Novel Functional Peptide for Next-Generation Vital Pulp Therapy.

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography-tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

The chemokine receptor CCR5 plays a role in post-traumatic cartilage loss in mice, but does not affect synovium and bone.

OBJECTIVE: C-C chemokine receptor type 5 (CCR5) has been implicated in rheumatoid arthritis and several inflammatory diseases, where its blockade resulted in reduced joint destruction. However, its role in modulating cartilage and bone changes in post-traumatic osteoarthritis (OA) has not yet been investigated. In this study, we investigated changes in articular cartilage, synovium and bone in a post-traumatic OA model using CCR5-deficient (CCR5(-/-)) mice. METHOD: Destabilization of the medial meniscus (DMM) was performed on the right knee of 10-week old CCR5(-/-) and C57BL/6J wild-type (WT) mice to induce post-traumatic OA. The contralateral left knee served as sham-operated control. Knee joints were analyzed at 4-, 8- and 12-weeks after surgery to evaluate cartilage degeneration and synovitis by histology, and bone changes via micro-CT. RESULTS: Our findings showed that CCR5(-/-) mice exhibited significantly less cartilage degeneration than WT mice at 8- and 12-weeks post-surgery. CCR5(-/-) mice showed some altered bone parameters 18- and 22-weeks of age, but body size and weight were not affected. The effect of CCR5-ablation was insignificant at all time points post-surgery for synovitis and for bone parameters such as bone volume/total volume, connectivity density index (CDI), structure model index (SMI), subchondral bone plate thickness, and trabecular bone number, thickness and spacing. CONCLUSION: These findings suggest that CCR5(-/-) mice developed less cartilage degeneration, which may indicate a potential protective role of CCR5-ablation in cartilage homeostasis. There were no differences in bone or synovial response to surgery suggesting that CCR5 functions primarily in cartilage during the development of post-traumatic OA.

Akt phosphorylation in human chondrocytes is regulated by p53R2 in response to mechanical stress.

OBJECTIVE: The p53 tumor-suppressor protein p53R2 is activated in response to various stressors that act on cell signaling. When DNA is damaged, phosphorylation of p53 at its Ser 15 residue induces p53R2 production. The role of p53R2 in chondrocytes remains poorly understood. In this study, we evaluated in chondrocytes, p53R2 expression and its regulation in response to mechanical stress. Furthermore, we investigated the function of p53R2 in relation to mechanotransduction. METHODS: Osteoarthritis (OA) cartilage obtained from total knee replacements and normal cartilage obtained from femoral neck fractures was used to measure p53R2 expression by using immunohistochemistry, western blotting, and real-time polymerase chain reaction (PCR). The OA chondrocytes were subjected to a high magnitude of cyclical tensile strain by using an FX-2000 Flexercell system. Next, sulfated glycosaminoglycan (sGAG) production was quantified in these cells. Protein expression of p53R2, and phosphorylation of Akt, p38MAPK, ERK1/2, and JNK was also detected using western blotting. Moreover, Akt phosphorylation was detected after transfecting the cells with p53R2-specific small interfering RNA (siRNA). RESULTS: Expression of p53R2 was significantly increased in OA chondrocytes and in chondrocytes after applying 5% tensile strain to the cells. However, Akt phosphorylation was down-regulated in OA chondrocytes after the strain, and was up-regulated after transfection of p53R2. sGAG protein as well as collagen type II and aggrecan mRNA was increased following transfection of p53R2-specific siRNA after 5% tensile strain. CONCLUSIONS: p53R2 could regulate matrix synthesis via Akt phosphorylation during chondrocyte mechanotransduction. Down-regulation of p53R2 may be a new therapeutic approach in OA therapy.

Cellular expression of the monocarboxylate transporter (MCT) family in the placenta of mice.

