Visualizing the stimulation of encephalitogenic T cells in gut associated lymphoid tissue as a trigger of autoimmunity

Autoantigen-specific encephalitogenic T cells exist in the healthy immune repertoire. In case of CNS autoimmunity, such as multiple sclerosis (MS), cells penetrate into the central nervous system (CNS), where they get activated by local antigen presenting cells, and induce inflammation. However, the triggering mechanisms that provoke CNS infiltration of pre-existing autoreactive T cells are largely unknown. Recent studies have shown evidence that microbiota induce proliferation of encephalitogenic T cells in gut associated lymphatic tissues (GALT) before CNS infiltration in the spontaneous experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In this study, the activation and subsequent behaviour of encephalitogenic T cells in the GALT are investigated. Myelin oligodendrocyte glycoprotein (MOG) specific T cells from transgenic mice are retrovirally transduced with FRET-based calcium activation sensors and adoptively transferred to recipient mice. T cells in the lamina propria and Peyer’s patches are imaged in vivo with two-photon microscopy and the calcium fluctuation is quantified to detect T cell activation. Moreover, migrating T cells in the efferent lymphatic vessels of GALT were analysed in order to elucidate phenotypic changes due to in vivo stimulation. Intravital imaging reveals that encephalitogenic T cells, but not polyclonal T cells, display continuous calcium signaling in the lamina propria. In contrast, encephalitogenic T cells in the Peyer’s patch show only brief calcium signaling. The continuous calcium signaling is diminished by administration of anti-MHC class II blocking antibody. This observation suggests that the calcium signaling of encephalitogenic T cells is mediated by antigen presenting cells. Additionally, the role of commensal microbiota is highlighted through the fact encephalitogenic T cells do not show continuous calcium signaling in germ free mice, which suggests the influence of microbiota. The increased number of IL17A and IFN producing T cells were detected in efferent lymph from mesenteric lymph nodes, further suggests the stimulation of T cells in the GALT. This phenotype, in addition to the enhanced number of CD44+ encephalitogenic T cells, suggests that microbiota induced stimulation in the GALT influences the migration of encephalitogenic T cells in CNS autoimmunity. In summary, the following study reveals pre-existing encephalitogenic T cells are capable of being stimulated in the lamina propria of ileum. The stimulation is dependent upon an intact gut microbiota compartment and may enhance migration of encephalitogenic T cells

Immunocytochemical Identification of the Peripolar Cell and Macula Densa Cell Types

The three "classical" components of the juxtaglomerular apparatus are the myoepithelioid cells, the macula densa cells and the lacis cells, which have been studied for over half a century. This complex multicellular structure is situated in the periglomerular position of every glomerulus, and is responsible for the intrinsic renal control of glomerular filtration rate and tubular reabsorption of sodium, and the regulation of body fluid volume and blood pressure. Complex cellular interactions occur within the juxtaglomerular apparatus. These cells also exhibit physiological relationships with other tissues of the body; but detailed mechanism are still unclear. Ten years ago, a distinctive type of cell, the peripolar cell, was recognised in close anatomical relationship with the juxtaglomerular apparatus. Morphological evidence indicates that the peripolar cell is a secretory type of cell but the substance of the secretory product is unknown. The available data suggests that the peripolar cell might be a previously unrecognised but integral component of the juxtaglomerular apparatus. Its secretory product may be responsible for the hormonal control of renal proximal tubular reabsorption, although this remains speculative

Topical delivery of DNA oligonucleotide to induce p53 generation in the skin via thymidine dinucleotide (pTT)-encapsulated liposomal carrier

Yi-Ping FangDepartment of Biotechnology, Yuanpei University, Hsinchu, TaiwanIntroduction: Transcription factor p53 has a powerful tumor suppressing function that is associated with many cancers. Since the molecular weight of p53 is 53 kDa, it is difficult to transport across cell membranes. Thymidine dinucleotide (pTT) is an oligonucleotide that can activate the p53 transcription factor and trigger the signal transduction cascade. However, the negative charge and high water solubility of pTT limit its transport through cellular membranes, thereby preventing it from reaching its target in the nucleus. A suitable delivery carrier for pTT is currently not available.Objective: The purpose of this study was to employ a nanoscale liposomal carrier to resolve the delivery problem, and increase the bioavailability and efficiency of pTT.Methodology: The approach was to employ liposomes to deliver pTT and then evaluate the particle size and zeta potential by laser light scattering (LLS), and permeation properties of pTT in vitro in a Franz diffusion assembly, and in vivo in a murine model using confocal laser scanning microscopy (CLSM).Results: We found that dioleoylphosphatidylethanolamine (DOPE) combined with cholesterol 3 sulfate (C3S) were the best ingredients to achieve an average desired vehicle size of 133.6 ± 2.8 nm, a polydispersity index (PDI, representing the distribution of particle sizes) of 0.437, and a zeta potential of −93.3 ± 1.88. An in vitro penetration study showed that the liposomal carrier was superior to the free form of pTT at 2–24 hours. CLSM study observed that the penetration depth of pTT reached the upper epidermis and potential of penetration maintained up to 24 hours.Conclusion: These preliminary data demonstrate that nanosized DOPE/C3S liposomes can be exploited as a potential carrier of drugs for topical use in treating skin diseases.Keywords: thymidine dinucleotide, p53, liposome, permeation ability, confocal laser scanning microscop