Mineral trioxide aggregate suppresses pro-inflammatory cytokine expression via the calcineurin/nuclear factor of activated T cells/early growth response 2 pathway in lipopolysaccharide-stimulated macrophages.

AIM: To elucidate mechanisms by which mineral trioxide aggregate (MTA) suppresses pro-inflammatory cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. METHODOLOGY: Mineral trioxide aggregate extracts were prepared by immersing set ProRoot MTA in culture medium. RAW264.7 cells were cultured in the presence of LPS and MTA extracts. mRNA expression levels of interleukin (IL)-1α, IL-6, early growth response 2 (Egr2), suppressor of cytokine signalling 3 (Socs3) and IL-10 were quantified with reverse transcription-quantitative polymerase chain reaction. Phosphorylation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells was analysed by Western blotting. Intracellular calcium imaging was performed with Fluo-4 AM. The activity of nuclear factor of activated T cells (NFAT) was determined by luciferase assays. Enforced expression and silencing of Egr2 in RAW264.7 cells were carried out using an expression vector and specific RNAi, respectively. In vivo kinetics of Egr2+ cells in MTA-treated rat molar pulp tissues were examined using immunohistochemistry. Data were analysed by one-way analysis of variance, followed by the Tukey-Kramer test (P < 0.05). RESULTS: Exposure to MTA extracts resulted in reduced mRNA expression levels of IL-1α and IL-6, as well as reduced expression of phosphorylated NF-κB, in LPS-stimulated RAW264.7 cells. Exposure to MTA extracts induced Ca2+ influx, which was blocked by NPS2143, an antagonist of calcium-sensing receptor (CaSR); Ca2+ influx then triggered activation of calcineurin/NFAT signalling and enhanced mRNA expression of Egr2. Enforced expression of Egr2 in RAW264.7 cells promoted the expression of both IL-10 and Socs3. In vivo application of MTA onto rat molar pulp tissue resulted in the appearance of Egr2-expressing cells that coexpressed CD163, a typical M2 macrophage marker. CONCLUSIONS: Mineral trioxide aggregate extracts induced downregulation of IL-1α and IL-6 in LPS-stimulated RAW264.7 cells via CaSR-induced activation of calcineurin/NFAT/Egr2 signalling and subsequent upregulation of IL-10 and Socs3.

Preoperative administration of rebamipide significantly lowers body temperature and circulating interleukin-6 in gastric cancer patients after gastrectomy.

BACKGROUND/AIMS: It has recently been reported that rebamipide (OPC-12759) inhibits inflammatory cytokines and activation of neutrophils. The aim of the present study was to investigate the effects of preoperative administration of rebamipide on parameters of systemic inflammatory response syndrome (SIRS) and serum levels of inflammatory cytokines in gastric cancer patients after gastrectomy. PATIENTS AND METHODS: We measured the parameters of SIRS, circulating cytokines and acute phase reactants in patients after (i). distal gastrectomy with D2 lymph node dissection (group 1, n = 10); (ii). distal gastrectomy with D2 lymph node dissection following administration of rebamipide (group 2, n = 10), and (iii). laparoscopy-assisted distal gastrectomy (LADG) with D1 lymph node dissection (group 3, n = 10). Group 2 was administered 100 mg of rebamipide 3 times/day after meals for 7 days before surgery. RESULTS: Among the parameters of SIRS, a difference was observed in body temperature on day 3. The mean body temperature of group 2 was significantly lower than group 1 (p = 0.006), and was similar to group 3. In proinflammatory cytokines, a significant difference was observed in the serum levels of interleukin (IL)-6. On day 1 the IL-6 levels of group 2 were significantly lower than those of group 1 (p < 0.001). The changes in the IL-6 level of group 2 were similar to group 3, except in the very early postoperative phase. However, other proinflammatory cytokines, such as IL-8 and IL-10, were not detected, and there was no difference in C-reactive protein between the 3 groups. CONCLUSION: Preoperative administration of rebamipide significantly decreased postoperative body temperatures and circulating IL-6 in gastric cancer patients after gastrectomy to levels similar to those of patients with LADG.

Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3).

Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1alpha,25-dihydroxyvitamin D(3). We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50-55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation.

Nd:YAG laser influence on sound dentin bond strength.