Lactate plays an important role as an alternative energy substrate, especially in conditions with a decreased utility of glucose. Proton-coupled monocarboxylate transporters (MCTs) are essential for the transport of lactate, ketone bodies, and other monocarboxylates through the plasma membrane and may contribute to the net transport of lactate through the placental barrier. The present study examined the expression profile and subcellular localization of MCTs in the mouse placenta. An in situ hybridization survey of all MCT subtypes detected intense mRNA expressions of MCT1, MCT4, and MCT9 as well as GLUT1 in the placenta from gestational day 11.5. The expression of MCT mRNAs decreased in the intensity at the end of gestation in contrast to a consistently intense expression of GLUT1 mRNA. Immunohistochemically, MCT1 and MCT4 showed a polarized localization on the maternal side and fetal side of the two cell-layered syncytiotrophoblast, respectively. The membrane-oriented localization of MCTs was supported by the coexistence of CD147 which recruits MCT to the plasma membrane. However, the subcellular arrangement of MCT1 and MCT4 along the trophoblastic cell membrane was completely opposite of that in the human placenta. Although we cannot exactly explain the reversed localization of MCTs between human and murine placentas, it may be related to differences between humans and mice in the origin of lactate and its utilization by fetuses.

Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta.

We have shown previously that epimorphin (EPM), a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which EPM mediates luminal morphogenesis. Treatment of cells with EPM to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein (C/EBP)beta and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, since constitutive expression of LIP was sufficient to produce lumen formation, whereas constitutive expression of LAP blocked EPM-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which EPM expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for EPM in luminal morphogenesis through control of C/EBPbeta expression.

Immunological analysis in xenogeneic fetal porcine liver transplantation into a beagle.

BACKGROUND/AIMS: While allogeneic organ transplantation has been performed safely, a major barrier in xenogeneic transplantation is how to inhibit hyperacute rejection. METHODOLOGY: We challenged xenogeneic fetal liver transplantation from pig to dog. The graft was investigated by immunohistochemical analysis on recipient's IgG, IgM and C3. RESULTS: In 1 of 4 cases, the graft escaped hyperacute rejection for about 4 hours after transplantation, however, the recipient died next day due to hemorrhage from the torn capsule of the liver due to the arterial blood pressure of the recipient. Histologically, the parenchyma showed good countenance and no congestion nor hemorrhage was shown in the vessels. On immunohistochemical analysis, canine IgG, IgM and C3 were deposited on the sinusoidal epithelium of the fetal liver more moderately than that of adult control. Fetal porcine liver showed less expression of major histocompatability complex class I antigen than that of the adult one. CONCLUSIONS: We consider that the hyperacute rejection occurred more slowly in xenogeneic fetal liver transplantation than in the adult one due to not only less expression of major histocompatability complex class I, but also lower expression of the epitope recognized by a natural antibody of the recipient.

Primary leiomyosarcoma of the jejunal mesentery: report of a case.

The occurrence of a primary leiomyosarcoma of the mesentery is rare. A 61-year-old man was admitted to the hospital complaining of an abdominal mass. The findings of both abdominal ultrasonography and a computed tomography (CT) scan revealed an irregular and heterogeneous mass located in the mesentery. A laparotomy was performed and a 7.0 x 6.5 cm tumor was thus found within the jejunal mesentery. The tumor was successfully resected by a combined resection of 40 cm of the jejunum and end-to-end anastomosis of the jejunum. Pathological examination of the resected specimen revealed leiomyosarcoma. The patient had an uneventful postoperative course, but multiple liver metastases were discovered 1 year and 5 months after the initial operation. A second operation was performed, but the patient died due to hepatic failure and unexpected bleeding from the cut surface of the remnant liver. Preoperative imaging examinations, including abdominal ultrasonography and CT scan, were thus found to be useful tools for both identifying and diagnosing the origin and extension of a mesenteric mass. However, even using such diagnostic techniques an accurate diagnosis of intraabdominal leiomyosarcoma remains difficult.

Meal-related changes in plasma CCK bioactivity in patients with chronic pancreatitis.

In order to clarify whether there is a negative feedback mechanism for CCK secretion, we investigated plasma CCK bioactivity in patients suffering from chronic pancreatitis (CP) according to the characteristics of their pancreatic disease. Basal, meal-stimulated, and integrated release of plasma cholecystokinin (CCK) bioactivity was measured in 24 patients with CP and in 12 healthy controls. The values obtained were compared between the healthy control group and the CP group, and between subgroups of CP patients established on the basis of the presence/absence of several parameters: abnormal gastric emptying, abdominal pain, steatorrhea, pancreatic calcification, insulin-requiring diabetes mellitus, and impairment of pancreatic exocrine functions as indicated by secretin test. A bioassay method using pancreatic acini was used to measure plasma CCK bioactivity. In the control group, plasma CCK bioactivity increased from a basal value of 1.6 +/- 0.7 pmol/L to a maximal increase of 6.6 +/- 4.1 pmol/L, and the integrated CCK release following a test meal was 37.7 +/- 19.3 pmol/L.150 min. In the CP group, plasma CCK bioactivity increased from 1.6 +/- 0.9 pmol/L to a maximal increase of 8.2 +/- 8.7 pmol/L, and the integrated release of CCK was 43.0 +/- 37.7 pmol/L.150 min. None of the differences between them were significant. No significant differences in basal value, maximal increase, or integrated plasma CCK release were noted according to any of the parameters of the CP patients and the control group. Nor was there any correlation between impairment of pancreatic exocrine function and plasma CCK bioactivity. These results provide no evidence of a negative feedback mechanism between pancreatic exocrine dysfunction and CCK secretion.

Erythromycin derivative improves gastric emptying and insulin requirement in diabetic patients with gastroparesis.

OBJECTIVE: To evaluate the effect of the erythromycin derivative EM523L on gastric emptying and postprandial insulin requirement in insulin-dependent diabetic patients with severe gastroparesis. RESEARCH DESIGN AND METHODS: In six IDDM patients with severe gastroparesis (two men and four women, mean age 44.5 years [range 36-53]), the insulin infusion pattern during feedback control with an artificial endocrine pancreas device (Biostator) after intake of a test meal, the retention rate of residual isotope ([99m]Tc-labelled Sn-colloid) in the stomach, and the time-concentration curve of plasma acetaminophen as the marker for liquid emptying were studied with EM523L or a control placebo RESULTS: Time courses of insulin infusion rates peaked within 120 min after intake of the test meal in the EM523L phase, whereas no apparent peak rates were observed in the control phase. The total amount of insulin required in the first 90 min postprandial was significantly greater in the EM523L phase than in the control phase. EM523L significantly decreased the residual isotope ratio in the stomach at > or =50 min postprandial and increased the plasma acetaminophen concentrations at 30-120 min postprandial, compared with respective values in the control phase. CONCLUSIONS: Preliminary results obtained from a small number of patients suggest that EM523L or erythromycin analogs, which have agonistic activity to motilin receptors as well as no antibacterial effect, may be useful to accelerate gastric emptying and improve insulin requirement patterns, thereby establishing more stable glycemic control.

Serum concentrations of soluble HLA-class I and CD8 forms in patients with viral hepatic disorders.

Soluble HLA-class I and CD8 molecules were determined by sandwich ELISA in patients with viral-induced hepatic disorders. As a whole, the patients with hepatic disorders (acute hepatitis: AH; chronic hepatitis: CH; liver cirrhosis: LC; hepatocellular carcinoma: HCC) showed higher sHLA-class I and sCD8 levels than normal controls (P < 0.001). AH patients had the highest sHLA-class I levels (mean, 3513 +/- 2112 ng/ml), followed by CH (2896 +/- 1290 ng/ml), LC (2293 +/- 1266 ng/ml), and HCC (2221 +/- 1212 ng/ml) sCD8 levels wer highest in AH, followed by HCC, LC, and CH, in that order. Among histologically defined C virus-positive patients, sHLA-I levels were higher in those with chronic active hepatitis (CAH) 2A (3802 +/- 1124 ng/ml) than in those with chronic persistent hepatitis (CPH; 2200 +/- 711 ng/ml; P < 0.01), the levels then decreased as the disease progressed (CAH2B, 3564 +/- 1783 ng/ml, LC, 2376 +/- 1265 ng/ml). In contrast, sCD8 values showed little difference among the disorders. sHLA-class I levels showed a positive correlation with sCD8 values both in whole patients and in patients with AH (P < 0.01), but no correlation was shown, in any patients, with biochemical parameters such as GPT and GOT. These findings, taken together, suggest that hepatic destruction is not the only cause of sHLA-class I production, but that sHLA-class I levels, together with sCD8 levels, may reflect immunological activity in hepatic disorders.

A comparative study of adrenocorticotropin-releasing activity of prostaglandins E1, E2, F2 alpha and D2 in the rat.

