Richieri-Costa-Pereira syndrome: Expanding its phenotypic and genotypic spectrum.

Richieri-Costa-Pereira syndrome is a rare autosomal recessive acrofacial dysostosis that has been mainly described in Brazilian individuals. The cardinal features include Robin sequence, cleft mandible, laryngeal anomalies and limb defects. A biallelic expansion of a complex repeated motif in the 5' untranslated region of EIF4A3 has been shown to cause this syndrome, commonly with 15 or 16 repeats. The only patient with mild clinical findings harbored a 14-repeat expansion in 1 allele and a point mutation in the other allele. This proband is described here in more details, as well as is his affected sister, and 5 new individuals with Richieri-Costa-Pereira syndrome, including a patient from England, of African ancestry. This study has expanded the phenotype in this syndrome by the observation of microcephaly, better characterization of skeletal abnormalities, less severe phenotype with only mild facial dysmorphisms and limb anomalies, as well as the absence of cleft mandible, which is a hallmark of the syndrome. Although the most frequent mutation in this study was the recurrent 16-repeat expansion in EIF4A3, there was an overrepresentation of the 14-repeat expansion, with mild phenotypic expression, thus suggesting that the number of these motifs could play a role in phenotypic delineation.

Gingival epithelial cells support osteoclastogenesis by producing receptor activator of nuclear factor kappa B ligand via protein kinase A signaling.

BACKGROUND AND OBJECTIVE: Periodontal disease is dental plaque-induced inflammatory disease of the periodontal tissues that results in bone loss in the affected teeth. During bone resorption, receptor activator of nuclear factor kappa B ligand (RANKL) is an essential factor that regulates osteoclastogenesis. Recently, we found that gingival epithelial cells (GECs) in periodontal tissue produce RANKL, the expression of which is regulated by tumor necrosis factor-α and protein kinase A signaling. In this study, we asked whether RANKL-producing GECs induce bone marrow macrophages (BMMs) to form osteoclasts in a co-culture system. MATERIAL AND METHODS: Ca9-22 GECs and osteoclast precursor BMMs were co-cultured with or without the protein kinase A signaling activator forskolin or inhibitor H89 to examine whether the RANKL-producing GECs could be induced to form osteoclasts, as determined using a pit formation assay. RESULTS: Osteoclasts formed spontaneously in co-cultures of Ca9-22 cells and BMMs, even in the absence of RANKL. The cells were cultured on bone slices for 14 d, at which time resorption pits were observed. Forskolin treatment significantly increased osteoclast numbers in these co-cultures, but forskolin alone did not induce osteoclast formation by BMMs. CONCLUSION: GECs producing RANKL are able to support osteoclastogenesis in an in vitro co-culture system using GECs and BMMs, in a process promoted by forskolin.

Differences in the developmental origins of the periosteum may influence bone healing.

BACKGROUND AND OBJECTIVE: The jaw bone, unlike most other bones, is derived from neural crest stem cells, so we hypothesized that it may have different characteristics to bones from other parts of the body, especially in the nature of its periosteum. The periosteum exhibits osteogenic potential and has received considerable attention as a grafting material for the repair of bone and joint defects. MATERIAL AND METHODS: Gene expression profiles of jaw bone and periosteum were evaluated by DNA microarray and real-time polymerase chain reaction. Furthermore, we perforated an area 2 mm in diameter on mouse frontal and parietal bones. Bone regeneration of these calvarial defects was evaluated using microcomputed tomography and histological analysis. RESULTS: The DNA microarray data revealed close homology between the gene expression profiles within the ilium and femur. The gene expression of Wnt-1, SOX10, nestin, and musashi-1 were significantly higher in the jaw bone than in other locations. Microcomputed tomography and histological analysis revealed that the jaw bone had superior bone regenerative abilities than other bones. CONCLUSION: Jaw bone periosteum exhibits a unique gene expression profile that is associated with neural crest cells and has a positive influence on bone regeneration when used as a graft material to repair bone defects. A full investigation of the biological and mechanical properties of jaw bone as an alternative graft material for jaw reconstructive surgery is recommended.

Tumor necrosis factor-α enhances RANKL expression in gingival epithelial cells via protein kinase A signaling.

