Effect of fibroblast growth factor-1 on the expression of early growth response-1 in human periodontal ligament cells.

BACKGROUND AND OBJECTIVES: Early growth response-1 is a nuclear transcription factor implicated in regulating cell proliferation. Fibroblast growth factor-1 is the prototypic fibroblast growth factor involved in the proliferation and differentiation of various cell types. Expression of early growth response-1 induced by fibroblast growth factor-1 thus may be very important for cell growth, during both development and wound healing in oral tissue. However, little is known about the expression and kinetics of early growth response-1 in fibroblast growth factor-1-stimulated oral cells. The aim of this study was to investigate the effects of fibroblast growth factor-1 on the expression of early growth response-1 in human periodontal ligament cells. MATERIAL AND METHODS: Periodontal ligament cells were cultured in medium containing 1, 10 or 100 ng/mL of fibroblast growth factor-1 for 45 min or with 10 ng/mL of fibroblast growth factor-1 for 15, 30, 45, 60 or 120 min. The proliferation of periodontal ligament cells was evaluated by measuring 5-bromo-2'-deoxyuridine incorporation. The expression of early growth response-1 mRNA and protein, and the localization of early growth response-1 protein, were examined by western blotting, northern blotting and immunocytostaining. RESULTS: 5-Bromo-2'-deoxyuridine incorporation correlated directly with increases in fibroblast growth factor-1 concentration, and 5-bromo-2'-deoxyuridine incorporation peaked 45 min after starting treatment. Early growth response-1 protein was expressed in response to a concentration of fibroblast growth factor-1 as low as 1 ng. Peak expression of early growth response-1 mRNA was observed at 15 min and that of early growth response-1 protein at 60 min. The 140-kDa early growth response-1 protein was not detected in the nuclear fraction, and the peak expression of the 80-kDa early growth response-1 protein occurred at 60 min. Early growth response-1 localized in or around the nucleus at 30 min. CONCLUSION: These results show that a concentration of fibroblast growth factor-1 as low as 1 ng induces the expression of early growth response-1 protein, and that the 80-kDa early growth response-1 protein functions in the nucleus of periodontal ligament cells treated with fibroblast growth factor-1.

Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines.

To develop a novel therapeutic strategy for ovarian cancer, we constructed a recombinant adenovirus which highly expresses pro-apoptotic Bax protein and examined its therapeutic effect on a series of ovarian cancer cell lines: A2780, A2780/cDDP, OVCAR-3 and SK-OV-3. A recombinant adenovirus carrying the Bax-alpha gene (AxCALNKYbax) induced high expression of the Bax-alpha protein in all the cell lines. The cytotoxic effect of Bax was observed in three ovarian cancer cell lines: the per cent reduction in the number of cells was 40.0% for cisplatin-sensitive A2780, 50.0% for cisplatin-resistant A2780/cDDP, and 64.8% for marginally cisplatin-resistant OVCAR-3. In contrast, it was only 12.3% for cisplatin-resistant SK-OV-3. Cisplatin-resistant A2780/cDDP had a p53 mutation and exhibited attenuated Bax induction after cisplatin treatment, which may explain why supplementation of Bax was effective in this chemoresistant ovarian cancer. Combination with cisplatin or paclitaxel enhanced the cytotoxic effect of Bax induction in all but one cell line including cisplatin-resistant A2780/cDDP. It appears that adenovirus-mediated Bax induction, with or without combination with conventional chemotherapy, useful strategy for the treatment of ovarian cancer.

Acylated anthocyanins from fruits of Sambucus canadensis.

Two new acylated anthocyanin glycosides were isolated from fruits of Sambucus canadensis. The structures were determined to be cyanidin 3-O-(6-O-Z-p-coumaroyl-2-O-beta-D-xylopyranosyl)- beta-D-glucopyranoside)-5-O-beta-D-glucopyranoside and cyanidin 3-O-(6-O-E-p-coumaroyl-2-O-beta-D-xylopyranosyl)-beta-D- glucopyranoside.

Hepatitis C virus RNA and hepatitis C virus antibody in the serum of patients with abnormal liver function.

In order to elucidate the relation between hepatitis C virus (HCV) RNA and antibody to HCV (anti-HCV) in serum, we examined samples of serum collected from 228 HBsAg-negative patients, with abnormal alanine aminotransferase (ALT) values, for HCV-RNA by nested polymerase chain reaction (PCR) assay and for anti-HCV using C100 protein as the antigen. HCV-RNA was detected in 99 (92.5%) of 107 anti-HCV-IgG-positive samples, regardless of ELISA optical density cut-off value (ELISA ratio), and in 34 (28.1%) of 121 anti-HCV-IgG-negative samples in which the frequency of the presence of HCV-RNA became higher in proportion to the ELISA ratio. Among 42 discordant cases (34 anti-HCV-IgG-negative, RNA-positive cases and eight anti-HCV-IgG-positive, RNA-negative cases), 10 were positive for anti-HCV-IgM (8/34 and 2/8, respectively) irrespective of clinical status. These findings suggest that in patients with abnormal ALT values, even if they are anti-HCV-IgG negative, HCV infection cannot be excluded. Furthermore, PCR assay for detecting HCV-RNA may be more suitable for identifying patients with infectious virus than is detection of anti-HCV-IgG. Detection of anti-HCV-IgM may also be useful.

