical conductance for 2.5 h. After ex vivo luminal exposure to Salmonella serovar Choleraesuis, JPP responded with an increase in mRNA expression of IL-1beta and IL-8, but not TNFalpha. Increased IL-1beta and IL-8 expression were dependent on efficient Salmonella adhesion and internalization, whereas mutant Salmonella did not induce inflammatory cytokine expression. Commensal enteric bacteria, present in some experiments, also did not induce inflammatory cytokine expression. These findings indicate that Salmonella uptake by Peyer's patch is important in the induction of an innate response involving expression of IL-1beta and IL-8, and that ex vivo intestinal immune tissue explants provide an intact tissue model that will facilitate investigation of mucosal immunity in swine.