Expression of duodenase-like protein in epitheliocytes of Brunner’s glands in human duodenal mucosa

A duodenase, a protease structurally related to human cathepsin G, was found earlier in bovine duodenal mucosa. It was demonstrated that under the influence of duodenase an enteropeptidase zymogen is activated in vitro showing the possible participation of duodenase in the cascade of activation of digestive enzymes. To identify a duodenase functional analog in human duodenum, an immunofluorescence study of duodenal mucosa was conducted by confocal microscopy using antibodies to human cathepsin G and to bovine duodenase. The previously unknown place of synthesis and secretion of cathepsin G — Paneth cells located at the bottom of Lieberkuhn crypts — was revealed. Binding of cathepsin G-specific antibodies in a rough endoplasmic reticulum zone and in the cryptal duct was observed. Duodenase-specific immunofluorescence but not that of cathepsin G was found in the epitheliocytes and secretory ducts of Brunner’s glands, which are characteristic sites of duodenase biosynthesis in cattle. Binding of CD14-specific antibodies in the Brunner’s glands, where the antibodies co-localized with the antibodies to duodenase, was also demonstrated. These data indicate the presence of a protein immunologically similar to duodenase in the human duodenal mucosa. Our study demonstrated the absence of its colocalization with cathepsin G in Brunner’s glands.     

Association of Shh and Ptc with keratin localization in the initiation of the formation of circumvallate papilla and von Ebner’s gland

The development of gustatory papillae in mammalian embryos requires the coordination of a series of morphological events, such as proliferation, differentiation and innervation. In mice, the circumvallate papilla (CVP) is a specialized structure that develops in a characteristic spatial and temporal pattern in the posterior region of the tongue dorsal surface. The distinct expression patterns of Shh and Ptc, which play important roles in the development of other epithelial appendages, have been localized in the trench wall that gives rise to von Ebner’s gland (VEG). To define the cellular mechanisms responsible for morphogenesis and differentiation during early development of CVP and VEG, the localization patterns of keratins (cytokeratins) K7, K8, K18, K19, K14 and connexin-43, which are dependent on Shh expression in other developmental systems, have been examined in detail. The distinct localization of keratins K7, K8, K18, K19, K14 and connexin-43 in the epithelium giving rise to the CVP and VEG suggests that cytodifferentiation is established prior to morphological changes. Interestingly, the localization of proliferating cell nuclear antigen, a marker for cell proliferation, is similar to that of Shh. An understanding of the regulatory roles of cell-cell interactions and signalling molecules in orchestrating a mutual network will bring us nearer to defining the molecular and cellular mechanisms underlying morphogenesis in mammalian taste bud development.M.-J. Lee and J.-Y. Kim contributed equally to this work.This work was supported by grant no. R01-2003-000-11649 from the Korea Science and Engineering Foundation.     

Young’s modulus of elasticity of Schlemm’s canal endothelial cells

Schlemm’s canal (SC) endothelial cells are likely important in the physiology and pathophysiology of the aqueous drainage system of the eye, particularly in glaucoma. The mechanical stiffness of these cells determines, in part, the extent to which they can support a pressure gradient and thus can be used to place limits on the flow resistance that this layer can generate in the eye. However, little is known about the biomechanical properties of SC endothelial cells. Our goal in this study was to estimate the effective Young’s modulus of elasticity of normal SC cells. To do so, we combined magnetic pulling cytometry of isolated cultured human SC cells with finite element modeling of the mechanical response of the cell to traction forces applied by adherent beads. Preliminary work showed that the immersion angles of beads attached to the SC cells had a major influence on bead response; therefore, we also measured bead immersion angle by confocal microscopy, using an empirical technique to correct for axial distortion of the confocal images. Our results showed that the upper bound for the effective Young’s modulus of elasticity of the cultured SC cells examined in this study, in central, non-nuclear regions, ranged between 1,007 and 3,053 Pa, which is similar to, although somewhat larger than values that have been measured for other endothelial cell types. We compared these values to estimates of the modulus of primate SC cells in vivo, based on images of these cells under pressure loading, and found good agreement at low intraocular pressure (8–15 mm Hg). However, increasing intraocular pressure (22–30 mm Hg) appeared to cause a significant increase in the modulus of these cells. These moduli can be used to estimate the extent to which SC cells deform in response to the pressure drop across the inner wall endothelium and thereby estimate the extent to which they can generate outflow resistance.     