Prevalence of blood-borne viruses among intravenous drug users and alcoholics in Hiroshima, Japan.

We investigated the prevalence of human immunodeficiency viruses-1 and 2 (HIV-1 and HIV-2), human T-lymphotropic virus type I and II, hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus among intravenous drug users (IVDU) in Hiroshima, Japan, where little is known about their present levels. From June to December 1993, serum samples were collected from 47 IVDU and 98 alcoholics in Hiroshima, Japan, and examined for markers of virus infection. The prevalence of antibody to HCV (anti-HCV) and/or HCV-RNA was significantly higher in IVDU than alcoholics (74.5% vs 20.4%, 44.7% vs 10.2% respectively, P < 0.001). In contrast, the prevalence of antibody to hepatitis B surface antigen and/or core antigen (anti-HBs and/or anti-HBc) showed no significant difference between the 2 groups (57.4% vs 66.3%). HIV-1 infection was found in one (2.1%) IVDU and genome analysis indicated that it was subtype B according to Myers' classification. Thus, an extremely low level of HIV infection and a high level of HCV infection was found in IVDU. Careful follow-up of this group is thought to be needed to minimize an outbreak of HIV-1 infection in Japan.

Segmental mediolytic arteritis [correction of arteries]: a case report with review of the literature.

Segmental mediolytic arteritis is a very rare vascular disease which causes sudden intraabdominal hemorrhage. The disease is characterized by degeneration of the arterial media, followed by aneurysmal dilatation and rupture of the involved artery. Up to now, only 13 cases have been reported, and this unique disease is not fully recognized among general pathologists and physicians. Here, we present a case of segmental mediolytic arteritis involving the propria hepatic artery, which resulted in intraabdominal hemorrhage, and consequently hypovolemic circulatory disturbance. Histologically, the rupture focus showed degeneration and desquamation of the intima and media with fibrin-like material covering the exposed adventitia. Inflammatory infiltrates were only noted in the rupture focus as a secondary reactive change. Other than the rupture focus, there were two foci showing similar findings. This disease has rarely been reported and is seldom recognized as a cause of arterial rupture. In cases of sudden intraabdominal hemorrhage, segmental mediolytic arteritis should be considered as a possible cause in addition to atherosclerotic and mycotic aneurysm, traumatic injury and vasculitis syndromes.

Characteristics and localization of rat submandibular gland proteoglycans.

The submandibular gland proteoglycans were investigated biochemically and immunohistochemically in male Sprague-Dawley rats. Proteoglycans were extracted with 4 M guanidine-HCl, followed by ultracentrifugation in a CsCl density gradient, and fractionated by ion-exchange chromatography and gel filtration. The molecular weight of PGs was estimated by SDS-PAGE and immunoblot analysis with monoclonal antibodies (HepSS-1 or 6-B-6). The glycosaminoglycan side-chains in the proteoglycan fractions were identified by electrophoresis on cellulose acetate membrane. Three proteoglycan fractions were obtained. One was a heparan sulphate proteoglycan that migrated as a diffuse band of about 210 kDa. The other two fractions contained at least two dermatan sulphate proteoglycans of 70-85 kDa and 40-50 kDa. Digestion of these two proteoglycans with chondroitinase ABC, but not heparitinase, produced two bands of 50 and 21 kDa, which were core proteins. The smaller dermatan sulphate proteoglycan may be a portion of the other, as the core protein of both bound to 6-B-6 antibody, and sugar chains of both were the same (20-30 kDa). Heparan sulphates recognized by antibody HepSS-1 were observed widely in the basement membrane, fibrous connective tissue, and striated and excretory ductal cells, while dermatan sulphate proteoglycans recognized by antibody 6-B-6 were located in the connective tissue surrounding striated and excretory ducts.

Laparoscopic surgery using a newly designed suction lifter.

The authors describe a simple and available abdominal wall-lift technique for endoscopic surgery that involves a new suction lifter that they designed for laparoscopic surgery. Since July 1998, the authors have used this technique successfully in eight patients with appendicitis, in one patient with a perforated duodenal ulcer, and in one patient with an adenoma of the ascending colon.

Type IV collagen serum and vitreous fluid levels in patients with diabetic retinopathy.