There is substantial evidence to indicate that prostaglandin (PG) E2 exerts a stimulatory effect on the hypothalamo-pituitary-adrenal axis in rodents. However, little is known regarding the possibility that other PGs play a similar role in regulating the endocrine axis. Therefore, in this study we compared the effects of intravenous administration of PGs E1, E2, F2 alpha and D2 on adrenocorticotropin (ACTH) secretion in conscious male rats. Each PG was administered at two doses of 0.1 and 1.0 mg/kg body weight, and blood samples were collected sequentially up to 120 min postinjection. Although PGD2 was without effect on ACTH secretion at either dose, PGs E1, E2 and F2 alpha all significantly stimulated the hormonal response at both doses. Interestingly, PGs E1, E2 and F2 alpha were largely equipotent in stimulating ACTH release. To the best of our knowledge, this is the first study to demonstrate significant ACTH-releasing activity of intravenously administered PGs E1 and F2 alpha in the rat. These results suggest that PGE2 might not be the only prostanoid playing a role in regulating the hypothalamo-pituitary-adrenal axis and, thus, multiple PGs may be involved in the endocrine axis.

Correlation between pancreatic endocrine and exocrine function and characteristics of pancreatic endocrine function in patients with diabetes mellitus owing to chronic pancreatitis.

CONCLUSION: Pancreatic endocrine capacities are remarkably disturbed in patients with pancreatic diabetes owing to calcific pancreatitis as opposed to those owing to noncalcific pancreatitis. Insulin secretion in calcific pancreatitis resembled that in insulin-dependent diabetes mellitus (IDDM), whereas insulin secretion in noncalcific pancreatitis resembled that in non-IDDM (NIDDM). The involvements of acinar cell and ductal cell function closely correlate with endocrine function (insulin and glucagon secretions) in chronic pancreatitis (pancreatic diabetes). BACKGROUND: We sought to clarify the differences of pancreatic endocrine function between pancreatic diabetes and primary diabetes, and to verify the correlations between pancreatic exocrine and endocrine dysfunction in patients with chronic pancreatitis. METHODS: Urinary C-peptide (CPR) excretion and fasting plasma glucagon levels in patients with pancreatic diabetes owing to calcific pancreatitis (19 cases) and owing to noncalcific pancreatitis (14 cases) were studied in comparison with those in patients with insulin-dependent diabetes mellitus (IDDM, 23 cases), noninsulin-dependent diabetes (NIDDM, 18 cases), and in healthy controls (11 cases). In addition, pancreatic exocrine function was investigated in patients with chronic pancreatitis (calcific and noncalcific) and in healthy controls. The correlation between pancreatic exocrine and endocrine function was studied. RESULTS: The urinary CPR excretion in controls was 94.9 +/- 20.5 micrograms/d. The urinary CPR excretion in calcific pancreatitis was 12.8 +/- 7.4 micrograms/d and it resembled that in IDDM (9.4 +/- 5.8 micrograms/d). The urinary CPR excretion in noncalcific pancreatitis was 41.5 +/- 30.1 micrograms/d, being similar to that in NIDDM (49.3 +/- 21.0 micrograms/d). The plasma glucagon level in calcific pancreatitis was 64.1 +/- 15.9 rho g/mL, which was significantly lower than the values in IDDM (111.2 +/- 50.2 rho g/mL) and NIDDM (96.7 +/- 21.9 rho g/mL). The plasma glucagon level in calcific and noncalcific pancreratitis (88.4 +/- 29.6 rho g/mL) were significantly lower than that in controls (129.8 +/- 21.6 rho g/mL). The residual capacities of acinar cells and ductal cells were strongly correlated with urinary CPR excretion and plasma glucagon concentration.

Xenogeneic (pig to rat) fetal liver fragment transplantation using macrocapsules for immunoisolation.

Acute liver failure caused by viral infection, surgical resection of a large part of the liver or by drug use has a high mortality. For its treatment, hepatocyte or liver tissue transplantation is useful. We report here the beneficial effects of xenogeneic fetal liver fragment (FLF) transplantation with an immunoisolation macrocapsule. The macrocapsules were made of a microporous polypropylene membrane. Pig FLFs (1 mL) was inserted into each capsule to serve as a graft in LEW rats. Acute liver failure was induced by 90% liver resection on day 0. Group 1: transplantation of encapsulated FLF into the omentum 2 days before liver resection (n = 17). Group 2: FLF transplantation into the omentum on day -2 (n = 11). Group 3: liver resection (control) (n = 19). The survival rate, the histology of the grafts and the biochemical parameters [blood sugar (BS), GPT, and GOT] were evaluated. The survival rates of groups 1, 2, and 3 on day 7 were 70.6, 0, and 11.1%, respectively. There were significant differences in BS, GPT, and GOT levels between groups 1 and 3 on day 1 (p < 0.05). On day 28, the histological analyses of the grafts of encapsulated FLFs revealed that the hepatocytes appeared viable, but that the haematopoietic cells had degenerated. Xenogeneic FLFs with macrocapsules survived more than 1 mo, and supported the host's liver function.

