Morphofunctional analysis of antigen uptake mechanisms following sublingual immunotherapy with beads in mice

BackgroundRecently, sublingual immunotherapy (SLIT) has been used as a safe and efficient method for the treatment of and immunization against asthma and various allergies. However, the routes of antigen uptake through the mucosa of the oral cavity remain incompletely understood, as do the roles of sex and age in the process. For this purpose, to elucidate the mechanism and efficacy of SLIT among different sexes and ages microbeads were dripped into the sublingual region to mimic antigen uptake by the sublingual mucosa.MethodsTwenty microliters of either phosphate buffered saline (PBS) or fluorescently labelled microbeads (latex and silica beads) were placed under the tongue of both male and female C57BL/6 mice at young (3 months) and old (6 months) ages. The lower jaw was examined 30 min after administration, and beads were detected with a fluorescence stereomicroscope. Morphological observations of the mucosa of the fluorescent areas were made with scanning electron microscopy (SEM) and an all-in-one light fluorescence microscope (LM). Fluorescence intensity was compared between both sexes and ages.ResultsStereomicroscopic observation revealed fluorescent illuminations in three compartments of the sublingual mucosa: the sublingual caruncles (SC), the oral rostral mucosa (OR) and the buccal mucosa (BM). Interestingly, the fluorescence intensity tended to be higher among females than among males in the SC region in particular. However, there were no significant age-related differences. SEM and LM revealed beads in the lumina of both mandibular ducts and sublingual ducts (Sd). Additionally, the apical cytoplasm of some Sd cells contained silica beads. However, there were no specification in the OR mucosa or BM.ConclusionsThis study reveals the major role Sd play in local immunity via the antigen uptake mechanisms. Furthermore, our data suggest that the efficacy of SLIT in humans could be affected by sex.

In vivo performance of a dual genetic marker, manA-gfp, in transgenic bentgrass

A dual-marker combination, manA-gfp, comprising 2 independent expression cassettes of genes encoding an Escherichia coli phosphomannose isomerase (PMI) and a synthetic green fluorescent protein (GFP), was incorporated into the binary vector pPZP201. Agrobacterium tumefaciens-mediated transfer was used to introduce the manA-gfp into the mature-seed derived calli of Agrostis stoloifera L. 'Crenshaw'. The putative transgenic bentgrass calli were screened in Murashige and Skoog medium containing 15 g mannose/L, in conjunction with a visual examination of the GFP expression with a fluorescence stereomicroscope. Calli with GFP fluorescence grew well on the mannose selection media. A total of 24 transgenic plants derived from a single piece of callus lobe were studied for the genomic integration, expression, and function of the transgene. Genomic integration of the dual markers manA and gfp was confirmed by Southern blotting analysis, and the expression of manA also was validated by using PMI-specific antiserum. The inheritance and expression of the dual marker, manA-gfp, was demonstrated in the T1 generation. This study on the environmentally friendly markers further documented the feasibility of using alternative selection methods without using herbicide- or antibiotic-resistance markers.Key words: bentgrass, Agrobacterium tumefaciens-mediated transformation, chlorophenol red assay, phosphomannose isomerase (PMI).

Acupuncture Meridian and Intravascular Bonghan Duct

Current anatomical theory does not recognize the existence of an extended floating threadlike structure inside the blood vessels. Nonetheless, this study developed a new method for observing such an intravascular threadlike structure. The key technique involves injecting acridineorange into the femoral vein to circulate along the blood vessels and stain the nuclei of the intravascular threads inside the blood vessels. In-situ observations were then made under a fluorescence stereomicroscope after saline-perfusion. Confocal microscope images revealed a distinctive characteristic pattern of nucleus distribution that was clearly distinguishable from fibrin, capillaries, small venules, arterioles, or lymph vessels. Accordingly, it is suggested that the identified intravascular threads are part of the Bonghan's circulatory network that is distributed throughout the body, including inside the blood vessels.

In vivo imaging of graft-versus-host-disease in mice

We have developed a mouse system by which to track the migration and homing of cells in a setting of bone marrow transplantation (BMT)-induced graft-versus-host disease (GVHD) after systemic infusion using enhanced green fluorescence protein (eGFP) transgenic (Tg) cells and a simple application of a fluorescence stereomicroscope outfitted with a color charge-coupled device (CCD) camera. Whole body images of anesthetized mice taken at various time points after cell infusion revealed the early migration of allogeneic cells to peripheral lymphoid organs, with later infiltration of GVHD target organs. Localization of eGFP Tg cells could be seen through the skin of shaved mice, and internal organs were easily discernible. After allogeneic or syngeneic eGFP Tg cell infusion, representative mice were dissected to better visualize deeper internal organs and tissues. Infusion of different cell populations revealed distinct homing patterns, and this method also provided a simple way to identify the critical time points for expansion of the transplanted cells in various organs. This simple application of the fluorescence stereomicroscope will be valuable for GVHD and graft-versus-tumor studies in which visualization of cellular migration, expansion, and cell-cell interactions will be more informative when analyzed by such an intravital method. (Blood. 2004;103:3590-3598)