OBJECTIVE: In the present study, the authors evaluated tensile bond strength of composite resin to dentin treated with Nd:YAG laser before and after bonding procedures. SUMMARY BACKGROUND DATA: Lasers have been widely used in dentistry and have contributed to the development of new technologies. Adhesive systems have been indicated for most dental procedures and have produced good results. Studies concerned with the combined use of this adhesive system was the subject of this study. METHODS: Thirty noncarious human anterior teeth, freshly extracted, for periodontal reasons, were used. After grinding the buccal dentinal surface until its exposition, specimens were separated into three groups and received the following treatments: (group 1) control--acid etching plus primer plus bond; (group 2) laser plus acid etching plus primer plus bond; (group 3) acid etching plus primer plus bond plus laser. Specimens of composite resin (Z100, 3M, Saint Paul, MN) were constructed on an inverted truncated 3-mm diameter cone mold. Tensile bond strength was performed using Inströn Universal machine, at 0.5 mm/min speed. RESULTS: Analysis of variance (ANOVA) (p < 0.05) determined that the type of dentinal treatments used had an influence on tensile bond strength. Tukey's test, however, showed that group 1 (15.46) and 3 (15.67) had similar results both of which were higher than group 2 (4.57). CONCLUSIONS: Based on the results obtained, one can conclude that group 1 (without laser) and group 3 (laser after bonding) had similar results, both higher than those observed for group 2 (laser before bonding). These results indicate that more research is needed about how a hybrid layer is formed when laser radiation is used.

[Clinical evaluation of Azasetron Hydrochloride: a new selective 5-HT3 receptor antagonist--antiemetic profile and plasma concentration in transcatheter arterial chemoembolization using CDDP for unresectable hepatocellular carcinoma].

We performed a clinical evaluation on the antiemetic profile and the plasma concentration of Azasetron Hydrochloride (a new selective 5-HT3 receptor antagonist), in transcatheter arterial chemoembolization using CDDP for unresectable hepatocellular carcinoma. Antiemetic effects were examined in 32 patients in the serotone group (administration of serotone 10 mg + methylprednisolone 125 mg) and in 77 patients of the control group (administration of metoclopramide 20-30 mg + methylprednisolone 500 mg). The response rate and the CR ratio in serotone group was 97% and 66%, respectively. These results were statistically higher than in the control group. Although all patients had chronic liver diseases, no side effects and complications related to administration of serotone were observed. The average area under the concentration (AUC) curve of plasma serotone in five patients with liver cirrhosis was 531 ng.h/ml, which was greater than that of a healthy volunteer. In conclusion, serotone is a new, safe and useful antiemetic drug in TACE therapy for hepatocellular carcinoma.

Positions of disulfide bonds in yam (Dioscorea japonica) acidic class IL (class IV) chitinase.

Yam acidic class I chitinase belongs to a low molecular weight subclass of class I (class IL; corresponds to class IV) chitinase. The positions of disulfide bonds in this chitinase were examined. Chitinase protein was digested with acid protease and thermolysin, and the resulting disulfide bond containing peptides were separated by reversed-phase HPLC and detected using the SBD-F (7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid ammonium salt) method. Four intradisulfide bonds containing peptides were purified and three disulfide bonds in the catalytic domain were identified as Cys-66 and Cys-115, Cys-128 and Cys-136, and Cys-218 and Cys-250. Location of disulfide bonds in the catalytic domain was identical to that of barley class II chitinase but different from rye class II chitinase at the C-terminal. Conservation of S-S bonds at the N-terminal half of the catalytic domain between class I and class II chitinases strongly suggests that this region is important for formation of the active site.

[Pulmonary lymphangiomyomatosis with tuberous sclerosis and endolymphatic stromal myosis of the uterus].

Pulmonary lymphangiomyomatosis is a rare disorder that affects only women of reproductive age. The lung shows numerous cystic changes and striking proliferation of smooth muscle around the airways, blood vessels, and lymphatic channels. We encountered a patient with pulmonary lymphangiomyomatosis who had repeated pneumothorax and bloody sputum over 10 years. The patient also had other lesions, including intracranial calcification, renal angiomyolipoma and endolymphatic stromal myosis of the uterus. The first two findings are known as tuberous sclerosis, and lymphangiomyomatosis is considered to be a form fruste of tuberous sclerosis. However, we know of no previous reports of uterine lesions in this disease. Lymphangiomyomatosis may be a systemic disorder of smooth muscle.

Resident gynecologists and total hysterectomy.

We examined the specific number of surgeries necessary for a three-year obstetrics and gynecology resident to acquire proficiency in two types of hysterectomies. Improvement in the technical skills of the residents was assessed using surgical time and blood loss, and resected tumor weight was chosen as the factor representing the difficulty of the surgery. Regarding abdominal total hysterectomy (ATH), early residents (less than 25 ATH experience) performed relatively easier surgeries, and improvement in technical skill was manifested as reduced blood loss by mid residents (25 to 49 ATH) and as shortened surgical time by later residents (75 or more ATH). Regarding vaginal total hysterectomy (VTH), blood loss for earlier residents (less than 15 VTH) was greater than that for the staff, and there was a significant difference between staff surgical time and that for each resident group. These data suggest that performing more than 75 ATH during the residency period of three years is adequate to establish proficiency in this type of surgery, but that the execution of 25 VTH is insufficient and that residents require more training to learn VTH.

