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.

Hypoxia-inducible factor 1α promotes interleukin 1ß and tumour necrosis factor α expression in lipopolysaccharide-stimulated human dental pulp cells.

AIM: To elucidate the role of HIF1α in pro-inflammatory cytokine mRNA expression from lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). METHODOLOGY: mRNA expression of interleukin (IL) 1ß and tumour necrosis factor (TNF) α in LPS-stimulated hDPCs was determined by quantitative RT-PCR. Expression of nuclear factor kappa B (NFκB) p65 and phospho-NFκB p65 was analysed by Western blotting. Activation of NFκB signalling was measured by luciferase assay using a reporter vector containing an NFκB response element. Enforced expression of HIF1α was induced by transfection of expression vectors with native or constitutively active forms of HIF1α. Expression of HIF1α protein in hDPCs was evaluated by immunocytochemistry and Western blotting. One-way analysis of variance and the Tukey-Kramer test were performed to determine a significant difference (P < 0.05). RESULTS: mRNA expression of IL1ß and TNFα, protein expression of phospho-NFκB p65 and LPS-induced NFκB signalling activity were promoted in low oxygen conditions (1% O2 ; P < 0.05). These findings were replicated following enforced expression and stabilization of HIF1α in hDPCs. Dimethyloxalylglycine, an inhibitor of prolyl hydroxylase (a HIF1α degrading enzyme), promoted IL1ß and TNFα mRNA expression and NFκB signalling in LPS-stimulated hDPCs (P < 0.05). HIF1α expression was detected in hDPCs cultured in low oxygen conditions (1% O2 ). LPS stimulation further enhanced HIF1α expression in hDPCs, especially within their nuclei. CONCLUSION: HIF1α promoted mRNA expression of IL1ß and TNFα via NFκB signalling in LPS-stimulated hDPCs, suggesting that HIF1α is involved in the progress of inflammation in dental pulp.

Transient pulmonary edema following adrenal infarction in a patient with primary anti-phospholipid syndrome.

We report a patient with primary anti-phospholipid syndrome (APS) who developed pulmonary edema following sudden-onset pain in the left, lower back of the chest. Radiological examinations demonstrated fresh infarction of the left adrenal gland but no obvious thrombi in pulmonary arteries. The patient quickly recovered from pulmonary edema with anti-coagulation therapy alone. Primary APS may have caused adrenal infarction in the patient, leading to transient pulmonary edema via microthrombosis and/or excessive release of catecholamine.

Pulp ablation therapy by inductive heating: heat generation characteristics in the pulp cavity.

OBJECTIVE AND METHODS: This study was performed to clarify the usefulness of inductive heating system for the new endodontic therapy. Dextran magnetite complex (DM) suspensions were injected into the root canal of a permanent tooth, and the tooth was heated up to about 55.0 degrees C by alternating-current magnetic field. RESULTS AND CONCLUSION: The time until the temperature in the pulp cavity reached 55.0 degrees C was 328 +/- 26 s (mean +/- s.d., n = 8) in the 56 mg as Fe ml(-1) of DM concentration. The temperature in the pulp cavity could be maintained at 53.5-59.0 degrees C for 1200 s by changing the magnetic field intensity safely, while temperature elevations of the dental surface on the coronal and apical sides were 4.9 degrees and 3.7 degrees C, respectively. Thus, this inductive heating system, which has the possibility of selective heating, might be useful for eliminating residues of pulp as a new ablation therapy.

Antitumor effect of new local hyperthermia using dextran magnetite complex in hamster tongue carcinoma.