Gene expression profiling of jejunal Peyer’s patches in juvenile and adult pigs

Peyer's patches are organized lymphoid tissues of the small intestine that play a critical role in disease resistance and oral tolerance. Peyer's patches in the jejunum contain lymphocytes, dendritic cells, macrophages, villous epithelium, and specialized follicle-associated epithelium. Little is known about the mechanisms and processes by which cells of the Peyer's patches discriminate food nutrients and commensal microflora from pathogenic microbiota. We hypothesize that the jejunal Peyer's patches express genes that mediate and regulate its essential functions. Expression patterns of approximately 2600 cDNAs from a porcine Peyer's patch subtracted library were examined by microarray profiling. Individual mRNAs of interest were further examined by quantitative RT-PCR. Innate immunity-associated genes, including complement 3 and lysozyme, and the genes for epithelial chloride channel and trappin 1 were highly expressed by jejunal Peyer's patch in both juvenile and adult pigs. The growth- and apoptosis-associated genes CIDE-B, GW112, and PSP/Reg I (pancreatic stone protein or regenerating gene) were differentially expressed in juvenile pig Peyer's patches. Many sequences which were highly expressed in jejunal Peyer's patches have previously been described with functions in epithelial cells. Animal-to-animal variation in basal jejunal Peyer's patch gene expression was considerable and reflects the dynamic physiological environment of the gut in addition to genetic, epigenetic, and microbiological variation in the small intestine.     

Analysis of DNA damage induced by aflatoxin B1 in Dunkin–Hartley guinea pigs

Aflatoxin B1 (AFB1) is among the most potent naturally occurring carcinogens and classified as a group I carcinogen. Since the ingestion of aflatoxin-contaminated food is associated with several liver diseases, the aim of the present study was to evaluate the effect of 2, 20, and 200 ppb of AFB1 on DNA damage in peripheral blood lymphocytes and liver cells in Dunkin-Hartley guinea pigs. The animals were divided into four groups according to the given diet. After the treatment the lymphocytes and liver cells were isolated and DNA damage determined by Comet assay. The levels of DNA damage in lymphocytes were higher animals treated with 200 ppb of AFB1-enriched diet (P = 0.02). In the liver cells there were a relationship between the levels of DNA damage and the consumption of AFB1 in all studied groups. These results suggest that Comet assay performed on lymphocytes is a valuable genotoxic marker for high levels of exposure to AFB1 in guinea pig. Additionally our results indicate that the exposure to this toxin increases significantly and increases the level of DNA damage in liver cells, which is a key step on liver cancer development. We also suggest that the Comet assay is an useful tool for monitoring the genotoxicity of AFB1 in liver.     

Expression of pro-inflammatory TACE-TNF-α-amphiregulin axis in Sjögren’s syndrome salivary glands

The tumor-necrosis-factor-converting-enzyme (TACE)-TNF-α-Amphiregulin (AREG) axis plays an important pathogenic role in inflammatory and autoimmune disorders. However, the pathological roles of these proteins in the chronic autoimmune disease Sjögren’s syndrome (SS) remain to be elucidated. It is known that the TACE–AREG axis is clearly part of a larger cascade of signals that starts with the activation of Furin, responsible for maturation of TACE that, in turn, determines the production of active TNF-α, directly involved in the up-regulation of AREG expression. This study showed that Furin, TACE, TNF-α, and AREG proteins, detected in acinar and ductal cells of human salivary glands from SS patients, increased remarkably in comparison with biopsies of labial salivary glands from healthy controls. The changes in Furin, TACE, TNF- α, and AREG proteins’ level detected in salivary glands biopsies of SS patients could be responsible for pro-inflammatory cytokines overexpression characterizing Sjögren’s syndrome.     

Hair cortisol: a parameter of chronic stress? Insights from a radiometabolism study in guinea pigs

Measurement of hair cortisol has become popular in the evaluation of chronic stress in various species. However, a sound validation is still missing. Therefore, deposition of radioactivity in hair and excretion into feces and urine after repeated injection of (3)H-cortisol was studied in guinea pigs (n?=?8). Each animal was given intraperitoneally 243.6?kBq (3)H-cortisol/day on 3 successive days. After the first injection, all voided excreta were collected for 3?days. After the second injection, hair was shaved off the animals' back and newly grown hair was obtained on day 7. Following methanol extraction, radiolabeled and unlabeled glucocorticoid metabolites (GCM) in fecal and hair samples were characterized by high-performance liquid chromatography (HPLC) and enzyme immunoassays (EIA). In feces, maximum radioactivity was reached 8?h (median) post each injection, whereas maxima in urine were detected in the first samples (median 2.5?h). Metabolites excreted into feces (13.3?%?±?3.7) or urine (86.7?%) returned nearly to background levels. HPLC of fecal extracts showed minor variation between individuals and sexes. In hair, small amounts of radioactivity were present. However, two EIAs detected large amounts of unlabeled GCM, including high levels at the position of the cortisol standard; radioactivity was absent in this fraction, demonstrating that (3)H-cortisol was metabolized. Furthermore, large amounts of immunoreactivity coinciding with a radioactive peak at the elution position of cortisone were found. These results show for the first time that only small amounts of systemically administered radioactive glucocorticoids are deposited in hair of guinea pigs, while measurement of large amounts of unlabeled GCM strongly suggests local production of glucocorticoids in hair follicles.     

Method for assessing IFN-γ responses in guinea pigs during TB vaccine trials

Aims: We sought to develop a new method that enables the assessment of the immune response of guinea pigs during TB vaccine evaluation studies, without the need to cull or anaesthetize animals. Method and Results: Guinea pigs were vaccinated with five different formulations of oral BCG. One week prior to challenge with Mycobacterium bovis, blood (50–200 μl) was taken from the ears of vaccinated subjects. Host RNA was isolated and amplified following antigenic restimulation of PBMCs for 24 h with 30 μg of bovine PPD. The up‐ or down‐regulation of γ‐interferon (IFN‐γ), a key cytokine involved in protection against tuberculosis, was assessed using real‐time PCR. The relative expression of prechallenge IFN‐γ mRNA in the vaccinated groups (n = 5) correlated (P < 0·001) with protection against M. bovis challenge. Conclusion: We have demonstrated that it is possible to take blood samples and track IFN‐γ responses in guinea pigs that then go on to be exposed to M. bovis, thus providing prechallenge vaccine uptake information. Significance and Impact of the Study: This methodology will also be applicable for tracking the immune responses of vaccinated guinea pigs over time that then go on to be challenged with M. tuberculosis during human TB vaccine evaluation studies.     

Precipitation Increases the Occurrence of Sporadic Legionnaires’ Disease in Taiwan