OBJECTIVE AND BACKGROUND: Periodontitis is an inflammatory disorder of the supporting tissue of teeth, which is composed of gingival soft tissue, cementum covering the tooth root, alveolar bone and periodontal ligament. The receptor activator of nuclear factor kappa B ligand (RANKL) is known to be an essential factor for osteoclastogenesis. Recent clinical studies indicate that levels of RANKL in the gingival crevicular fluid are increased while levels of its decoy receptor, osteoprotegerin (OPG), are decreased in patients with periodontitis. Although the gingival sulcus is composed of gingival tissue, RANKL and OPG expression in gingival epithelial cells is not fully understood. The aim of this study is to investigate the expression of RANKL and OPG in gingival tissue and which factors regulate RANKL expression in gingival epithelial cells. MATERIAL AND METHODS: Reverse transcriptase polymerase chain reaction analysis, western blotting and immunohistochemistry were performed to confirm RANKL and OPG expression in gingival epithelial cells (GECs) and in gingival tissue. Immunostaining was also examined to confirm tumor necrosis factor (TNF)-α and TNF receptor type 1 (TNFR1) expression in gingival tissue. Ca9-22 cells, a human gingival epithelial cell line and human primary GECs were treated with TNF-α. Ca9-22 cells were treated by antibodies against TNF receptors, an inhibitor and an activator of protein kinase A (PKA) signaling and inhibitors of p38, Erk and NF-κB signaling to examine TNF-α-RANKL signaling pathways. RESULTS: RANKL mRNA and protein were expressed in GECs. Immunohistochemistry also showed RANKL expression in gingival tissue. On the other hand, the reverse transcriptase polymerase chain reaction and immunohistochemistry assay showed that GECs did not express OPG. In addition, TNF-α and TNFR1 proteins were expressed in junctional epithelium. TNF-α increased RANKL expression in GECs. TNF-α-induced RANKL expression was inhibited by an antibody against TNFR1 and an inhibitor of PKA signaling. Surprisingly, forskolin, a PKA activator, increased TNF-α-induced RANKL expression. CONCLUSION: RANKL, TNF and TNFR1 were coexpressed in junctional epithelium of gingival tissue. TNF-α induced RANKL expression via TNFR1 and PKA signaling in GECs of junctional epithelium.

Lin28a is a putative factor in regulating cancer stem cell-like properties in side population cells of oral squamous cell carcinoma.

Cancer stem cells (CSCs) are among the target cells of cancer therapy because they are uniquely involved in both cancer progression and sensitivity to chemotherapeutic agents. We identified side population (SP) cells, which are known to be an enriched population of CSC, in five oral squamous cell carcinoma (OSCC) cells (SCC9, SCC25, TOSCC7, TOSCC17, and TOSCC23). The percentages of SP cells ranged from 0% to 3.3%, with TOSCC23 cells showing the highest percentages of SP cells (3.3% of the total cell population). The SP cells isolated from TOSCC23 cells also showed greater cell proliferation and invasion compared to non-SP (MP) cells. Therefore, our initial findings suggested that SP cells were enriched for CSC-like cells. Furthermore, DNA microarray analysis revealed that the expression of cell proliferation-related and anti-apoptotic genes was greater in SP cells compared to MP cells. We focused on Lin28a, which showed the highest expression (approximately 22-fold) among the upregulated genes. The overexpression of Lin28a in TOSCC23 cells increased their proliferation, colony formation, and invasion. These findings suggest that Lin28a is an appropriate CSC target molecule for OSCC treatment.

The distribution and expression of S100A8 and S100A9 in gingival epithelium of mice.

OBJECTIVE AND BACKGROUND: Gingival epithelium protects against bacterial infection by producing antimicrobial peptides such as calprotectin. Calprotectin consists of proteins S100A8 and S100A9. Although in vitro assay has shown that neutrophils and gingival epithelial cells express calprotectin, the expression of S100A8 and S100A9 and colocalization of both S100 proteins in gingival tissue in vivo are not fully understood. The aim of this study was to investigate the distribution of S100A8 and S100A9 expression in gingival epithelium of mice in the presence and absence of infection. MATERIALS AND METHODS: A quantitative analysis of S100A8 and S100A9 mRNA in junctional epithelium (JE) and oral gingival epithelium (OGE) of both germ-free mice and conventional mice was performed using laser microdissection and real-time polymerase chain reaction (PCR). Confirmation of S100A8 and S100A9 mRNA expression in the JE was conducted by fluorescent immunohistochemistry. RESULTS: Real-time PCR analysis indicated that S100A8 and S100A9 expressions were mainly detected in JE and only slightly or not detected in OGE. Levels of both S100A8 and S100A9 mRNA expression in JE of conventional mice were significantly higher than those in JE of germ-free mice. Additionally, fluorescent immunohistochemistry showed that S100A8 expression was observed in the JE of both conventional and germ-free mice, whereas S100A9 was expressed in the JE of conventional but not germ-free mice. CONCLUSION: S100A8 protein is expressed in JE cells of mice in the presence and in the absence of infection with oral bacteria. S100A9 expression in JE cells in the presence of microflora is significantly increased compared with the absence of microflora, which suggests that S100A9 expression may be induced by infection of microflora. The production of calprotectin in gingival epithelial cells may be mediated through S100A9 induction by bacterial infection.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up-regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin-1 receptor antagonist-deficient mice.

BACKGROUND AND OBJECTIVE: The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS: Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 µg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. RESULTS: The levels of IL-1α, IL-1ß, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice. CONCLUSION: IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.

Role of the junctional epithelium in periodontal innate defense and homeostasis.

BACKGROUND AND OBJECTIVE: The junctional epithelium provides the front-line defense against periodontal bacterial infection. The migration of neutrophils into the junctional epithelium might represent a protective reaction against bacterial infections. However, neutrophils penetrate into the junctional epithelium even under sterile conditions. In this study, we analyzed and compared the number of neutrophils and the cytokine expression related to neutrophil migration in the junctional epithelium in conventional and germ-free mice. MATERIAL AND METHODS: Germ-free and conventional ICR mice were used at 12 wk of age. Frozen sections were used for the detection of Gr-1, macrophage inflammatory protein-2 (MIP-2/CXCL2) and proliferating cell nuclear antigen-positive cells in the two groups of mice. Laser capture microdissection and RT-PCR analysis were used to evaluate the expression of keratinocyte-derived chemokine (KC/CXCL1), MIP-2, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) mRNAs in the two groups of mice. RESULTS: Morphometric examination indicated an increase in the area of the junctional epithelium upon bacterial infection. Immunohistochemical studies also detected an increased number of neutrophils in the junctional epithelium upon bacterial infection. Higher up-regulation of KC and MIP-2 were detected in the junctional epithelium of conventional mice than in germ-free mice, whereas the expression of Il-1ß and Tnfα mRNAs was not affected. CONCLUSION: Junctional epithelium cells constitutively expressed several types of chemokines and cytokines and the expression of chemokines was augmented by bacterial infection. Therefore, the constitutive expression of cytokines in junctional epithelium might be related to the morphological and functional homeostasis of the junctional epithelium in addition to the defense against the bacterial infection.

Characteristic genes in luminal subtype breast tumors with CD44+CD24-/low gene expression signature.

OBJECTIVE: Breast cancer cells with CD44+CD24-/low gene expression signature have been suggested to have stem cell-like tumor-initiating properties. The purpose of this study is to clarify the gene expression profiling of cells with CD44+CD24-/low gene expression signature in the luminal subtype. METHODS: Laser capture microdissection was used to select the isolation of cancer cells in 35 frozen tissues of breast cancer, and RNA extracted from these cells was examined by real-time RT-PCR to quantify CD44 and CD24 expressions. Human stem cell RT(2) Profiler PCR Array was used for gene expression analysis in the groups of CD44+CD24-/low and CD44+CD24+ gene expression signature. RESULTS: Thirty-five tumors were divided into 3 groups. Group A was composed of the CD44+CD24-/low type, in which the ratio of CD44/CD24 was >10.0. Group B was composed of the CD44+CD24+ type, in which the ratio was >0.1 and ≤10.0. In group C, composed of the CD44-/lowCD24+ type, the ratio was <0.1. The number of tumors in groups A, B, and C were 5, 28, and 2, respectively. Regarding the correlation of CD44/CD24 status with tumor characteristics, the tumors of group A were significantly associated with axillary lymph node metastasis compared with those of group B (p = 0.033). There were no significant differences in tumor size, nuclear grade, or HER2 status between the two groups. According to signaling pathways, the number of expression genes for the Notch pathway in group A was significantly greater than in group B (p = 0.028). Overexpressed genes for ALDH1 (p = 0.021) and SOX2 (p = 0.018) were noted in group A compared to group B. CONCLUSION: This study suggests that the Notch pathway may be an important signaling pathway in luminal subtype with CD44+CD24-/low gene expression signature. In addition, either ALDH1 or SOX2 may be a candidate marker for cancer stem cells in luminal subtype breast cancer.

Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis.

BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. MATERIAL AND METHODS: A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry. RESULTS: The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. CONCLUSION: We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.

Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor.

There is now evidence that the immune system, during times of infectious challenge, can stimulate the secretion of glucocorticoids, the adrenal steroids that mediate important aspects of the response to stress. Specifically, secretion of interleukin-1 (IL-1), a monocyte lymphokine secreted after infection, appears at least in part responsible for this effect. Glucocorticoids are secreted in response to a neuroendocrine cascade involving, first, the brain, then the pituitary, and finally the adrenal gland. In this report, human IL-1 is shown to activate the adrenocortical axis at the level of the brain, stimulating the release of the controlling hormone corticotropin-releasing factor (CRF) from the hypothalamus. Infusion of IL-1 induced a significant secretion of CRF into the circulation exiting the hypothalamus, whereas immunoneutralization of CRF blocked the stimulatory effect of IL-1 on glucocorticoid secretion. IL-1 appeared to have no acute direct stimulatory effects on the pituitary or adrenal components of this system. Furthermore, IL-1 did not cause a nonspecific release of other hypothalamic hormones. Thus, the lymphokine acts in a specific manner to activate the adrenocortical axis at the level of the brain; this effect appears to be unrelated to the known pyrogenic effects of IL-1 within the hypothalamus.

Impact of metabolic syndrome and C-reactive protein on outcome after coronary stenting.

Metabolic syndrome (MS) identifies cardiovascular risk; however, there is little information regarding the evolution of patients with MS after stent implantation. The aim of this single-center study is to evaluate the possible association between MS and clinical restenosis, after adjustment for highsensitivity C-reactive protein (hs-CRP) and angiographic predictors of restenosis. In a longitudinal study, 159 patients (89 with and 70 without MS) were studied. Criteria for MS were: elevated blood pressure (systolic >or=130 mmHg, diastolic >or=85 mmHg or drug treatment for hypertension; elevated fasting glucose (>100 mg/dl) or drug treatment for elevated glucose; reduced HDL-cholesterol (<40 mg/dl in men and <50 mg/dl in women) or drug treatment for reduced HDL-cholesterol; elevated triglycerides (>or=150 mg/dl) or drug treatment for elevated triglycerides; and obesity (body mass index >28.8 kg/m2). The primary end point was the rate of major adverse clinical events (MACE): cardiovascular death, myocardial infarction, or target lesion revascularization (TLR) during the 12-month follow-up period. The secondary end point was the rate of TLR. MS was neither identified as predictor of MACE [hazard ratio (HR): 0.844; 95% CI: 0.41-1.74; p=0.648], nor TLR (HR: 1.05; 95% CI: 0.44-2.50; p=0.91), even when controlled for hs-CRP levels and angiographic predictors of restenosis. Also, no significant interaction between MS and hs-CRP was found (p=0.135 and p=0.194, for MACE and TLR, respectively). This study shows that patients with MS do not have an additional risk of MACE, even when controlled for angiographic predictors of restenosis and hs-CRP.

An animal model of intrinsic dental erosion caused by gastro-oesophageal reflux disease.

OBJECTIVES: To explore the association between dental erosion and gastro-oesophageal reflux disease (GORD), we used an animal model of GORD. MATERIALS AND METHODS: We performed an operation to force gastro-duodenal contents reflux in male Wistar rats, and examined the teeth in the reflux rats at 15 or 30 weeks postoperatively. Dental erosion was evaluated based on a slightly modified index from a previous report. Estimation of pH was employed in the oesophageal and gastric contents. RESULTS: Macroscopically, dental erosion was only detected in the reflux rats. Histopathologically, dentin exposure was detected in three of the seven cases after 30 weeks. Alveolar bone destruction and osteomyelitis were also noted in severe cases. The pH of the oesophageal and stomach contents was 6.93 +/- 0.15 and 3.7 +/- 0.39, respectively. CONCLUSIONS: We confirmed the relationship between dental erosion and GORD. First step of dental erosion caused by GORD is the loss of surface enamel induced by regurgitation of an acidic liquid and acidic gas. Subsequently, further destruction of dental hard tissues and tooth supporting structure is accelerated by mixed juice with gastric and duodenal contents. The reflux animal model is a useful tool to examine the mechanism of dental erosion in GORD.

Association of the 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene with unstable angina.

The 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene consists of the substitution of a guanine base by a thymine at the 894th nucleotide of the gene. An association of this polymorphism with acute coronary syndromes has been described, only when in combination with other polymorphisms of this gene. The aim of the present study was to search for an association between this polymorphism and unstable angina in a southern Brazilian population. In a case-control study, 156 patients (group 1 (N = 83): unstable angina, group 2 (N = 73): stable angina) were genotyped by PCR and digestion of the product. Univariate analysis demonstrated that the minimal luminal diameter and the degree of stenosis of the culprit lesion differed between groups (P = 0.006 and 0.005, respectively). In addition, the frequencies of the T allele and of the T allele carriers (combined TT and TG genotypes) were significantly higher in the group with unstable angina (41.6 vs 28.8%; P = 0.025, Pearson chi-square test, and 73.5 vs 45.2%; P = 0.001, Pearson chi-square test, respectively). Multivariate logistic regression showed that the frequency of the T allele carriers was the only variable with a predictive value for unstable angina, when controlled for the other variables (6.1 (95% CI = 2.55-14.43); P < 0.001). Thus, in a homogenous group of patients, the endothelial constitutive nitric oxide synthase 894G>T polymorphism was associated with unstable angina. We suggest that this polymorphism may be a genetic risk factor for unstable angina.

rh-BMP2-induced ectopic bone for grafting critical size defects: a preliminary histological evaluation in rat calvariae.

The aim of this study was to evaluate histologically the biological usefulness of recombinant human BMP2 (rh-BMP2)-induced ectopically-formed bone as graft material to repair a bone defect model, compared with autogenous bone and frozen allogeneic bone. Forty-five male Wistar rats were used, which were divided into three graft groups. Each of the three graft groups was divided into three observation period groups (3, 6 and 9 weeks after graft). All rats underwent craniotomy to create a bone defect, and then received a bone graft. In the rh-BMP2-induced ectopic bone graft group, marked bone formation was seen from 3 weeks after graft. In the autogenous bone graft group, marked bone formation was seen from 6 weeks after graft. In the group that received a frozen allogeneic bone graft, marked bone formation was seen from 9 weeks after graft. At 3, 6 and 9 weeks after graft, newly formed bone area was significantly greater in the tissue engineered bone (TEB) group than in the auto or frozen allogeneic bone (FAB) group. rh-BMP2-induced ectopically-formed bone graft exhibited better osteoconductivity than autogenous bone graft and frozen allogeneic bone graft. These histological findings indicate that rh-BMP2-induced ectopically-formed bone is suitable as bone graft material.

Case Report of Extensive Isolated Spontaneous Celiac Trunk Dissection After Liver Transplantation.

