Macrophages control innate inflammation.

Macrophages play a critical role in the pathogenesis of metabolic diseases including gout and type 2 diabetes. The Nod-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) forms the inflammasome with apoptosis-associated speck-like protein containing a CARD (ASC), the adaptor protein, and mediates inflammatory responses by macrophages. By compound screening, we found that tubulin polymerization inhibitors suppress NLRP3 inflammasome activation. NLRP3 inflammasome inducers reduce the NAD(+) level to inactivate the α-tubulin deacetylase Sirtuin 2, resulting in accumulation of acetylated α-tubulin. Acetylated α-tubulin mediates mitochondrial transport and subsequent proximity of ASC on mitochondria to NLRP3 on the endoplasmic reticulum. Thus, microtubule-driven transport of mitochondria is required for NLRP3 inflammasome activation. Macrophages are comprised of two subsets, M1 (inflammatory) and M2 (anti-inflammatory). Trib1 is an adaptor protein involved in protein degradation of immune-related transcription factors. We found that Trib1 is critical for the differentiation of F4/80(+) MR(+) tissue-resident M2-like macrophages. Mice lacking Trib1 in haematopoietic cells show severe lipodystrophy owing to increased lipolysis, even on a normal diet. In response to a high-fat diet, the mice show hypertriglyceridaemia and insulin resistance, together with increased proinflammatory cytokine production. Thus, Trib1 is critical for adipose tissue maintenance and suppression of metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages.

A phase I study of full-dose gemcitabine and regional arterial infusion of nafamostat mesilate for advanced pancreatic cancer.

BACKGROUND: The primary end points of this study were to determine the dose-limiting toxic effects (DLTs), maximum tolerated dose, and a recommended phase II dose of a synthetic serine protease inhibitor, nafamostat mesilate, in combination with full-dose gemcitabine in patients with unresectable locally advanced or metastatic pancreatic cancer. The secondary end point was to assess therapeutic response. PATIENTS AND METHODS: Patients with previously untreated pancreatic cancer received gemcitabine (1 000 mg/m(2) i.v. for 30 min) on days 1, 8, and 15, with nafamostat mesilate (continuous regional arterial infusion for 24 h through a port-catheter system) on days 1, 8, and 15; this regimen was repeated at 28-day intervals. The initial dose of nafamostat mesilate was 2.4 mg/kg and was escalated in increments of 1.2 mg/kg until a dose of 4.8 mg/kg was achieved. A standard '3+3' phase I dose-escalation design was used. Therapeutic response and clinical benefit response were assessed. RESULTS: Twelve patients were enrolled in this study. None of the patients experienced DLTs, and nafamostat mesilate was well tolerated at doses up to 4.8 mg/kg in combination with full-dose gemcitabine. This combination chemotherapy yielded a reduction of a high serum level of the tumor marker CA19-9. Pain was reduced in three of seven patients without oral morphine sulfate. Overall survival was 7.1 months for all patients. CONCLUSION: This phase I study was carried out safely. This combination chemotherapy showed beneficial improvement in health-related quality of life. The recommended phase II dose of nafamostat mesilate in combination with full-dose gemcitabine is 4.8 mg/kg.

Protein microarray analysis identifies human cellular prion protein interactors.

AIMS: To obtain an insight into the function of cellular prion protein (PrPC), we studied PrPC-interacting proteins (PrPIPs) by analysing a protein microarray. METHODS: We identified 47 novel PrPIPs by probing an array of 5000 human proteins with recombinant human PrPC spanning amino acid residues 23-231 named PR209. RESULTS: The great majority of 47 PrPIPs were annotated as proteins involved in the recognition of nucleic acids. Coimmunoprecipitation and cell imaging in a transient expression system validated the interaction of PR209 with neuronal PrPIPs, such as FAM64A, HOXA1, PLK3 and MPG. However, the interaction did not generate proteinase K-resistant proteins. KeyMolnet, a bioinformatics tool for analysing molecular interaction on the curated knowledge database, revealed that the complex molecular network of PrPC and PrPIPs has a significant relationship with AKT, JNK and MAPK signalling pathways. CONCLUSIONS: Protein microarray is a useful tool for systematic screening and comprehensive profiling of the human PrPC interactome. Because the network of PrPC and interactors involves signalling pathways essential for regulation of cell survival, differentiation, proliferation and apoptosis, these observations suggest a logical hypothesis that dysregulation of the PrPC interactome might induce extensive neurodegeneration in prion diseases.