Legionnaires’ disease (LD) is an acute form of pneumonia, and changing weather is considered a plausible risk factor. Yet, the relationship between weather and LD has rarely been investigated, especially using long-term daily data. In this study, daily data was used to evaluate the impacts of precipitation, temperature, and relative humidity on LD occurrence in Taiwan from 1995–2011. A time-stratified 2:1 matched-period case-crossover design was used to compare each case with self-controlled data using a conditional logistic regression analysis, and odds ratios (ORs) for LD occurrence was estimated. The city, gender and age were defined as a stratum for each matched set to modify the effects. For lag day- 0 to 15, the precipitation at lag day-11 significantly affected LD occurrence (p<0.05), and a 2.5% (95% CIs = 0.3–4.7%) increased risk of LD occurrence was associated with every 5-mm increase in precipitation. In addition, stratified analyses further showed that positive associations of precipitation with LD incidence were only significant in male and elderly groups and during the warm season ORs = 1.023–1.029). However, such an effect was not completely linear. Only precipitations at 21–40 (OR = 1.643 (95% CIs = 1.074–2.513)) and 61–80 mm (OR = 2.572 (1.106–5.978)) significantly increased the risk of LD occurrence. Moreover, a negative correlation between mean temperature at an 11-day lag and LD occurrence was also found (OR = 0.975 (0.953–0.996)). No significant association between relative humidity and LD occurrence was identified (p>0.05). In conclusion, in warm, humid regions, an increase of daily precipitation is likely to be a critical weather factor triggering LD occurrence where the risk is found particularly significant at an 11-day lag. Additionally, precipitation at 21–40 and 61–80 mm might make LD occurrence more likely.     

Distribution of immune cells and expression of interleukin receptors in ileal Peyer’s patches of calves

Newborn calves lack a mature immune system. The immune system develops with age, but the role of the expression of cytokine receptors in the development of immune cells of Peyer’s patches (PPs) in the intestines of calves in the first 2 months has not yet been elucidated. In this study, the distribution of immune cells and the expression of interleukin (IL) receptors (R) in the ileal PPs of newborn and 2-month-old calves were investigated immunohistochemically with monoclonal antibodies against bovine CD4, CD8, IgM, γδTCR, T19, WC3, WC5, and WC6 antigens. The expression of ILRs was examined with antibodies against CD25 (IL-2Rα), IL-2Rγ, IL-4R, IL-6R, IL-10R, and IL-13R antigens. CD4⁺, CD8⁺, γδTCR⁺, T19⁺, and WC6⁺ cells were found to be more widely distributed in the ileal PPs of 2-month-old calves than in those of newborn calves. Moreover, the expression of CD25 (IL-2Rα), IL-4R, and IL-13R in the ileal PPs of 2-month-old calves was more prominent than that in newborn calves. These data suggest that the immune system of calves at 2 months of age is developed by reactions to foreign antigens and aging.     

Crohn’s disease of the colon: ultrastructural changes in submuscular interstitial cells of Cajal

Interstitial cells of Cajal (ICC) at the submuscular border of the human colon (ICC-SMP) are the proposed pacemaker cells of the musculature. In patients with Crohn’s disease (CD) of the colon, ICC-SMP showed characteristic cytological changes from controls. The changes comprised secondary lysosomes in connection with lipid droplets and cytoplasmic vacuoles or multiple empty, confluent and often outbulging vacuoles merging with cisterns of granular endoplasmic reticulum and clusters of glycogen granules. These changes were most pronounced in patients with macroscopical mucosal inflammation but were also demonstrable in uninvolved colonic segments. Relationships of ICC to other cells were undisturbed. The changes were selective to ICC-SMP, as glial cells, muscle cells and fibroblast-like cells at the submuscular border showed no cytological alterations compared with controls. Varicosities of the submuscular plexus were often empty and dilated. Fibroblast-like cells selectively encased macrophages and mast cells. The cytological changes in ICC-SMP in CD are thus similar to changes seen in ulcerative colitis and may be of pathophysiological significance with regard to the motility and sensory disturbances seen in patients with CD.     

Mitochondrial modulation of store-operated Ca2+ entry in model cells of Alzheimer’s disease

Mitochondrial malfunction and calcium dyshomeostasis are early pathological events considered as important features of the Alzheimer’s disease (AD) brain. Recent studies have suggested mitochondrion as an active regulator of Ca²⁺ signaling based on its calcium buffering capacity. Herein, we investigated the mitochondrial involvement in the modulation of store-operated calcium entry (SOCE) in neural 2a (N2a) transgenic AD model cells. Results showed that SOCE was significantly depressed in N2a cells transfected with wild-type human APP695 (N2a APPwt) compared with empty vector control (N2a WT) cells. Pharmacological manipulation with mitochondrial function blockers, such as FCCP, RuR, or antimycin A/oligomycin, could inhibit mitochondrial calcium handling, and then impair SOCE pathway in N2a WT cells. Furthermore, mitochondria of N2a APPwt cells exhibited more severe swelling in response to Ca²⁺, which is an indication of mitochondrial membrane permeability transition (MPT), than the wild-type controls. Additionally, treatment with cyclosporin A, a potent inhibitor of cyclophilin D, which can block MPT, could significantly restore the attenuated SOCE in N2a APPwt cells. Therefore, inhibition of cyclophilin D might be a therapeutic strategy for Alzheimer’s disease.     

Environmental Occurrence of the Whipple’s Disease Bacterium (Tropheryma whippelii)

Whipple’s disease is a systemic disorder in which a gram-positive rod-shaped bacterium is constantly present in infected tissues. After numerous unsuccessful attempts to culture this bacterium, it was eventually characterized by 16S rRNA gene analysis to be a member of the actinomycetes. The name Tropheryma whippelii was proposed. Until now, the bacterium has only been found in infected human tissues, but there is no evidence for human-to-human transmission. Here we report the detection of DNA specific for the Whipple’s disease bacterium in 25 of 38 wastewater samples from five different sewage treatment plants in the area of Heidelberg, Germany. These findings provide the first evidence that T. whippelii occurs in the environment, within a polymicrobial community. This is in accordance with the phylogenetic relationship of this bacterium as well as with known epidemiological aspects of Whipple’s disease. Our data argue for an environmental source for infection with the Whipple’s disease bacterium.Whipple’s disease, which was first described in 1907 as intestinal lipodystrophy (18), is a multisystem disorder characterized by the presence of gram-positive, rod-shaped bacteria in infected human tissues (2). Numerous attempts to culture the bacterium associated with Whipple’s disease have failed (2), and eventually its phylogenetic position within the actinomycetes was established by 16S rRNA gene analysis (11, 19). The name Tropheryma whippelii was proposed (11).The clinical presentation of Whipple’s disease is heterogeneous. Frequently, patients suffer for years from arthralgias, chronic diarrhea, and weight loss, and less often from dementia or cardiac insufficiency. If untreated, the disease is usually fatal, but with appropriate antibiotic therapy the prognosis is favorable (2). However, the pathogenesis of Whipple’s disease appears to involve more than just an infection with T. whippelii. Immunological abnormalities are presumed to play a necessary role as predisposing factors (2, 7, 8), a view which is strengthened by the detection of T. whippelii in association with AIDS (5).Two outstanding questions in the epidemiology of Whipple’s disease are the bacterium’s natural habitat and the route of infection. Until now, T. whippelii has never been found outside of infected human hosts, and although an oral route of infection has often been suspected (10), it has not been proven. There is no evidence for human-to-human transmission.A reassessment of its phylogeny revealed a close relationship of the Whipple’s disease bacterium to typical environmental bacteria, such as the cellulomonads and the rare group B peptidoglycan organisms (6). This would support the hypothesis that T. whippelii may be a soil or water inhabitant. This may also explain the difficulties involved with its culture, as an estimated 80 to 99% of bacteria occurring in such natural environments are not culturable on artificial media (14, 17) and uncultured members of the actinomycete line of descent have been found in different environments and geographical locations (12). Soil and water bacteria tend to concentrate in sewage to form communities in which a large variety of different species are found. Therefore, the search for the Whipple’s disease bacterium was concentrated at sewage treatment plants.From September 1995 to July 1996, a total of 38 effluent samples from the sedimentation ponds of five different sewage treatment plants were examined by PCR for the presence of the Whipple’s disease bacterium. The sewage treatment plants are located within a distance of 5 to 25 km from Heidelberg, Germany (Fig. ​(Fig.1).1). Effluent was sampled in 1,000-ml single-use plastic flasks and filtered as described previously (4) in portions of 25 ml through cellulose acetate prefilters (25-mm diameter, 5-μm pore size; Sartorius, Göttingen, Germany). It was then passed through polyvinylidene fluoride filters (25-mm diameter, 0.45-μm pore size; Millipore, Bedford, Mass.). The DNA from the 0.45-μm-pore-size filters was extracted with the EnviroAmp Legionella sample preparation kit (Perkin-Elmer, Norwalk, Conn.), with the addition of 0.2% bovine serum albumin to the water in the final extraction step. Distilled water for negative controls was sampled in the same flasks and treated in the same way as the sewage. Open in a separate windowFIG. 1Map of the area around Heidelberg, Germany, displaying the rivers Rhine (Rhein) and Neckar, the relevant townships (shaded areas with names in capital letters), and the five sewage treatment plants (dots) from which samples were taken. The inset in the figure shows the localization of the area within Germany.PCR amplification and detection were performed as described in detail elsewhere (15). Primers whip1 (5′-AGAGATACGCCCCCCGCAA) and whip2 (5′-ATTCGCTCCACCTTGCGA), which amplify a fragment of 267 bp from the 16S rRNA gene of the bacterium, were used. In addition to hybridization with oligonucleotide whip3 (5′-TGGTACAGAGGGTTGCAATA), a second hybridization in a separate reaction was performed with oligonucleotide whip4 (5′-GTAATGGCGGGGACTCACAG). The specificities of primers whip1 and whip2 and of probe whip3 have been tested previously (15), and testing of probe whip4 with the same set of 37 control bacteria gave the same results as those reported for whip3 (15).Of the 38 sewage samples tested, 25 were found to be positive by PCR and hybridization with both oligonucleotides (Table ​(Table1).1). Positive samples were found at each of the five sewage treatment plants. Most of the positive PCR products displayed relatively weak bands at the expected size of 267 bp. They also contained some extraneous bands, which were not of the expected size, probably due to the large amount of DNA from other organisms present in sewage. On hybridization, however, the PCR products displayed distinct bands, which were consistent between the two oligonucleotides (Fig. ​(Fig.2).2).

