Afa/Dr遺伝子を保有しない分散接着性大腸菌の病因学的意義に関する疫学的研究

博士(学術)大阪市立大

Association of IL-8-inducing strains of diffusely adherent Escherichia coli with sporadic diarrheal patients with less than 5 years of age

The role of diffusely adherent Escherichia coli (DAEC) in diarrheal disease has been controversial. However, DAEC strains were recently implicated in diarrheal disease in developing countries. To clarify whether DAEC are prevalent among sporadic cases of diarrheal illness in Osaka City, Japan, E. coli strains isolated between July 1997 and March 2000 during diarrheagenic E. coli (DEC) investigation were retrospectively examined. DAEC strains were recognized among 41 (4.4%) of 924 patients and formed the biggest subgroup of DEC. Previously, we reported that some DAEC strains caused epithelial cells to secrete as much IL-8 as enteroaggregative E. coli strains did. In this study, we attempted to evaluate epidemiologically whether the ability of DAEC to induce IL-8 was involved in the pathogenesis. Relationship among patient age, symptoms, Afa adhesins, season and IL-8 induction were examined. The subgroup of DAEC that possessed Afa genes and/or induced a high level of IL-8 was significantly prevalent among patients age 1 to 4 years; however total DAEC was not significantly high among the children compared to other age group. IL-8 inducing DAEC seems to play a role in causing sporadic diarrheal illnesses, particularly in pediatric fields. Investigations highlighting the relationship between IL-8 induction and enteropathogenicity are clearly necessary to confirm the role of DAEC in infectious enteritis

Characterization of Free and Porous Silicon-Encapsulated Superparamagnetic Iron Oxide Nanoparticles as Platforms for the Development of Theranostic Vaccines

Tracking vaccine components from the site of injection to their destination in lymphatic tissue, and simultaneously monitoring immune effects, sheds light on the influence of vaccine components on particle and immune cell trafficking and therapeutic efficacy. In this study, we create a hybrid particle vaccine platform comprised of porous silicon (pSi) and superparamagnetic iron oxide nanoparticles (SPIONs). The impact of nanoparticle size and mode of presentation on magnetic resonance contrast enhancement are examined. SPION-enhanced relaxivity increased as the core diameter of the nanoparticle increased, while encapsulation of SPIONs within a pSi matrix had only minor effects on T2 and no significant effect on T2* relaxation. Following intravenous injection of single and hybrid particles, there was an increase in negative contrast in the spleen, with changes in contrast being slightly greater for free compared to silicon encapsulated SPIONs. Incubation of bone marrow-derived dendritic cells (BMDC) with pSi microparticles loaded with SPIONs, SIINFEKL peptide, and lipopolysaccharide stimulated immune cell interactions and interferon gamma production in OT-1 TCR transgenic CD8+ T cells. Overall, the hybrid particle platform enabled presentation of a complex payload that was traceable, stimulated functional T cell and BMDC interactions, and resolved in cellular activation of T cells in response to a specific antigen

Activation of the Inflammasome and Enhanced Migration of Microparticle-Stimulated Dendritic Cells to the Draining Lymph Node

Porous silicon microparticles presenting pathogen-associated molecular patterns mimic pathogens, enhancing internalization of the microparticles and activation of antigen presenting dendritic cells. We demonstrate abundant uptake of microparticles bound by the TLR-4 ligands LPS and MPL by murine bone marrow-derived dendritic cells (BMDC). Labeled microparticles induce concentration-dependent production of IL-1β, with inhibition by the caspase inhibitor Z-VAD-FMK supporting activation of the NLRP3-dependent inflammasome. Inoculation of BALB/c mice with ligand-bound microparticles induces a significant increase in circulating levels of IL-1β, TNF-α, and IL-6. Stimulation of BMDC with ligand-bound microparticles increases surface expression of costimulatory and MHC molecules, and enhances migration of BMDC to the draining lymph node. LPS-microparticles stimulate in vivo C57BL/6 BMDC and OT-1 transgenic T cell interactions in the presence of OVA SIINFEKL peptide in lymph nodes, with intact nodes imaged using two-photon microscopy. Formation of in vivo and in vitro immunological synapses between BMDC, loaded with OVA peptide and LPS-microparticles, and OT-1 T cells are presented, as well as elevated intracellular interferon gamma levels in CD8⁺ T cells stimulated by BMDC carrying peptide-loaded microparticles. In short, ligand-bound microparticles enhance (1) phagocytosis of microparticles; (2) BMDC inflammasome activation and upregulation of costimulatory and MHC molecules; (3) cellular migration of BMDC to lymphatic tissue; and (4) cellular interactions leading to T cell activation in the presence of antigen

