Tolerability and safety of a new elimination diet for pediatric eosinophilic gastritis and duodenitis.

BACKGROUND: Non-esophageal eosinophilic gastrointestinal disorders (non-EoE EGIDs) are chronic inflammatory disorders with massive infiltration of eosinophils into the gastrointestinal tract. Food elimination diets are potentially effective treatments. But the existing dietary therapies have various weak points. We developed a new regimen to compensate for the shortcomings of the elemental diet and 6-food elimination diet. The new regimen consists of an amino-acid-based formula, potatoes, vegetables, fruits and restricted seasonings. We named it the "Rainbow Elimination Diet (ED)." The aims of this study were to evaluate the tolerability and safety of this diet. METHODS: A retrospective medical record examination was conducted at the National Center for Child Health and Development covering the period from January 2010 through December 2018. The medical records of patients (age 2-17 y) with histologically diagnosed non-EoE EGIDs were reviewed. The tolerability, nutritional intake, symptoms, and blood test findings were evaluated. RESULTS: Nineteen patients were offered several kinds of food-elimination diets. Seven patients (eosinophilic gastritis: 5; gastroenteritis: 1; duodenitis: 1) were treated with Rainbow ED. Six patients were compliant with this diet. The median duration of the diet induction phase was 15 days (range 14-30). All 5 patients who had had symptoms just before the induction phase became symptom-free. The body weight decreased in 5 patients (median -0.6 kg), probably because the serum protein increased, resulting in reduced edema. All 5 patients with hypoproteinemia had elevated serum albumin (median 2.9-3.5 g/dL). The ingested nutritional elements were calculated, and most of them were sufficient, except for fat and selenium. CONCLUSIONS: The Rainbow ED was well-tolerated and safe for pediatric non-EoE EGIDs.

Response of TGF-ß isoforms in epithelial-mesenchymal transition of enamel epithelial cells.

OBJECTIVE: During enamel formation, transforming growth factor-beta (TGF-ß) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF-ß isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells. DESIGN: Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-ß isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-ß pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-ß receptor gene in response to the binding affinity of the TGF-ß isoform were analyzed. RESULTS: EMT was observed in mHAT9d cultured in the presence of TGF-ß1 and ß3 but not TGF-ß2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-ß signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-ß1 or ß3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-ß signaling in mHAT9d cells reduced following stimulation with each TGF-ß isoform. In contrast, endoglin levels increased following TGF-ß1 or ß3 stimulation, but no change was noted in response to TGF-ß2. CONCLUSIONS: We propose that in TGF-ß-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-ß isoforms were due to their endoglin-mediated binding affinity for the TGF-ß receptor.

Porcine keratin 75 in developing enamel.

OBJECTIVE: To provide in vivo biochemical evidence for the isolation, identification, and characterization of porcine keratin 75 (K75) in developing enamel. METHODS: Immunolocalization of K75 was observed in mandibles from mice at postnatal days 5 and 11. K75 gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction using enamel organ epithelium (EOE) of incisors from pigs at 5 months of age. Enamel protein was extracted and isolated from both immature and mature enamel of second molars from 5-month-old pigs, and the K75 antibody-positive fraction was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). In vitro protease digestion of K75-antibody-positive fraction was carried out using porcine kallikrein 4 (pKLK4) or recombinant human enamelysin (rhMMP20) and their degradation patterns were characterized by both SDS-PAGE and western blotting. RESULTS: Specific immunostaining for K75 was restricted to the layers of stratum intermedium and the enamel side of ameloblasts in mice at postnatal day 5, and to the papillary layer at postnatal day 11. Porcine K75 was expressed throughout enamel formation, but its transcript levels were significantly higher in the transition EOE than in the secretory- and maturation-stage EOE. Porcine K75 was extracted from the neutral soluble fraction from both immature and mature enamel. It was identified by LC-MS/MS analysis, and was found not to be degraded by either pKLK4 or rhMMP20. CONCLUSION: We propose that K75 is present in the developing enamel and undergoes different processing/degradation compared to other enamel proteins.

Potential function of TGF-ß isoforms in maturation-stage ameloblasts.

OBJECTIVES: To investigate potential functions of transforming growth factor-beta (TGF-ß) isoforms in maturation-stage ameloblasts during amelogenesis. METHODS: In vivo activation of TGF-ß was characterized by using matrix metalloproteinase 20 null (Mmp20-/-) and wild-type (Mmp20+/+) mice. Using mHAT9d cells cultured in the presence of each TGF-ß isoform, (1) cell proliferation was determined by MTS assay, (2) immunostaining with anti-cleaved caspase-3 monoclonal antibody was performed and apoptotic indices were measured, (3) gene expression was analyzed by RT-qPCR, and (4) the uptake of amelogenin into mHAT9d cells was directly observed using a fluorescence microscope. RESULTS: TGF-ß1 and TGF-ß3 were present in the enamel matrix of developing teeth which were activated by MMP20 in vivo. A genetic study revealed that the three TGF-ß isoforms upregulate kallikrein 4 (KLK4) mRNA levels but downregulate carbonic anhydrase II. Moreover, TGF-ß1 and TGF-ß2 significantly upregulated the mRNA level of amelotin, whereas TGF-ß3 dramatically downregulated the mRNA levels of odontogenic ameloblast-associated protein (ODAM), family with sequence similarity 83 member H (FAM83H), and alkaline phosphatase (ALP). Immunostaining analysis showed that the apoptosis of mHAT9d cells is induced by three TGF-ß isoforms, with TGF-ß3 being most effective. Both TGF-ß1 and TGF-ß3 induced endocytosis of amelogenin. CONCLUSIONS: We propose that TGF-ß is regulated in an isoform-specific manner to perform multiple biological functions such as gene expression related to the structure of basal lamina/ameloblasts, mineral ion transport, apoptosis, and endocytosis in maturation-stage ameloblasts.

TGF-ß and Physiological Root Resorption of Deciduous Teeth.

The present study was performed to examine how transforming growth factor ß (TGF-ß) in root-surrounding tissues on deciduous teeth regulates the differentiation induction into odontoclasts during physiological root resorption. We prepared root-surrounding tissues with (R) or without (N) physiological root resorption scraped off at three regions (R1-R3 or N1-N3) from the cervical area to the apical area of the tooth and measured both TGF-ß and the tartrate-resistant acid phosphatase (TRAP) activities. The TGF-ß activity level was increased in N1-N3, whereas the TRAP activity was increased in R2 and R3. In vitro experiments for the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that proteins from N1-N3 and R1-R3 enhanced the TRAP activity in RAW264 cells. A genetic study indicated that the mRNA levels of TGF-ß1 in N1 and N2 were significantly increased, and corresponded with levels of osteoprotegerin (OPG). In contrast, the expression level of RANKL was increased in R2 and R3. Our findings suggest that TGF-ß is closely related to the regulation of OPG induction and RANKL-mediated odontoclast differentiation depending on the timing of RANKL and OPG mRNA expression in the root-surrounding tissues of deciduous teeth during physiological root resorption.

CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells.

Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated-spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells.