Tmem2 Deficiency Leads to Enamel Hypoplasia and Soft Enamel in Mouse.

Teeth consist of 3 mineralized tissues: enamel, dentin, and cementum. Tooth malformation, the most common craniofacial anomaly, arises from complex genetic and environmental factors affecting enamel structure, size, shape, and tooth eruption. Hyaluronic acid (HA), a primary extracellular matrix component, contributes to structural and physiological functions in periodontal tissue. Transmembrane protein 2 (TMEM2), a novel cell surface hyaluronidase, has been shown to play a critical role during embryogenesis. In this study, we demonstrate Tmem2 messenger RNA expression in inner enamel epithelium and presecretory, secretory, and mature ameloblasts. Tmem2 knock-in reporter mice reveal TMEM2 protein localization at the apical and basal ends of secretory ameloblasts. Micro-computed tomography analysis of epithelial-specific Tmem2 conditional knockout (Tmem2-CKO) mice shows a significant reduction in enamel layer thickness and severe enamel deficiency. Enamel matrix protein expression was remarkably downregulated in Tmem2-CKO mice. Scanning electron microscopy of enamel from Tmem2-CKO mice revealed an irregular enamel prism structure, while the microhardness and density of enamel were significantly reduced, indicating impaired ameloblast differentiation and enamel matrix mineralization. Histological evaluation indicated weak adhesion between cells and the basement membrane in Tmem2-CKO mice. The reduced and irregular expressions of vinculin and integrin ß1 suggest that Tmem2 deficiency attenuated focal adhesion formation. In addition, abnormal HA accumulation in the ameloblast layer and weak claudin 1 immunoreactivity in Tmem2-CKO mice indicate impaired tight junction gate function. Irregular actin filament assembly was also observed at the apical and basal ends of secretory ameloblasts. Last, we demonstrated that Tmem2-deficient mHAT9d mouse ameloblasts exhibit defective adhesion to HA-containing substrates in vitro. Collectively, our data highlight the importance of TMEM2 in adhesion to HA-rich extracellular matrix, cell-to-cell adhesion, ameloblast differentiation, and enamel matrix mineralization.

Retinoic Acid Deficiency Underlies the Etiology of Midfacial Defects.

Embryonic craniofacial development depends on the coordinated outgrowth and fusion of multiple facial primordia, which are populated with cranial neural crest cells and covered by the facial ectoderm. Any disturbance in these developmental events, their progenitor tissues, or signaling pathways can result in craniofacial deformities such as orofacial clefts, which are among the most common birth defects in humans. In the present study, we show that Rdh10 loss of function leads to a substantial reduction in retinoic acid (RA) signaling in the developing frontonasal process during early embryogenesis, which results in a variety of craniofacial anomalies, including midfacial cleft and ectopic chondrogenic nodules. Elevated apoptosis and perturbed cell proliferation in postmigratory cranial neural crest cells and a substantial reduction in Alx1 and Alx3 transcription in the developing frontonasal process were associated with midfacial cleft in Rdh10-deficient mice. More important, expanded Shh signaling in the ventral forebrain, as well as partial abrogation of midfacial defects in Rdh10 mutants via inhibition of Hh signaling, indicates that misregulation of Shh signaling underlies the pathogenesis of reduced RA signaling-associated midfacial defects. Taken together, these data illustrate the precise spatiotemporal function of Rdh10 and RA signaling during early embryogenesis and their importance in orchestrating molecular and cellular events essential for normal midfacial development.

Lansoprazole increases serum IgG and IgM in H. pylori-infected patients.

Proton-pump inhibitors have been reported to influence the human immune system, we therefore evaluated the effect of lansoprazole, a proton-pump inhibitor, on humoral immunity. Patients with gastric ulcer received lansoprazole 30 mg/day for 8 weeks, and serum immunoglobulins were evaluated before and upon completion of the treatment. There were 79 patients with gastric ulcer; 51 were H. pylori-infected and 28 were H. pylori-uninfected. Eighteen patients positive for H. pylori were receiving at least one non-steroidal anti-inflammatory drug, and 12 patients negative for H. pylori received one non-steroidal anti-inflammatory drug. H. pylori-infected patients showed significant increases in serum immunoglobulins G and M 8 weeks after the start of lansoprazole treatment (P<0.001 for IgG and P<0.01 for IgM), but uninfected patients did not. Even when H. pylori-infected patients receiving a non-steroidal anti-inflammatory drug or low-dose aspirin were analyzed separately, these increases were seen (P<0.001 for IgG and P<0.005 for IgM). Lansoprazole elevated serum levels of immunoglobulins G and M in gastric ulcer patients with H. pylori infection, particularly in those receiving non-steroidal anti-inflammatory drugs. Deducing from these observations, lansoprazole might alter the Th1 shift in the immune response induced by H. pylori infection.

Prevalence of Helicobacter pylori in NSAID users with gastric ulcer.

OBJECTIVE: Regarding the interaction of Helicobacter pylori and non-steroidal anti-inflammatory drugs (NSAIDs), we cannot accept unanimous conclusions in inducing gastric ulcer. We therefore evaluated the role of Helicobacter pylori and NSAIDs in inducing gastric ulcer. METHODS: Dyspeptic patients receiving NSAIDs underwent endoscopic examination. Gastric ulcer formation and H. pylori status were investigated. Biopsy specimens from the antrum and lower body of the stomach were prepared for the rapid urease test and pathological evaluation. Anti-H. pylori antibody was measured by enzyme-linked immunosorbent assay. RESULTS: Two hundred and twenty-six patients receiving NSAIDs (220 chronic and six on-demand users) underwent gastrofibrescopic examination. There were 110 patients with gastric ulcer and 111 non-ulcer patients with gastritis. The remaining five patients had neither. NSAID users with gastric ulcer showed a low prevalence of H. pylori compared with those without them [55/110 (50.0%) vs 79/111 (71.2%), P < 0.01]. The same tendency was seen when patients receiving low-dose aspirin and those with rheumatoid arthritis were analysed separately [13/29 (44.8%) vs 50/62 (80.6%), P < 0.01, and 11/33 (33.3%) vs 16/26 (61.5%), P < 0.06 with Yates' correction, respectively]. CONCLUSION: Helicobacter pylori infection appeared to be a risk factor for developing gastritis, but we found no evidence that it increases gastric ulcer formation in NSAID users with dyspepsia.

Tranilast inhibits cell proliferation and collagen synthesis by rabbit corneal and Tenon's capsule fibroblasts.

PURPOSE: To determine whether tranilast, N-(3,4-dimethoxycinnamoyl) anthranilic acid, influences cell proliferation and collagen synthesis by rabbit Tenon's capsule fibroblasts (TFs) and corneal stromal fibroblasts (CFs). METHODS: Rabbit TFs and CFs (7000 cells/well) were cultured in F-12 nutrient mixture supplemented with 1% FBS, plus 0, 3, 30, or 300 microM tranilast, and the number of cells was counted 72 hrs later. To determine the effect of tranilast on collagen synthesis, cells at confluence were cultured in a medium containing 0, 3, 30, or 300 microM tranilast and labeled with 3H-proline, and the amount of radioactivity incorporated into collagenase-sensitive proteins was measured. RESULTS: At 300 microM, tranilast decreased the number of TFs by about 27% and the number of CFs by about 45%, but had no effect on cell viability. The same concentration of tranilast reduced TFs collagen synthesis and CFs collagen synthesis. CONCLUSIONS: Tranilast may inhibit scar formation after trabeculectomy for glaucoma and after excimer laser photorefractive keratectomy.

Effect of rabbit aqueous humor obtained after cataract surgery on collagen gel contraction induced by bovine lens epithelial cells.

We evaluated the effect of specimens of pre- and postoperative aqueous humor on the contraction of collagen gels, and the effect of transforming growth factor-beta(2) (TGF-beta(2)) in postoperative aqueous humor. Rabbit aqueous humor was collected preoperatively and on postoperative day 7. Bovine lens epithelial cells (LECs) were cultured in collagen gel in F-12 nutrient mixture supplemented with 5% fetal bovine serum that contained 10% aqueous humor obtained under various conditions. Gel area was determined on day 4. Gels cultured with the medium that contained phosphate-buffered saline showed a statistically significant contraction after 4 days. Aqueous humor from aphakic or pseudophakic eyes significantly increased contraction, with both specimens having a similar effect. Approximately 60% of the contractile effect of the postoperative aqueous humor was neutralized by anti-TGF-beta(2) antibody. However, the promoting effect of the aqueous humor sampled postoperatively was less than that sampled preoperatively. Although the aqueous humor obtained postoperatively increased the contractility of the LECs, with the level of TGF-beta(2) apparently responsible for much of its effect, the effect was less than that observed in the aqueous humor obtained preoperatively.

Extracellular matrixes influence alpha-smooth muscle actin expression in cultured porcine lens epithelial cells.

PURPOSE: To determine whether extracellular matrix (ECM) influences the expression of alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, in cultured porcine lens epithelial cells (LECs). METHODS: Porcine LECs were cultured for 6 days in an F-12 nutrient mixture supplemented with 5% fetal bovine serum on wells that were coated with laminin, fibronectin, type I collagen, or type IV collagen. LECs cultured on uncoated wells served as a control. Alpha-SMA was detected immunocytochemically with a mouse monoclonal antibody, and the ratio of the number of alpha-SMA-positive cells to the total number of cells, the P/T ratio, was calculated. RESULTS: The P/T ratio of the LECs on the uncoated dishes was about 5%. LECs cultured on wells coated with laminin or type IV collagen significantly reduced the ratio, whereas fibronectin or type I collagen had no effect. CONCLUSIONS: The ECM influences alpha-SMA expression in cultured porcine LECs.

Fibrous membrane formation at the capsular margin in capsule contraction syndrome.

PURPOSE: To determine whether the pathogenesis of capsule contraction syndrome involves the outgrowth of the fibrous membrane from the anterior capsule margin. SETTING: Department of Ophthalmology, Keio University School of Medicine, Tokyo, and the Ando Eye Clinic, Kanagawa, Japan. METHODS: A retrospective review of medical records and slitlamp photographs was conducted in 12 eyes (10 patients) that had required treatment for a narrowed anterior capsule opening after cataract surgery. All patients had had continuous curvilinear capsulorhexis and phacoemulsification with implantation of an intraocular lens in the capsular bag. Specimens of surgically removed fibrous membrane were examined by histopathological methods. RESULTS: Fibrous membrane on the inner surface of the anterior capsule and the linear folds of the anterior capsule were present in each eye. In 10 eyes of 8 patients, the fibrous membrane was on the outer surface of the anterior capsule and covered the capsular folds at its margin. Pathological study showed that this fibrous membrane consisted of the flattened lens epithelial cells that proliferated on the inner and outer surfaces of the shrunken anterior capsule. The outgrowth of this membrane from the margin of the anterior capsule to the center of the opening of the anterior capsule was noted. CONCLUSION: In this study, capsule contraction syndrome involved contraction of the fibrous membrane as well as its outgrowth from the capsule margin.

[Extracellular matrix influencees proliferation of cultured porcine lens epithelial cells].

PURPOSE: To investigate whether extracellular matrix (ECM) influences the proliferation of cultured lens epithelial cells (LECs). MATERIAL AND METHODS: Porcine LECs were cultured in F-12 nutrient mixture supplemented with 5% fetal bovine serum (FBS) for 24 or 96 hours on the dishes coated with laminin, fibronectin, type I collagen, or type IV collagen. As a control, LECs were cultured on uncoated dishes. Twenty-four or ninety-six hours later, the number of cells was determined. We determined the proliferation ratio of the number of cells 96 hours after plating to the number of cells 24 hours after plating. This ratio was used to assess the cell proliferation. RESULTS: The ratio of the LECs on the uncoated dishes was 2.3. Dish coating with type I or type IV collagen, and fibronectin significantly increased this ratio (4.0, 3.5, and 3.0, respectively), whereas coating with laminin did not affect this ratio (2.5). CONCLUSION: These findings suggest that ECM influences cultured porcine LEC proliferation.

Central nervous system action of melatonin on gastric acid and pepsin secretion in pylorus-ligated rats.

We recently demonstrated that centrally administered melatonin at low doses inhibits the induction of gastric lesions by water-immersion restraint stress. To investigate the mechanism of the potent anti-ulcer action of melatonin, the central nervous system (CNS) effects of melatonin on gastric acid and pepsin secretion were studied in conscious pylorus-ligated rats. Intracisternal (i.c.) melatonin (1-100 ng) dose-dependently decreased acid and pepsin output, while a higher i.p. dose (1 microg) had no inhibitory effect. The i.c. melatonin did not change serum gastrin concentrations. Serum melatonin concentrations at 1 and 4 h after i.c. administration of 10-100 ng melatonin did not differ from those in rats receiving i.c. vehicle. The present results suggest that melatonin administered centrally modulates the secretion of gastric acid and pepsin which may explain, at least in part, the protective, anti-stress role of melatonin in the gastric mucosa observed in our previous study.

Transforming growth factor-beta 1 promotes contraction of collagen gel by bovine corneal fibroblasts through differentiation of myofibroblasts.

PURPOSE: To determine whether the ability of transforming growth factor-beta (TGF-beta) to influence the contractile activity of corneal fibroblasts depends on their differentiation into myofibroblasts. METHODS: Bovine corneal fibroblasts were cultured on collagen gel in MED 5 medium (F-12 nutrient mixture supplemented with 5% fetal bovine serum) with or without TGF-beta 1 (0.01-10 ng/ml). To evaluate the corneal fibroblast-derived contraction of collagen gel, the thickness of the gel was measured daily for 6 days. The total number of cells on the gel was counted with a Coulter counter. The detection of alpha-smooth muscle actin (alpha-SMA); a marker for myofibroblasts, on these cells was performed immunocytochemically by using a mouse monoclonal antibody against alpha-SMA. The number of myofibroblasts (alpha-SMA-positive cells) was determined. RESULTS: The control gels containing bovine corneal fibroblasts that were cultured with the MED 5 medium alone significantly contracted to 72.3 +/- 1.2% of their original thickness after 6 days. TGF-beta 1 increased the contraction of collagen gel mediated by bovine corneal fibroblasts in a dose-dependent manner. Approximately 0.2% of the cells on the control gels cultured with MED 5 medium alone were alpha-SMA positive. TGF-beta 1 significantly increased the expression of alpha-SMA in a dose-dependent manner. There was no significant correlation between the thickness of the collagen gel and the total number of cells. However, there was a significant negative correlation between the thickness of collagen gel and the number of myofibroblasts. CONCLUSIONS: TGF-beta 1 increased the contractile activity of bovine corneal fibroblasts and their ability to differentiate into myofibroblasts. Because contractile activity was correlated with differentiation, the influence of TGF-beta 1 on corneal fibroblast-induced collagen gel contraction may depend on the promotion of myofibroblast differentiation.

Central nervous system action of melatonin on gastric acid and pepsin secretion in pylorus-ligated rats.

We recently demonstrated that centrally administered melatonin at low doses inhibits the induction of gastric lesions by water-immersion restraint stress. To investigate the mechanism of the potent anti-ulcer action of melatonin, the central nervous system (CNS) effects of melatonin on gastric acid and pepsin secretion were studied in conscious pylorus-ligated rats. Intracisternal (i.c.) melatonin (1-100 ng) dose-dependently decreased acid and pepsin output, while a higher i.p. dose (1 microg) had no inhibitory effect. The i.c. melatonin did not change serum gastrin concentrations. Serum melatonin concentrations at 1 and 4 h after i.c. administration of 10-100 ng melatonin did not differ from those in rats receiving i.c. vehicle. The present results suggest that melatonin administered centrally modulates the secretion of gastric acid and pepsin which may explain, at least in part, the protective, anti-stress role of melatonin in the gastric mucosa observed in our previous study.

Central effect of melatonin against stress-induced gastric ulcers in rats.

We investigated the role of melatonin in the induction of gastric lesions induced by water immersion restraint stress or centrally administered thyrotropin-releasing hormone (TRH). Melatonin (0.1-1 ng) injected intracisternally (i.c) 30 min prior to stress dose-dependently inhibited the induction of gastric lesions by water immersion restraint stress, while 100 micrograms/kg, i.p. failed to protect the gastric mucosa. Preadministration of melatonin (1 ng, i.c.) significantly reduced (83%) the severity of gastric lesions induced by a TRH analogue (500 ng, i.c.). Serum melatonin concentrations 30 min after administration of 1 ng melatonin i.c. did not differ from those of rats receiving i.c. vehicle. These results suggest that melatonin plays a protective, anti-stress, role in the gastric mucosa via a mechanism involving the central nervous system.

Efficacy of lansoprazole against peptic ulcers induced by non-steroidal anti-inflammatory drugs: endoscopic evaluation of ulcer healing.

Beyond the obvious step of limiting use of non-steroidal anti-inflammatory drugs (NSAIDs), the treatment of ulcers induced by NSAIDs remains controversial. We evaluated the efficacy of the proton-pump inhibitor lansoprazole on NSAID-induced ulcers. Ulcers were endoscopically diagnosed in 47 NSAID users. These patients received 30 mg/day lansoprazole, orally, for 6 or 8 weeks (6 weeks for duodenal ulcers and 8 weeks for other ulcers). Ulcer healing was assessed using an established classification system. The presence of immunoglobulin G antibody against Helicobacter pylori was also evaluated. The antibody was present in the sera of 51% of patients (24/47). Most of the ulcers reached scarring stages S1 (healing) or S2 (good healing), and the S2 healing rate was 35%. Two H. pylori seropositive patients did not reach these stages; their ulcers were improved by H. pylori eradication therapy, followed, in one case, by medication with misoprostol. Lansoprazole seemed to be useful for most patients with NSAID-induced ulcers, but a few needed additional treatments.

Lansoprazole elevates the ratio of serum pepsinogen I v.s. pepsinogen II.

In order to investigate the mechanism by which proton pump inhibitor increases serum pepsinogen levels, we evaluated the effects of ulcer location and IgG antibody against Helicobacter pylori on lansoprazole-induced elevations. Patients with endoscopically proven peptic ulcer received lansoprazole 30 mg/day for 6 or 8 weeks; pepsinogen I and II levels, along with antibody to H. pylori, were measured in fasting blood samples. We found that whether or not antibody to H. pylori was present, pepsinogen I and II levels and the I/II ratio rose significantly in lansoprazole-treated patients. Patients with stomach-body ulcers showed smaller increases in both pepsinogens than did those with ulcers in the gastric angle/antrum or in the duodenum. In conclusion, lansoprazole increases serum levels of both pepsinogens I and II, although a larger increase in pepsinogen I elevates the pepsinogen I/II ratio. The relatively small increases seen in patients with stomach-body ulcers suggest atrophic changes in the gastric mucosa in patients with stomach-body ulcer.

[Newly developed detection system for serum Helicobacter pylori antibodies using ELISA and its clinical significance].

Using ELISA, a specific and sensitive system that detects serum immunoglobulin G antibodies against all soluble antigens of Helicobacter pylori (Hp) has been developed. This system is used at three different concentrations of Hp antigens coated on the solid phase of the ELISA. Anti-Hp antibodies were judged positive when the difference in the ELISA values between high and middle or middle and low concentrations of antigen were over the specific values. For the ELISA system, called the three point antigen method, sensitivity and specificity of urease activity were 86.9% and 70.8%, respectively. Positive rate for antibodies to serum Helicobacter pylori in dyspeptic patients was higher than that in healthy subjects.