TABLE 1

Results of PCR for the Whipple’s disease bacterium from sewage samples

Rapid and significant induction of TRAIL-mediated type II cells in apoptosis of primary salivary epithelial cells in primary Sjögren’s syndrome

Expressions of the effector molecules of Fas-mediated apoptosis in primary cultured salivary gland epithelial cells (SGEC) of primary Sjögren’s syndrome (pSS) remain to be clarified. We focused on Fas-mediated caspase cleavage compared to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Induction of apoptosis was performed by anti-Fas antibody coupled with PI3K inhibitor, or TRAIL. Activation of caspases, cytochrome C, and apoptotic protease activating factor-1 (Apaf-1) was determined by western blotting or immunofluorescence observed by confocal microscopy. Fas-mediated apoptosis and activation of caspase 3/8 were induced in the presence of LY294002. TRAIL-induced apoptosis in SGEC, which was stronger than that induced by anti-Fas antibody. TRAIL-induced caspase 9 cleavage accompanied by activation of cytochrome C and Apaf-1 were not mediated by anti-Fas antibody. Our results suggest that death receptor-dependent apoptosis in primary cultured SGEC is regulated by the engagement of type II cells in pSS.     

Differentiation of Boettcher’s cells during postnatal development of rat cochlea

Contrary to the highly specialized epithelial cells of the mammalian auditory organ, little is known about the surrounding cells and, in particular, Boettcher’s cells (BC). Our morphological studies show that, in rats, these cells began their differentiation around postnatal day 8 (P8) reaching maturity around P20, when they are completely covered by Hensen’s and Claudius’ cells. Tight junctions were noted near the apex of BC, providing that they were in direct contact with the endolymphatic space, between approximately P8 and P16. We observed gap junctions between BC and adjacent cells before the end of the covering process suggesting the additional involvement of BC in potassium recycling into the endolymph. Adherens junctions were also seen between BC throughout their maturation. Importantly, we noticed cytoplasmic secretory granules and an accumulated material, probably a secretion, in the intercellular space, between P8 and P25. These results indicate that BC could basally take part in the secretion of the extracellular matrix of the basilar membrane. Finally, we show that the basolateral interdigitations of BC are longer and more tighlty grouped at maturity and harbour urea transporters as early as P18. Our observations thus support the view that BC perform several functions.     