Xenogeneic iso-skin graft and mixed lymphocyte reaction studies using HLA-DP transgenic mice.

To elucidate the cellular responses against xeno-MHC antigens, in vitro mixed lymphocyte culture (MLC) and in vivo skin grafting (SG) studies were conducted using HLA-DP transgenic mice (B6-DP mice). Xenogenic iso-(B6-DP to B6 mice) MLC showed positive but much lower responses compared to allo-MLC responses. Nevertheless, B6-DP skin grafts were rejected in a similar time course as allo-skin grafts. To examine mechanisms underlining skin graft rejection, both in vitro cytotoxic lymphocyte (CTL) responses and delayed-type hypersensitivity (DTH) reactions were tested. The studies showed that DTH but not CTL reactions were involved for the graft rejection. SG was again conducted after the administration of anti-CD4 and/or CD8 monoclonal antibody (mAb). Mice treated with both CD4 and CD8 mAb accepted B6-DP SG for as long as up to 60 days and those treated with either CD4 or CD8 mAb alone rejected skin grafts on its own most of the time (75% in anti-CD4 mAb treated mice, 88.9% in anti-CD8 mAb treated mice), which suggests that the strict T cell restrictions for xeno-DP antigens do not exist. Even in these finally rejected cases, longer median survival time and final rejection time were observed, and in the other mice (25% in anti-CD4 mAb treated, and 11.1% in anti-CD8 mAb treated mice), graft acceptance was found. Therefore, it was suggested that the immunological reactions leading to the graft rejection occurs most efficiently when both T cell subsets are present. The above results indicate that xenogeneic HLA-DP antigens could act as significant transplantation antigens equivalent to alloantigens despite their lower stimulative activity in vitro, and also support the interpretation that DP antigens act like a minor histocompatibility antigen beyond the difference of species. Monomorphic anti-HLA class II antibodies were detected in recipients' sera as early as 2 weeks and even at 6 months, indicating that xeno-MHC antigens are prone to be memorized to B cells. It was concluded that HLA transgenic mice are useful for the investigation of cellular responses across xeno-MHC barriers.

Effects of gender and gonadectomy on ACTH response to interleukin-1beta in the rat: comparison with the modulation of ACTH response to immobilization stress.

We examined the influence of gender and gonadectomy on the plasma adrenocorticotropin (ACTH) response to intravenous administration of human recombinant interleukin (IL)-1beta (3 microg/kg) in the rat. For comparison, we also examined whether gender and gonadectomy affect ACTH secretion after immobilization stress (a 30-min period), which is a nonimmunological stressor. IL-1beta induced a significantly higher ACTH response in females than in males, and this sexual difference was abolished by gonadectomy in both sexes. By contrast, ACTH secretion after immobilization was statistically the same in males and females, but tended to be higher in gonadectomized males than in gonadectomized females. These results may suggest a dissociative regulation by gonadal steroids of IL-1beta- and immobilization-induced ACTH responses in the rat. The sexual difference in ACTH response to IL-1beta may represent another example of the sexually dimorphic immunological activity, which is known to be higher in females than in males.

Study of gastric emptying in patients with pancreatic diabetes (chronic pancreatitis) using acetaminophen and isotope.

The gastric emptying function tests were carried out in eight patients with pancreatic diabetes, who were classified into two groups according to the coefficient of variation in the R-R interval in ECG (C.V. R-R) on the normal subjects: < or = the mean - 2SD (the autonomic nerve dysfunction group: AND+ group) and > the mean - 2SD (the autonomic nerve normal group: AND- group). Both the gastric emptying of liquid food by the acetaminophen method and that of solid food by the isotope method were significantly reduced in the AND+ group than in the AND- and normal groups. In addition, a significant correlation was found between the C.V. R-R and the serum acetaminophen concentration (a 45 min value) and the % gastric retention of isotope (a 120 min value). The above results demonstrated that even pancreatic diabetes might be complicated by gastroparesis diabeticorum among autonomic nerve dysfunction. There was a close relation of delayed gastric emptying to the C.V. R-R in ECG or an index of the vagus nerve function.