Visualization of right atrial thrombus associated with constrictive pericarditis by indium-111 oxine platelet imaging.

A right atrial thrombus is not often seen and only a few reports of visualization have been described. We report a 44-yr-old man who had a large atrial thrombus associated with constrictive pericarditis. Two-dimensional echocardiography and computed tomography showed a large right atrial mass. Indium-111 oxine platelet deposition was demonstrated on the surface of thrombus by platelet imaging. Platelet imaging was useful for differential diagnosis from cardiac tumor, and as an indication for surgical treatment, since right atrial thrombus may have a high risk of pulmonary embolism or severe right heart failure.

Possible association of nm23 gene-expression and ki-ras point mutations with metastatic potential in human pancreatic cancer-derived cell-lines.

Nm23 gene expression and Ki-ras point mutations of eight human pancreatic cancer-derived cell lines with different metastatic capabilities were studied. Nm23 gene expression was significantly reduced in the cell lines with a high metastatic potential as compared with those with a low meta static potential (p<0.05). The same mutation at codon 12 of the Ki-ras gene (Gly12 to Asp12) was found in all cell lines with a high metastatic potential. These findings suggest a possible association of metastatic potential with Nm23 gene expression as well as the mutation of Ki-ras gene in human pancreatic cancer.

Endoscopic ultrasonographic detection of carcinomatous invasion and of lymph nodes in the thoracic esophagus.

The use of endoscopic ultrasonography (EUS) for diagnosing the depth of carcinomatous invasion into the esophageal wall and in detecting mediastinal lymph nodes in patients with esophageal carcinoma was assessed. EUS was performed before surgery in 33 patients who underwent subtotal esophagectomy with lymph node dissection in our department of surgery between January 1987 and February 1989. The findings of EUS prospectively correlated with intraoperative macroscopic findings and histopathologic findings of the resected specimens. An accurate diagnosis of the depth of invasion into the esophageal wall was made in 30 of the 33 patients (90.1%). Visualization rates of mediastinal lymph nodes were 92.9%, 53.1%, and 1.0% when the nodes were greater than 10 mm in maximum diameter, 5 to 9 mm, and less than 5 mm, respectively. Although EUS had no diagnostic value for patients in whom the ultrasonic probe could not be inserted beyond the tumor, it is an excellent method for evaluating the depth of invasion and detecting lymph nodes greater than 10 mm in diameter. Detection is not feasible when the lymph node is less than 5 mm in diameter. EUS provides the surgeon with one more tool for the preoperative determination of curability.

Mitomycin C cross-resistance induced by adriamycin in human ovarian cancer cells in vitro.

We prepared Adriamycin-resistant cancer cells by exposing an ovarian serous cystadenocarcinoma cell line to the drug. The resistant cells also showed cross-resistance to a wide variety of other compounds, including vincristine, vinblastine, actinomycin D, daunorubicin, mitomycin C and carboquone. Against vincristine, the cells showed a greater than 5,000-fold increase in resistance, far surpassing their resistance to the selection drug. The resistant cells displayed a decrease in intracellular Adriamycin content and an increase in the mRNA of the mdr-1 gene coding for P-glycoprotein, with no amplification of the DNA. In revertant cells, resistance to Adriamycin was lost, but that to mitomycin C was maintained. Adriamycin resistance was partially overcome by the addition of verapamil or cyclosporin A, but cross-resistance to mitomycin C was not influenced at all. These results strongly suggest that the resistance to mitomycin C observed in our Adriamycin-resistant cells was due to some other mechanism than that causing multidrug resistance.

Development of systemic immunologic responses against hepatic metastases during gene therapy for peritoneal carcinomatosis with retroviral HS-tk and ganciclovir.

Gene therapy with retroviral mediated gene transfer of the herpes simplex thymidine kinase (HS-tk) gene into a tumor mass confers sensitivity of the tumor cells to ganciclovir (GCV). Tumor-specific immunologic responses may develop following treatment of the primary tumor with retroviral HS-tk and GCV. In the present study we assessed whether GCV treatment of HS-tk transduced colon cancer (TK+) implanted in the peritoneal cavity induced a systemic antitumor response that would inhibit growth of a second wild-type (TK-) tumor implanted in the liver. DHDK12 rat colon cancer cells were transduced in vitro with the retroviral HS-tk vector and established as a permanent cell line (TK+ cells). TK+ or TK- DHDK12 cells (6x10(6) cells) were injected intraperitoneally on day 0 into BD-IX rats. On day 10, TK- cells (3x10(6) cells) were injected into the liver in all the groups. The animals were then treated with GCV (150 mg/kg) for 13 days. TK+ peritoneal tumors underwent significant regression during therapy with GCV (0.05+/-0.004 g; n=7) compared to wild-type (TK-) tumors (2.2+/-0.7g; n=6) (P<0.05). The volume of TK- tumors in the liver was significantly lower in GCV-treated rats with TK+ peritoneal tumors (12.5+/-8.3 mm3) compared to rats with TK- peritoneal tumors (96.7+/-18.1 mm3) (P<0.05). Histology of the liver tumors in the TK+ groups showed a dense monocytic infiltrate with fibrosis and only occasional viable tumor cells. Gene therapy with retroviral HS-tk vectors may provide a novel approach to treatment of gastrointestinal cancer by both direct cytotoxicity and an indirect mechanism that may include enhanced immuno logic responses against disseminated disease.

Direct inhibitory effect of calcitonin gene-related peptide and atrial natriuretic peptide on gastric smooth muscle cells via different mechanisms.

Smooth muscle cells isolated from the gastric muscle layers of the guinea pig were used to determine whether calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) can inhibit the contractile response produced by 10(-6) M carbachol by exerting a direct action on muscle cells. In addition, the inhibitory effect of 2', 5'-dideoxyadenosine, an inhibitor of adenylate cyclase, on the CGRP-induced or ANP-induced relaxation of gastric smooth muscle cells were examined. CGRP and ANP inhibited the contractile response produced by carbachol in a dose-dependent manner, and the values of IC50 were 3 nM and 2 nM, respectively. 2',5'-dideoxyadenosine significantly inhibited the relaxation produced by CGRP. On the other hand, 2',5'-dideoxyadenosine did not have any significant effect on the relaxation produced by ANP. These results demonstrate the difference between intracellular mechanism responsible for gastric smooth muscle relaxation by CGRP and the mechanism responsible for muscle relaxation by ANP, and strongly suggest that the action of CGRP is mediated by adenosine 3',5'-cyclic monophosphate.

Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine.

We examined the direct effect of motilin on longitudinal and circular smooth muscle cells isolated from the guinea pig small intestine. In addition, the effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8, an inhibitor of intracellular Ca(2+)-release), verapamil (a voltage-dependent Ca(2+)-channel blocker), and removal of extracellular Ca2+ were investigated to evaluate the role of intracellular Ca2+ stores and extracellular Ca2+ on the muscle contraction induced by motilin. The effects of atropine (a muscarinic receptor antagonist), spantide (a substance P receptor antagonist) and loxiglumide (a CCK-receptor antagonist) were also examined to determine whether the motilin-induced contraction was independent of those receptors. Motilin induced a contraction of the longitudinal and circular smooth muscle cells in a dose-dependent manner with the maximal effect attained after 30 seconds of incubation. The ED50 values were 0.3 nM and 0.05 nM, respectively. TMB-8 suppressed completely the motilin-induced contraction of both types of smooth muscle cells. Verapamil had only a slight suppressive effect. Removal of extracellular Ca2+ did not have any significant influence on motilin-induced contraction. The contractile response to motilin was not affected by atropine, spantide or loxiglumide. Our findings showed that:1) motilin has a direct contractile effect on both longitudinal and circular smooth muscle cells; 2) this contractile effect is not evoked via muscarinic, substance P or CCK receptors, and 3) the intracellular release of Ca2+ plays an important role in the contractile response to motilin on both types of smooth muscle cells.

The direct contractile effect of gastrin releasing peptide on isolated gastric smooth muscle cells of the guinea pig.

Smooth muscle cells isolated from the gastric muscle layers of the guinea pig were used to determine whether gastrin releasing peptide (GRP) can cause contraction by exerting a direct action on muscle cells. In addition, the inhibitory effect of 8-( N,N-diethylamino )-octyl-3,4,5-trimethoxybenzoate hydrochloride ( TMB-8 ), an inhibitor of intracellular Ca2+ release, and verapamil, a Ca2+ channel blocker, on the GRP-induced contraction of gastric smooth muscle cells were examined. GRP elicited a contractile response of gastric muscle cells in a dose-dependent manner. The ED50 was 13 pM. TMB-8 significantly inhibited the contractile effect of GRP in gastric muscle cells. These results demonstrate the direct action of GRP on the gastric smooth muscle cells of the guinea pig, and the importance of Ca(2+)-release from intracellular calcium, stores in the contractile response to GRP.

Modulation of multidrug resistance by immunosuppressive agents: cyclosporin analogues, FK506 and mizoribine.

It has recently been reported that an immunosuppressive agent, cyclosporin A, shows a potent overcoming effect on multidrug resistance (MDR). We studied the presence of such a modulating effect of cyclosporin analogues and other immunosuppressive agents, FK506 and mizoribine, in human multidrug-resistant ovarian cancer cells (TAOV/A0.2). The intensity of the overcoming effect of cyclosporin analogues against adriamycin resistance was found to be in the other of cyclosporin D greater than A greater than C greater than H. It was found that cyclosporin D, which has relatively weak immunosuppressive activity, overcame adriamycin resistance in the multidrug-resistant ovarian cancer cells to a remarkable degree. On the other hand, it was found that FK506, a new potent immunosuppressant, could also distinctly modulate adriamycin-resistance. It was found that FK506 conferred chemosensitization upon adriamycin with reincreasing intracellular adriamycin accumulation in MDR cells which was far less than the parent strain. However, in the case of mizoribine, no modulation of drug resistance existed. Such modulation was not necessarily accompanied by immunosuppressive activity and the two functions were thought to be based on different mechanisms.

Ultrasonographic assessment of the risk of injury to branches of the middle hepatic vein during laparoscopic cholecystectomy.

BACKGROUND: Although hemorrhage from the gallbladder bed during laparoscopic cholecystectomy is one of main reasons for conversion to open cholecystectomy, the cause of this life-threatening complication is unclear. PATIENTS AND METHODS: Color Doppler ultrasound was used to examine the cause of venous hemorrhage from the gallbladder bed during laparoscopic cholecystectomy in 4 patients postoperatively and to examine the anatomic relationship between the gallbladder bed and branches of the middle hepatic vein in 50 healthy volunteers. RESULTS: Injury to a large branch of the middle hepatic vein adjacent to the gallbladder bed was diagnosed in all 4 patients. One patient required conversion to open cholecystectomy while the bleeding in 2 patients was immediately controlled by direct pressure with the gallbladder. The branch of the middle hepatic vein was completely adherent to the gallbladder bed in 5 of the 50 volunteers, and in 1 the diameter of the branch was as large as 3.5 mm. In 3 volunteers branches 3.0 to 3.8 mm in diameter traversed as close as 1.0 mm from the gallbladder bed. CONCLUSIONS: Patients with large branches of the middle hepatic vein close to the gallbladder bed are at risk of hemorrhage during laparoscopic cholecystectomy and should be identified preoperatively with ultrasound.

Rectal carcinoid tumor: endoscopic ultrasonographic detection and endoscopic removal.

Eight patients with rectal carcinoid tumors were examined by endoscopic ultrasonography (EUS) to assess the depth of invasion of the rectal wall. All resected tumors were contained within the submucosa and their depth of invasion was correctly diagnosed by EUS before treatment. Perirectal lymph nodes were not delineated by EUS. Four patients who were treated only with endoscopic polypectomy were completely cured as were four patients who had a surgical resection. EUS is useful in selecting the candidates for endoscopic removal or local resection and thus may help to avoid unnecessary radical surgery.

[Establishment of a human colon cancer-derived cell line (KC-1) which produces CEA, CA 19-9 and sialyl SSEA-1(SLX)].

A human colon cancer-derived cell line, KC-1, was established from the surgical specimen of mucinous adenocarcinoma of the colon. The cells grew as monolayers, showing formation of irregular aggregation of cells and pleomorphic nuclei. The doubling time in vitro was 56.6 hours. The cells produced CEA, CA19-9 and sialyl SSEA-1(SLX). Chromosome numbers were distributed between 79 and 83 with many structural abnormalities. A point mutation of the Ki-ras gene in codon 61 (CAA-->CAT) was found. The cells have been subcultured 13 times during these three years. This cell line can be useful for investigations of colon cancer.

How to decrease false-positive cases of ovarian cancer screening by transvaginal sonography.

Screening for ovarian cancer is an important step in improving ovarian cancer mortality. This is a report of screening for ovarian cancer using transvaginal sonography (TVS) on 2,095 asymptomatic women during the past four years. Ovarian tumors over 30 mm in diameter were detected in 87 women (4.2%). Fourteen tumors were morphologically classified as complex type and 73 as cystic type. Only 12 tumors were palpable on bimanual vaginal examination. Seven women with complex type tumors underwent laparotomy and their pathological diagnoses revealed 3 dermoid cysts, 3 endometrioid cysts and 1 ovarian cancer. Two women with cystic type tumors underwent laparotomy and their pathological diagnoses revealed 1 paraovarian cyst and 1 endometrioid cyst. Many ovarian tumors were detected at prolonged cycle of menstruation and 69% of them disappeared in repeated TVS. Sixty percent of ovarian cystic tumors under 50 mm in diameter also disappeared in repeated TVS. Close attention to menstrual cycle and morphologic findings is needed to decrease false-positive cases in TVS screening for ovarian cancer.

Hyper-adrenocorticotropinemia in a patient with Addison's disease after treatment with corticosteroids.

A 40-year-old man with Addison's disease due to adrenal tuberculosis retained high levels of adrenocorticotropic hormone (ACTH) after conventional hydrocortisone replacement. Plasma ACTH levels were completely suppressed by usual replacement with hydrocortisone (20 mg at 8:00 and 10 mg at 21:00) but rebounded to abnormally high levels the following morning. Administration of 2 mg or 8 mg of dexamethasone suppressed ACTH and cortisol. Magnetic resonance imaging of the brain showed a low-intensity lesion of the pituitary gland. Pituitary hyperplasia or microadenoma with preserved regulation of ACTH was considered to be the cause of the high plasma ACTH levels. The combination of hydrocortisone and dexamethasone reduced plasma ACTH levels.

First sporadic cases of non-pneumonic legionellosis, Pontiac fever in Japan.

Pontiac fever has rarely been found in sporadic cases. Here, we report the first sporadic cases of non-pneumonic legionellosis, Pontiac fever in Japan. Case 1. A 53-year-old man with spinocerebellar degeneration was presented to our hospital. He had an acute onset of high fever and consciousness disturbance. A chest X-ray film on admission was normal, but transient bilateral pleural effusions were revealed on hospital day 14. Case 2. A 77-year-old woman with gastric ulcer was presented to our hospital. She had an acute onset of high fever. A chest X-ray film on admission was normal, but transient bilateral pleural effusions were revealed on hospital day 7. High fever, resistant to beta-lactam antibiotics, continued in both cases. Both had serologic confirmation of legionellosis by indirect fluorescent antibody assay for Legionella pneumophila without seroconversion for Mycoplasma pneumoniae, and Chlamydia pneumoniae, and had a good prognosis. Both were thought to be sporadic community-acquired cases rather than epidemics.

Effects of prenatal aluminum treatment on development and behavior in the rat.

Developmental and behavioral effects of aluminum administration were studied in THA rats. To three groups of pregnant rats were administered single dose of 0, 900 or 1,800 mg/kg of aluminum chloride on the day 15 of gestation by gavage. Significant differences were observed between the aluminum treated offspring and controls in terms of body weight, timing of pinna detachment and eye opening, and appearances of auditory startle. Slower learning acquisition was observed in treated groups. The longer latency and more rearings in the open field test were observed in 1,800 mg/kg treated females. These results suggest that single dose of aluminum chloride during prenatal period affects the development and behavior in rats.