Tissue-type plasminogen activator predicts endocrine responsiveness of human pancreatic carcinoma cells.

BACKGROUND: Estrogen receptor (ER) has been found in human pancreatic carcinoma, but the potential benefit of endocrine therapy never has been assessed adequately. The aim of this study was to determine whether the presence of ER can be used as an indicator of hormone responsiveness, and whether modulation of tissue-type plasminogen activator (t-PA) by ER can identify hormone-responsive pancreatic carcinomas. METHODS: The authors investigated ER status and hormonal regulation of t-PA in nine human pancreatic carcinoma cell lines, AsPC-1, BxPC-3, Capan-1, Capan-2, Hs-700T, Hs-766T, MiaPaCa-2, PANC-1, and SUIT-2. Furthermore, to examine whether estrogen dependency of t-PA production in pancreatic carcinoma cells correlated with responsiveness to endocrine therapy, the in vivo effects of various endocrine agents on the growth of the nine pancreatic cell lines transplanted into nude mice were examined. RESULTS: In a 17 beta-estradiol (E2)-binding assay, three of the nine pancreatic carcinoma cell lines (BxPC-3, Capan-2, and MiaPaCa-2) contained measurable levels of estradiol binding sites in vitro and in vivo using tumors transplanted into nude mice. Although t-PA was present in the culture medium in eight of the nine pancreatic carcinoma cell lines (not in Hs-700T), t-PA production was regulated by estrogen via an ER system in vitro only in the Capan-2 cell line. E2 injection into tumor-bearing mice showed acceleration of tumor growth only in Capan-2 tumors. Administration of a competitive ER antagonist, toremifene, and a luteinizing hormone-releasing hormone analogue, leuprorelin acetate (LEU), to Capan-2-bearing mice significantly reduced the rate of tumor growth, although there was no actual shrinkage of tumor mass. These agents failed to exert any antitumor effect on the other eight pancreatic cell lines. Although aromatase inhibitors, CGS 20267 and vorozole did not modify the in vivo growth of the nine pancreatic carcinoma cell lines significantly, the combined use of aromatase inhibitors with LEU exhibited a synergistic antitumor effect on Capan-2-bearing mice. Medroxyprogesterone acetate treatment significantly reduced the tumor volume of Capan-2 and also caused delayed growth in two other cell lines, AsPC-1 and MiaPaCa-2. CONCLUSIONS: The estrogen dependency of t-PA production may clarify the functional state of ER in human pancreatic carcinoma cells. This finding may aid in planning endocrine therapy for patients with this lethal cancer.

Immunoreactive hepatocyte growth factor is a strong and independent predictor of recurrence and survival in human breast cancer.

Hepatocyte growth factor (HGF) is a stromally derived modulator of epithelial cell proliferation and motility. In the present study, we have measured immunoreactive (ir)-HGF concentration in tumor extracts of 258 primary human breast cancers using an enzyme-linked immunosorbent assay and have evaluated its association with disease-free and overall survival. The median value of ir-HGF concentration was 11.0 ng/100 mg protein (range, 1.4-566.7 ng/100 mg protein). Correlation analyses between ir-HGF concentration and clinicopathological factors showed that the ir-HGF level was correlated only with tumor size (P = 0.05). No significant associations were found between ir-HGF content and age, menopausal status, nodal status, histological type, histological grade, vessel involvement, estrogen receptor, progesterone receptor, type of surgery, or postoperative adjuvant therapy. Breast cancer patients with high ir-HGF concentration had a significantly shorter relapse-free (P = 0.001) and overall survival (P = 0.001) rate when compared to those with low ir-HGF concentration at the cutoff point of 21.7 ng/100 mg protein, which was determined in another group of 82 patients. In multivariate analysis, ir-HGF level was found to be the most important independent factor in predicting relapse-free and overall survival, of greater import than lymph node involvement. The putative role of HGF in breast cancer growth and metastasis is hereby strengthened.

The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase.

The complete amino acid sequence of acidic chitinase from yam (Dioscorea japonica) aerial tubers was determined. The protein is composed of a single polypeptide chain of 250 amino acid residues and has a calculated molecular mass of 27,890 Da. There is an NH2-terminal domain, a hinge region, and a main structure, typical for class I chitinases (Shinshi, H., Neuhaus, J.-M., Ryals, J., and Meins, F., Jr. (1990) Plant Mol. Biol. 14, 357-368). We have obtained the first evidence for an acidic class I chitinase. Comparison with sequences of other class I chitinases revealed approximately 40% sequence similarity, a value lower than that for other class I chitinases (70-80%). We assume that there is a local conformational change in the molecule; cysteine residues that probably form disulfide bonds are completely conserved, with the exception of Cys-178. The difference in structure between this chitinase and other basic class I chitinases suggests that acidic and basic isoforms should be grouped into subclasses; this protein is an ethylene- or a pathogen-independent chitinase produced by a gene that is inherent in the tuber.