Multivalent Presentation of MPL by Porous Silicon Microparticles Favors T Helper 1 Polarization Enhancing the Anti-Tumor Efficacy of Doxorubicin Nanoliposomes

<div><p>Porous silicon (pSi) microparticles, in diverse sizes and shapes, can be functionalized to present pathogen-associated molecular patterns that activate dendritic cells. Intraperitoneal injection of MPL-adsorbed pSi microparticles, in contrast to free MPL, resulted in the induction of local inflammation, reflected in the recruitment of neutrophils, eosinophils and proinflammatory monocytes, and the depletion of resident macrophages and mast cells at the injection site. Injection of microparticle-bound MPL resulted in enhanced secretion of the T helper 1 associated cytokines IFN-γ and TNF-α by peritoneal exudate and lymph node cells in response to secondary stimuli while decreasing the anti-inflammatory cytokine IL-10. MPL-pSi microparticles independently exhibited anti-tumor effects and enhanced tumor suppression by low dose doxorubicin nanoliposomes. Intravascular injection of the MPL-bound microparticles increased serum IL-1β levels, which was blocked by the IL-1 receptor antagonist Anakinra. The microparticles also potentiated tumor infiltration by dendritic cells, cytotoxic T lymphocytes, and F4/80⁺ macrophages, however, a specific reduction was observed in CD204⁺ macrophages.</p></div

pSi microparticle association with BMDC and biocompatibility.

<p>a) Scanning electron micrographs show pSi microparticles of varying dimensions (bars 500 nm). b) SEM images show cellular association of 2500×400 nm pSi microparticles with BMDC at various stages of uptake (top row) and cellular association with rod-shaped (left, bottom row) and smaller discoidal particles [600 nm (middle) and 1000 nm (right)]. c) BMDCs, treated with five D25.4 microparticles/cell for three hr, were imaged using confocal microscopy. d) TEM images show BMDC with internalized TLR4-ligand (LPS) bound pSi microparticles four hr after microparticle introduction. Seven 1000×400 nm discoidal microparticles are seen in the cell in the upper image (boxed regions; bar 2 µm) and in the magnified regions below (bars 1 and 0.5 µm). e) TEM images show control (left) and alum-treated BMDC four hr after introduction of 2 µg/ml alum. The boxed region showing internalized alum is magnified in the image to the right. f) BMDCs were incubated with LPS (10 ng/ml), alum (25, 12.5 and 6.25 µg/ml), or pSi microparticles (20 particles/cell) for 24 hr. Cellular necrosis was evaluated using the LIVE/DEAD® Aqua dead cell stain.</p

Combined chemo and MPL-pSi therapy inhibits cellular proliferation and stimulates tumor infiltration by immune cells which is partly IL-1 dependent.

<p>a) Excised tumors from mice treated with low dose DOX-NPs with or without pSi-MPL microparticles were stained with antibodies specific for Ki67 (red), CD8 (red), F4/80 (green), CD204 (red), or 33D1 (red). Nuclei were stained with Prolong Gold Antifade Reagent with DAPI (blue). b) The percentage of the population comprised of each cell type is shown graphically as the mean of 3–4 regions in randomly selected tissues representing at least two animals per group. *p<0.05; **p<0.01. c) Plasma IL-1β level six hr after intravenous injection of free MPL or 5×10⁸ control or MPL-pSi microparticles (10 µg MPL equivalents; n = 3/group; ***p<0.001; *p<0.05). d) Effect of anakinra on MPL-pSi microparticle-induced changes in plasma IL-1β six hr after injection (30 mg, 3×/week; *p<0.05; n = 3/group). e) Tumor growth of mice injected with MPL-pSi microparticles (weekly as indicated by arrows) with and without anakinra (30 mg, 3×/week; n = 3/group). Control verses MPL-pSi, *p<0.05; **p<0.01; ***p<0.001.</p

Injection of pSi microparticles with the TLR4 agonist MPL induces a synergistic recruitment of inflammatory cells.

<p>Female wild-type C57BL/6 mice were injected with PBS, MPL (50 µg/mouse), pSi microparticles (5×10⁸) or MPL plus pSi microparticles. The mice were sacrificed 24 hr later and PECs were isolated. PECs were stained with various markers to identify cell populations in the peritoneum at this time point, specifically the number of neutrophils (a; CD11b⁺ SiglecF⁻ Gr-1⁺), M1-like macrophages (b; CD11b⁺ SiglecF⁻ F4/80⁺ ), M2-like macrophages (c; CD11b^high F4/80^high), eosinophils (d; Gr-1⁻ ckit⁻ SiglecF⁻) and mast cells (e; ckit⁺ SiglecF⁻). Indicated variable versus MPL+pSi, *p<0.05, ***p<0.001, n = 3/group.</p