OBJECTIVE: This study was performed to clarify the usefulness of Dextran magnetite (DM) for the oral cancer hyperthermia. METHODS: Tumors were induced in golden hamster tongue by 9,10-dimethyl 1-1,2-benzanthracene (DMBA) application. DM suspension was locally injected into the tumor-bearing tongue and tongues were heated up to 43.0-45.0 degrees C, by AC magnetic field of 500 kHz. RESULTS: The average time taken for the temperature to rise to 43.0 degrees C or above was 162 s (n = 17) at the margin of the tumor and 420 s (n = 17) at the center of the tumor. According to the tumor volume, the time required for an increase in the central temperature of tumor to 43.0 degrees C tended to be prolonged. Both temperatures could be maintained at approximately 43.0-45.0 degrees C for 30 min. The inhibition of the growth of tongue carcinoma in the four-time heating group was significantly greater than in the control group (P < 0.01). Moreover, the survival rate was significantly higher in the heated groups than in the control group (P < 0.01). Histological examination revealed a brown uniform DM accumulation at the stroma in the margin of the tumors. Many of tumor cells disappeared at the site adjacent to this accumulation. CONCLUSION: These results strongly suggest the usefulness of this local hyperthermic system in the oral region that is accessible to this treatment.

New local hyperthermia using dextran magnetite complex (DM) for oral cavity: experimental study in normal hamster tongue.

The possibility of dextran magnetite complex (DM) as a new hyperthermic material was examined in this study. DM suspension of 56 mg ml(-1) iron concentration was locally injected into the normal tongue of golden hamster. DM injected tongues were heated by 500 kHz alternating current (AC) magnetic field and its serial changes in temperature were recorded at 30-s intervals. The temperature of DM injected tongue was maintained at about 43.0-45.0 degrees C for 30 min by changing the AC magnetic field intensity. While temperature elevations of the contralateral tongue and the rectum were only of minor degree. In experiment on the extent of heating area, there was correlation between volume of black stain area and amount of the injected DM suspension (Y = - 18.1 + 1.94X, r = 0.931, P < 0.0001, n = 9 ). Histological examination after heating revealed brown uniform DM accumulation in the connective tissue between fibers of the tongue muscle. Except for vascular dilatations, no tissue damage was seen in the heated tongue. Thus, DM which has the possibility of selective and uniform heating in local hyperthermia might be useful for oral cancer therapy.

Nitric oxide-mediated antitumor activity induced by the extract from Grifola frondosa (Maitake mushroom) in a macrophage cell line, RAW264.7.

We have investigated D-fraction (MDF) extracted from Grifola frondosa (Maitake mushroom) on the inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production in RAW264.7 (RAW) cells, a murine monocyte/macrophage cell line, with special reference to antitumor activity of MDF against human hepatoma-derived huH-1 cells. MDF could induce iNOS mRNA expression in RAW cells in a dose range of more than 30 microg/ml, but the effect of 10 microg/ml of MDF was negligible. The iNOS mRNA expression induced by 100 microg/ml of MDF was 6 hrs later, but lasted for a longer time than that of lipopolysaccharide (LPS), a representative iNOS inducer. Although iNOS mRNA levels in MDF-stimulated cells were almost equal to LPS-stimulated cells at the peak time, the cumulative amount of nitrite was only about 50% compared with that of LPS-treated cells. When huH-I cells were cultured in MDF containing media in a 24-well plate with inserted porous bottom in the presence or absence of RAW cells, the viability of huH-1 cells decreased significantly only in the presence of RAW cells in MDF dose-dependent manner. This antitumor activity of RAW cells in the presence of MDF was abolished or attenuated by the addition of L-NAME, a NOS inhibitor, confirming that this phenomenon is due to iNOS-mediated NO production by RAW cells, but not direct cytotoxic activity of MDF against huH-1 cells. These data suggest that MDF is a novel inducer for iNOS which contributes at least in part to antitumor activity of MDF.

[Carotid endarterectomy: how we do it].

Carotid endarterectomy (CEA) clearly benefits high stroke-risk patients with coronary or peripheral vascular disease, but its value of procedures is not still recognized for thoracic surgeons in Japan. Therefore we reviewed our technique of CEA and presented its usefulness and necessity.

Synergistic antiproliferative effect of delta 12-prostaglandin J2 (delta 12-PGJ2) and hyperthermia on human esophageal cancer cell lines.

delta 12-PGJ2, one of the cyclopentenone prostaglandins and the ultimate metabolite of prostaglandin D2, has been reported to have potent antiproliferative activity on various tumor cells in vitro and in vivo. In this study, the combined effect of delta 12-PGJ2 and hyperthermia on six established cell lines of human esophageal carcinoma (SGF series) was analyzed by an in vitro assay, and the degree of apoptosis induced by this combination was examined to clarify the mechanism of supra-additive effects. In five SGF cell lines, except SGF-7 cells, combination therapy with delta 12-PGJ2 and hyperthermia showed synergistic antiproliferative effects. The supra-additive combined effect of delta 12-PGJ2 and hyperthermia on esophageal cancer cells is attributed to the synergistic induction of apoptosis. delta 12-PGJ2 induced G1 accumulation and apoptosis was induced by delta 12-PGJ2 from G1 phase. Hyperthermia induced G1 accumulation and apoptosis was induced by hyperthermia during all cell phases. Both augmented G1 arrest followed by G1 phase-selective induction of apoptosis and increased apoptotic induction without cell-cycle specificity are responsible for the synergism of combined treatment with delta 12-PGJ2 and hyperthermia.

The combined effect against colon-26 cells of heat treatment and immunization with heat treated colon-26 tumour cell extract.

Cancer vaccines represent a promising new strategy for immunotherapy against cancer, but their effects are insufficiently understood. The effect of heat treatment against mouse colon adenocarcinoma cell line (colon-26), and combined effects of heat treatment and immunizing host animals with heat treated colon-26 cell extracts were investigated. Heat treatment of colon-26 cells induced heat-shock protein 70 (HSP70), but not other HSP. Immunization of BALB/cJ mice with heat treated colon-26 cell extract, which was enriched in HSP70, elicited antitumour immunity against subcutaneously injected colon-26 cells. Furthermore, combination therapy of heat treatment and immunization with heat treated colon-26 cell extract significantly reduced tumour volumes compared with heat treatment alone. Similar immunization enhanced the cytotoxic activity of mouse splenic lymphocytes against untreated and heat treated colon-26 cells in an in vitro assay, as well as against heat treated allogenic mouse lymphoma cell line (YAC-1). These findings suggest possible usefulness of heat treated cancer cell extract as a cancer vaccine, especially if given in combination with hyperthermia.

Tumor regression by inductive hyperthermia combined with hepatic embolization using dextran magnetite-incorporated microspheres in rats.

We formulated a novel preparation of microspheres incorporating dextran magnetite complex (DM-MS), which enable magnetic field-induced heating for the induction of hyperthermia. Using a 500 kHz magnetic field combined with arteriolar embolization of DM-MS, inductive hyperthermia was performed for the treatment of liver tumors in rats. Three days after treatment, the percentage increase of tumor volume in the embolo-hyperthermia, embolization alone, and control groups were 28%, 124%, and 385%, respectively, with the embolo-hyperthermia group differing significantly from the remaining groups. This study demonstrated the feasibility of combining embolization of DM-MS with hyperthermia as an antitumor therapy.

CEA immunohistochemical localization is correlated with growth and metastasis of human gallbladder carcinoma.

Carcinoembryonic antigen (CEA) is a good marker of colorectal cancer. Recent studies have demonstrated that CEA may function as a metastatic potentiator by different pathways; i.e., modulation of immune responses, facilitation of intercellular adhesion and cellular migration. However, expression patterns of CEA have not yet been established in human gallbladder carcinomas. In this study, we examined CEA expression in human gallbladder adenocarcinomas and its clinicopathological significance. CEA immunoreactivity was detected not only in the cancer cells (cytoplasmic type: 63.0%, 34/54) but also in the cancer stroma (stromal type: 29.6%, 16/54). According to TNM classification, 75.0% (30/40) of T2-4 gallbladder cancers showed cytoplasmic CEA, while 28.6% (4/14) of the T1 cancers were cytoplasmic CEA-positive (p<0.05). Stromal CEA expression was detected in 40.0% (16/40) and none (0/14) of the T2-4 and T1 cancers, respectively (p<0.05). Lymph node metastasis was frequently found in the cytoplasmic CEA- and stromal CEA-positive gallbladder cancers (44.1% and 62.5%, respectively). These observations suggested that CEA expression plays important roles in cancer cell growth and metastasis of human gallbladder adenocarcinomas.

An immunohistochemical and clinicopathological study of gastrointestinal stromal tumors.

Immunohistochemical and clinicopathological features of 58 gastrointestinal stromal tumors (GIST) were studied. One occurred in the esophagus, 41 in the stomach, nine in the small intestine, and seven in the large intestine. By using indirect immunoperoxidase staining for Cajal cell markers (c-kit protein and CD34), smooth muscle markers (alpha-smooth muscle actin, desmin, heavy caldesmon and calponin) and Schwann cell markers (S-100 protein and Leu 7), GIST were classified into five groups, such as Cajal cell type (n = 9), myogenic type (n = 5), Schwann cell type (n = 2), mixed cell type (n = 40) and undifferentiated type (n = 2). c-kit Protein (42/58; 72%) and CD34 (45/58; 78%) were commonly and diffusely expressed in GIST. Novel smooth muscle markers, caldesmon (29/58; 50%) and calponin (18/58; 31%), were useful in detecting myogenic characters of GIST. S-100 protein was expressed in 16 (28%) tumors, two of which were also reactive with Leu 7 (CD57). Three small bowel tumors with skeinoid fibers expressed the Cajal cell markers, and were categorizable in GIST. Clinicopathological analyses using aggressive (n = 21) and non-aggressive (n = 21) GIST indicated that the malignant potential was correlated with the intestinal location, large tumor size, high cellularity, necrosis, solid (non-interlacing bundled) pattern of growth, negativity of c-kit protein and/or CD34, high mitotic count, and high MIB-1 labeling.

Type VI collagen immunoreactivity in skeinoid fibers in small intestinal stromal tumors.

By immunoperoxidase analysis for types I to VI collagen, elastin, cytoskeletal components and some glycoproteins, we found type VI collagen immunoreactivity in amorphous eosinophilic deposits (skeinoid fibers) in three small intestinal stromal tumors. Negative results were obtained for types I, II, III, IV and V collagen, elastin, laminin, ubiquitin, intracellular filaments such as actin, desmin, vimentin, calponin and caldesmon, and glycoprotein such as lysozyme, factor XIIIa, beta2-microglobulin, alpha1-antitrypsin and alpha1-antichymotrypsin. In two lesions, the periodic acid-Schiff-positive skeinoid fibers were also focally labeled for amyloid P component.

Localization of prostate-specific antigen-like immunoreactivity in human salivary gland and salivary gland tumors.

Immunoreactivity of prostate-specific antigen (PSA), a kallikrein-like enzyme present in the seminal plasma, was demonstrated by indirect immunoperoxidase staining using a PSA antiserum in the apical cytoplasm along the luminal border of small-sized duct epithelial cells of the major salivary (parotid and submandibular) gland of both sexes (56/56, 100%). No PSA-like immunoreactivity was seen in large-sized duct epithelial cells and acinar cells. Minor salivary gland ducts were negative. When inflammatory and atrophic changes were observed, ductal expression of PSA-like immunoreactivity was decreased (12/37, 32%) and the site of intracellular localization often became diffusely cytoplasmic. The immunoreactivity was absorbed by human seminal plasma. Immunoreactivities of prostatic acid phosphatase and sex hormone receptors were undetectable in the salivary gland. Twenty-nine (34%) of 86 salivary gland tumors with ductal differentiation were immunoreactive for PSA mainly in the cytoplasm. A PSA monoclonal antibody ER-PR8 detected immunoreactivity in the prostate but not in the salivary glands or their tumors. Prostate-specific antigen-like immunoreactivity in small-sized (intercalated) duct epithelial cells of the major salivary gland and their tumors may be due to cross-reactivity of the antiserum with kallikrein-like substances.

Splenic artery aneurysm associated with systemic lupus erythematosus: report of a case.

We herein report on a 64-year-old Japanese female patient who presented with a splenic artery aneurysm (SAA) associated with systemic lupus erythematosus (SLE). The saccular aneurysm, which measured 3 cm in diameter, was located in the proximal third of the splenic artery from the pancreas with a portosystemic shunt. A double ligation of the splenic artery (the distal and proximal sides of the aneurysm) was performed without a splenectomy. The postoperative course showed acute pancreatitis without either splenic infarction or portal thrombus. To our knowledge, the closed association of SLE with an aneurysmal dilatation of the splenic artery has not been previously reported. Both the pathogenesis and the management of SAA associated with SLE are discussed following the presentation of this case. This is the first reported case of SAA associated with SLE.