Amino acid sequence of the N-terminal domain of yam (Dioscorea japonica) aerial tuber acidic chitinase. Evidence for the presence of a wheat germ agglutinin domain in matured acidic chitinase from unstressed tuber.

The amino acid sequence of the N-terminal domain of acidic chitinase from unstressed aerial tuber was determined and proved the presence of an N-terminal domain in acidic chitinase. The amino acid sequence was determined on a pyroglutamylaminopeptidase-treated N-terminal fragment of V8 protease and on chymotryptic peptides of this fragment. The sequence determined revealed 8 residues deletion and 2 residues insertion as compared with the N-terminal domain of tobacco basic chitinase. The N-terminal domain determined showed a homology of 40% and 52% with the N-terminal domain of tobacco basic chitinase and wheat germ agglutinin, respectively.

[Lectin binding of membrane proteins from human placental villi].

The binding of membrane proteins from human placental villi to lectins such as concanavalin A (Con A), wheat germ agglutinin (WGA) and Riccinus communis agglutinin (RCA) was examined. Five major components of 66K, 69K, 100K, 130K and 170K dalton showed specific binding to WGA, whereas two other major components of 43K and 40K dalton had only weak binding. There were no membrane proteins which bound specifically to either Con A or RCA. The WGA-Sepharose column is considered useful for the purification of human placental membrane proteins. Although the function of these proteins is still unknown, a diagnostic method not only for determining the pathological state of the placenta, but also for cancer will possibly be developed by taking advantage of them.

[Studies on immunotherapy of uterine cervical cancer by administration of schizophyllan (SPG) (author's transl)].

This study was designed to investigate effects of immunopotentiator on cervical cancer. We administered schizophyllan (SPG) as immunopotentiator to patients of cervical cancer therapy with irradiation, and examined successively immunological parameters such as blastformation of lymphocytes, skin reaction of PPD, lymphocyte counts, concentration of serum immunoglobulins, and other clinical inspections. Moreover we examined ability of interferon induction of SPG. We also calculated and compared 3 year relative survival rate of the cases administered SPG and historical controls therapy with only irradiation. In patients administered SPG the results revealed: 1) an enhancement of stimulation index of lymphocytes blastformation with PHA, 2) an enlargement of skin reaction to PPD, 3) no effects to number of lymphocytes, serum immunoglobulins, liver function and renal function, 4) an interferon activity on patients' sera of cervical cancer after administration of SPG, and 5) patients administrated SPG had significantly high relative survival rate in comparison with the historical control of cervical cancer cases treated solely by irradiation.

Clinical and histopathological study of carcinoma of the corpus uteri.

Clinical and histopathological investigations were carried out in 71 cases of carcinoma of the corpus uteri treated at the Department of Obstetrics and Gynecology, Tohoku University Hospital over the 10 year period from 1968 through 1977. The ratio of cases of the corpus uteri to those of the cervix was 6.5/100. The average age of the former group was 58 years and 30% of them were nulligravida. The rates of accompanying hyprtension and obesity were 21% and 20%, respectively. With regard to clinical stages, 75% were Stage I, 13% were Stage II, 10% were Stage III and 1% were Stage IV. The histological grade was G1 in 61%, G2 in 26%, and G3 in 13%. In Stage I, 80% of the cases had myometrial invasion into less than 1/3 of the uterine wall, but in Stage II invasion was limited to 1/3 in less than 25%. Metastasis to pelvic lymph nodes was found in 3 of 34 cases (9%): 1 of 24 cases of Stage I (4%) and 2 of 9 (cases of Stage II (22%). All three of these cases had myometrial invasion reaching deeper than 1/3 of the uterine wall. Those with deep myometrial invasion showed a diffuse invasive pattern, as did those of G2 or G3 lesions. Cases of G1 lesions showed a relatively shallow myometrial invasion, whereas there was a tendency for cases of G2 or G3 lesions to have deep myometrial invasion. The over-all 5 year relative survival rate was 83%, while that for the Stage I patients was 98%. All 12 of the patients who died of recurrence of carcinoma of the corpus uteri died within 2 years of treatment.

One case of Burkitt's tumor.

A 15-year-old Japanese female with complaints of abdominal tumor and exophthalmos underwent surgery. Histologically, the tumor showed a starry-sky pattern with the macrophages interspersed among undifferentiated lymphoblastoid cells. Considering the clinical aspects, distribution of tumor deposits, and cytological findings, the tumor was diagnosed as Burkitt's tumor. The patient expired on the 42nd day of hospitalization due to general dissemination.