Distribution and severity of spontaneous lesions in the neuroepithelium and Bowman’s glands in mouse olfactory mucosa: age-related progression

Age-related changes were examined in the distribution and severity of spontaneous lesions in the neuroepithelium and Bowman’s glands in mouse olfactory mucosa. The olfactory mucosa of female ICR mice at postnatal ages from 10 days to 16 months were investigated histologically by hematoxylin and eosin staining, high-iron diamine-Alcian blue (HID-AB) staining, and immunohistochemistry for olfactory marker protein (OMP), βIII tubulin (βIIIT), and Ki67. The lesions in the neuroepithelium and Bowman’s glands were quantitatively assessed by morphometric analyses of sections stained with anti-OMP antibody or HID-AB. The first appearance of neuroepithelial abnormality was observed in the dorsomedial portion of the olfactory mucosa in 5-month-old mice. The distribution and severity of lesions progressed with increasing age. In mildly affected epithelium in which OMP-positive olfactory receptor neurons (ORNs) were present but in smaller amounts, the numbers of βIIIT-positive and Ki67-positive neuroepithelial cells tended to be increased, indicating that neurogenesis was upregulated in these areas. In contrast, severely affected epithelium in which OMP-positive ORNs were virtually absent showed high variability in the numbers of βIIIT- and Ki67-positive cells among the areas examined, probably reflecting differences in the capacity of the basal cells remaining in the affected area to generate new neuronal cells. Histological analysis with HID-AB revealed that spontaneous lesions in Bowman’s glands also occurred in aged mouse olfactory mucosa. Lesions in the neuroepithelium and underlying Bowman’s glands tended to be spatially co-localized, suggesting a close association between pathogeneses in these two structures. Moreover, lesions in Bowman’s glands were associated with changes in the biochemical composition of mucus on the olfactory mucosa. This information should prove useful in improving the understanding of the pathogenetic mechanisms underlying age-related changes in the peripheral olfactory system. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (nos. 14770886, 16790987 and 18799002; K. Kondo) and a grant from the Japanese Ministry of Health, Labour, and Welfare (Comprehensive Research on Aging and Welfare, no. H13-choju-012; K. Nibu).     

Migration and differentiation of transplanted enteric neural crest-derived cells in murine model of Hirschsprung’s disease

Stem cell therapy offers the potential of rebuilding the enteric nervous system (ENS) in the aganglionic bowel of patients with Hirschsprung’s disease. P0-Cre/Floxed-EGFP mice in which neural crest-derived cells express EGFP were used to obtain ENS stem/progenitor cells. ENS stem/progenitor cells were transplanted into the bowel of Ret^−/− mouse, an animal model of Hirschsprung’s disease. Immunohistochemical analysis was performed to determine whether grafted cells gave rise to neurons in the recipient bowel. EGFP expressing neural crest-derived cells accounted for 7.01 ± 2.52 % of total cells of gastrointestinal tract. ENS stem/progenitor cells were isolated using flow cytometry and expanded as neurosphere-like bodies (NLBs) in a serum-free culture condition. Some cells in NLBs expressed neural crest markers, p75 and Sox10 and neural stem/progenitor cells markers, Nestin and Musashi1. Multipotency of isolated ENS stem/progenitor cells was determined as they differentiated into neurons, glial cells, and myofibloblasts in culture. When co-cultured with explants of hindgut of Ret^−/− mice, ENS stem/progenitor cells migrated into the aganglionic bowel and gave rise to neurons. ENS stem/progenitor cells used in this study appear to be clinically relevant donor cells in cell therapy to treat Hirschsprung’s disease capable of colonizing the affected bowel and giving rise to neurons.