[Extended resection of the great vessels for primary lung cancer and mediastinal tumor].

From 1973 to 1998, we resected and reconstructed the great vessels in 44 patients with primary lung cancer or mediastinal tumor. Among them, 39 patients (28 with lung cancer and 11 with mediastinal tumor) and 5 patients (all with lung cancer) underwent reconstruction of the superior vena cava (SVC) and aorta, respectively. The SVC was repaired by expanded polytetrafluoroethylene (EPTFE) graft (n = 8), prosthetic patch (n = 5) or direct suture (n = 26). The aorta was repaired with temporary subclavian artery-descending aorta (n = 3), or left atrium-femoral artery bypass (n = 2). No complication or operative death occurred after surgery. The survival rate of the patients with lung cancer who underwent SVC reconstruction at 3 year and 5 year were 26.2% and 11.2%, respectively. Five of 11 (45.5%) patients with mediastinal tumor are alive at 5 years. We concluded that extended resection for primary lung cancer or mediastinal tumor invading the SVC is acceptable operation method for some patients.

Active-oxygen scavenging activity of traditional nourishing-tonic herbal medicines and active constituents of Rhodiola sacra.

The active-oxygen scavenging activity of 70 traditional herbal medicines used in China and Japan as nourishing tonics were evaluated by electron spin resonance (ESR) technique, in order to evaluate their effectiveness for anti-aging and to search for new active-oxygen scavengers from natural resources. Most of the 70 herbal medicines showed scavenging activity with various intensities. Areca catechu (methanol extract), Dendrobium plicatile (methanol extract), Juglans regia (water extract), Paeonia lactiflora (methanol extract), Psychotria serpens (water and methanol extracts), Rhodiola sacra (water and methanol extracts) and Uncaria rhynchophylla (water extract) especially showed strong scavenging activity against superoxide anion radical (*O2-), while J. regia (water and methanol extracts), Morus alba (water extract) and Schisandra chinensis (water extract) revealed strong scavenging activity against hydroxyl radical (HO*). In addition, the active-oxygen scavenging activities of 19 compounds isolated from R. sacra were also examined, and hydroquinone (1), caffeic acid (3), protocatechuic acid (6), gallic acid (7), (-)-epigallocatechin 3-O-gallate (8), 3-O-galloylepigallocatechin-(4beta-->8)-epigallocatechin+ ++ 3-O-gallate (10), heterodendrin (17) and gallic acid 4-O-beta-D-glucopyranoside (19) were found to show mild or strong inhibitory activity against superoxide anion radical (*O2-), while 4-hydroxybenzoic acid (2), 3, 4-hydroxycinnamic acid (4), 6-8 and 19 inhibited hydroxyl radical (OH*). These active-oxygen scavengers may contribute, to different extents, to their anti-aging action.

A Case of Adenoid Squamous Cell Carcinoma of the Breast Skin.

We report a case of a 45-year-old Japanese women with adenoid squamous cell carcinoma (ASCC) of the left breast skin. The patient had showed a large mass in the left breast region with axillary swelling about 1 year before admission. Grossly, the tumor was an extensively ulcerated and elevated lesion measuring 15 x 16 x 5cm. Based on the tumor biopsy and cytologic examination of the axillary lymph nodes, squamous cell carcinoma (SCC) was diagnosed. No evidence of distant metastasis was identified. A modified radical mastectomy with left axillary node dissection was performed. Microscopically, the resected tumor showed on invasive proliferation of atypical squamous cells with marked keratinization. At the periphery of the tumor, an adenoid growth pattern was frequently seen with a transitional area showing squamous cell carcinoma and adenoid growth components. ASCC was diagnosed. A transition between the overlying squamous cell epithelium and squamous cell carcinoma component was also seen, thus the tumor was thought to haveoriginated from the breast skin. The patient died of respiratory failure due tomultiple lung metastasis about 1 month after the mastectomy. Tumor rarely originates at the breast region to include both the mammary glands and breast skin. The pathogenesis and management of ASCC are discussed following the presentation of this case.