Arterial dissection is a rare complication after liver transplantation (LT). We report a case of extensive isolated spontaneous celiac trunk dissection (ISCTD) up to the proper hepatic artery, left gastric artery, and splenic artery after living donor liver transplantation. A 48-year-old woman with cryptogenic liver cirrhosis underwent living donor liver transplantation. Intraoperative and postoperative Doppler ultrasound revealed sufficient flow in the hepatic artery, portal vein, and hepatic vein. On postoperative day (POD) 10, Doppler ultrasound showed reduction of hepatic arterial flow. On POD 16, a contrast-enhanced computed tomography scan showed that the ISCTD extended to the proper hepatic artery, left gastric artery, and splenic artery with an entry tear on the proximal side of the celiac trunk. Although the computed tomography scan showed ischemia of a small part of the liver, blood flow to the liver was kept to some extent. Because all false lumens were occluded by thrombi and the liver enzyme levels normalized, we chose conservative therapy with antiplatelet agents. The patient was discharged on POD 53. She remains well without any liver dysfunction after 18 months with reduction in all false lumens and a patent hepatic artery. Several cases of ISCTD have been reported apart from LT, most of which were treated with conservative therapy. We conclude that conservative therapy could be the first choice in ISCTD even after LT.

Single-channel recordings of TREK-1 K+ channels in periodontal ligament fibroblasts.

The periodontal ligament (PDL) works as a suspensory ligament when external mechanical stress is placed on the teeth. PDL fibroblasts, the principal cells in the PDL, are responsible for many PDL functions. We hypothesized that mechanosensitive ion channels are present in human PDL fibroblasts, which are capable of responding to mechanical stress during normal function of the tissue. Using patch-clamp techniques, we detected mechanosensitive TREK-1 K+ channels (a member of the two-pore-domain K+ channel family), whose single-channel conductance was 104 pS in symmetrical K+-rich solutions. The open probability of the channel was low in the quiescent state, but it was strongly increased by the induction of membrane stretch. Arachidonic acid also enhanced the channel activity. RT-PCR and immunocytochemical observations showed the expression of TREK-1 K+ channels in PDL fibroblasts. The results suggest that the activation of TREK-1 K+ channels by masticatory stress contributes to the hyperpolarization of PDL fibroblasts.

Stable maintenance of monkey embryonic stem cells in the absence of bFGF.

Monkey embryonic stem (ES) cells are useful tools in preclinical studies of gene therapy and tissue engineering as well as in primate developmental biology. However, their maintenance is not easy, requiring addition of bFGF to the medium. Herein, we have described a stable, cost-effective method that does not require bFGF. We used a high-density (1 to 1.5x10(5) cells/cm2) of mouse embryonic fibroblasts (MEF) as feeder cells to successfully maintain undifferentiated monkey ES cells for 2 years (approximately 150 passages). Furthermore, these ES cells were competent for electroporation of enhanced green fluorescent protein (EGFP) and subsequent drug selection procedures. We were able to establish EGFP-expressing cell lines using this culture condition. These cell lines expressed undifferentiated markers, such as alkaline phosphatase, SSEA-4, TRA-60, and TRA-81. In addition, strong EGFP expression was observed after differentiation into cardiomyocytes, neurons, or adipocytes, suggesting that these cell lines are a useful tool to study cell transplantation. This method simplifies the culture of monkey ES cells.

Effects of synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines, neurohypophysial peptides, and other substances on cultured corticotropic cells.

Synthetic ovine corticotropin-releasing factor (CRF) is a 41-residue peptide with high potency and intrinsic activity to stimulate the secretion of ACTH and beta-endorphin-like immunoactivity (beta-End-LI) by cultured adenohypophysial corticotropic cells. The action of CRF in vitro can be potentiated by the weaker secretagogues, vasopressin, oxytocin, epinephrine, norepinephrine, and angiotensin II. CRF-mediated secretion of ACTH and beta-End-LI is noncompetitively inhibited by pretreatment of cells with glucocorticoids. Long term exposure of adenohypophysial cells to CRF results in an increase in total medium plus cell ACTH in the cultures, suggesting that CRF can enhance rates of ACTH synthesis as well as release. CRF also stimulates the secretion of beta-End-LI by corticotropic cells cultured from the neurointermediate lobe. Higher concentrations of CRF are required to stimulate secretion by this cell type than by anterior lobe corticotropic cells. These in vitro results are consistent with CRF playing a major physiological role in the neuroregulation of secretion by anterior lobe corticotropic cells, where the peptide may interact with other modulators.