We compared the levels of type IV collagen (IV-C) in vitreous fluid and serum and the levels of glycosylated haemoglobin in 47 patients with proliferative diabetic retinopathy (DR) and 21 patients with non-inflammatory retinopathies. Levels of IV-C were found to be higher in the vitreous fluid in patients with DR than in patients with non-inflammatory retinopathy (53.2 +/- 14.9 microg/l versus 14.7 +/- 4.5 microg/l). Serum levels were likewise higher in patients with DR (349.7 +/- 106.2 microg/l versus 97.7 +/- 13.1 microg/l) as were glycosylated haemoglobin levels (8.3 +/- 0.3% versus 5.2 +/- 0.4%). In addition, levels of type IV collagen in the vitreous fluid were found to be higher in the patients who had been diabetic for > or = 10 years than in patients who had been diabetic for < 10 years (54.8 +/- 15.5 microg/l versus 16.8 +/- 4.6 microg/l). We conclude that accumulation of vitreous fluid IV-C may relate to high levels of glycosylated haemoglobin and long duration of diabetes. This suggests that the concentration of IV-C in vitreous fluid, and possibly also the serum levels of IV-C, reflects the progression of DR. Further investigation is needed to verify this and to investigate whether or not measuring IV-C levels is a useful means to assess therapeutic effects and/or prognosis of diabetic microangiopathy.

A long-term follow-up of five patients with hepatitis delta virus in Japan.

From 1973 to 1989 five patients with hepatitis delta virus having anti-hepatitis delta antibodies continuously in the serum for more than 5 years were identified among 1019 hepatitis B virus carriers who were being followed-up for more than 3 years (mean 8.9 years). Of the five patients with antibodies, three had a history of blood transfusion, in two cases the transfusion was massive, and one patient had been addicted to narcotics given intravenously 35 years before. In the remaining patient, the route of superinfection could not be determined. Hepatitis delta antigen was detected in hepatocyte nuclei of one of the three patients in whom liver biopsies were performed and there was chronic persistent hepatitis detected by an indirect immunoperoxidase technique. During the follow-up, hepatocellular carcinoma developed in one case but the clinical prognosis was favourable in the remaining four cases.

Persistence of hepatitis B e antigen for a long period after the clearance of hepatitis B surface antigen.

Four-hundred and seventy-eight hepatitis B virus (HBV) carriers were followed for more than 60 months. In thirty-six cases the hepatitis B surface antigen (HBsAg) was cleared from serum during the observation period. In one case hepatitis B e antigen (HBeAg) was detectable for more than 66 months even after the appearance of antibody against hepatitis B surface antigen (anti-HBs). HBeAg was detected not only by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), but also by reversed passive haemagglutination (RPHA) method which has been believed to be far less sensitive than RIA or ELISA tests. HBV DNA was demonstrated in the serum even after the appearance of anti-HBs. Therefore, it is suggested that HBV (Dane particles) remains or reappears intermittently in the serum after seroconversion of HBsAg antigenaemia to anti-HBs complexes.

[Detection of hepatitis delta virus (HDV) markers in HBV carriers].

To investigate the prevalence and clinical features of hepatitis delta virus (HDV) superinfection in hepatitis B virus (HBV) carriers, antibody to hepatitis delta antigen (anti-HD) was determined in the sera of 328 HBV carriers in Japan. 1) Of the 328 HBV carriers, six (1.8%) were seropositive for anti-HD by enzyme linked immunosorbent assay. IgM-antibody to hepatitis delta antigen was detected in 2/6 patients with a high anti-HDV titer. None of the patients was positive for hepatitis dealt antigen. 2) HBV carriers with chronic liver disease had a greater frequency of seropositivity of anti-HD than asymptomatic HBV carriers. These data indicate that HDV superinfection may be an etiologic agent of chronic liver disease in HBV carriers.

Long-term follow-up of a patient with hepatocellular carcinoma associated with triple hepatitis virus (HBV, HDV, HCV) infection.

A 69-year-old Japanese man with hepatocellular carcinoma (HCC) associated with triple hepatitis viruses [hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis D virus (HDV)] infection is reported. The patient had a past history of intravenous drug abuse and a tattoo on his back. A liver biopsy, performed in November 1989, showed HCC associated with cirrhosis. HBsAg and anti-HD antibody had been detected repeatedly starting in August 1984 and anti-HCV antibody was detected in 1990. By indirect immunoperoxidase staining the HD antigen was detected in the nuclei of hepatocytes of biopsy specimens and noncancerous liver cells obtained from autopsy specimens. Liver cirrhosis associated with triple hepatitis virus infection developed to hepatocellular carcinoma, and transcatheter arterial embolization treatment for HCC was effective. Despite having HCC and cirrhosis, the patient lived well beyond the expected time.

[Studies on hepatitis C virus infection in haemodialysis patients].

To examine the prevalence of hepatitis C virus (HCV) in haemodialysis patients without blood transfusion in Hiroshima Prefecture, antibody to HCV (anti-HCV) was studied by the Ortho ELISA Kit in sera from 393 consecutive haemodialysis patients and in sera from 510 age and sex matched healthy members of the general population (control). An additional confirmatory test was done by a recombinant immunoblot assay. 1) Anti-HCV was detected in 70 of the 393 dialysis patients and 3 of the 510 healthy controls (17.8% vs 0.6%, p less than 0.01). Prevalence of anti-HCV in haemodialysis patients sera was increased by the volume of blood transfusion, and even in dialysis patients who had no blood transfusion, the frequency of anti-HCV positivity (9.2%) was greater than the healthy controls (p less than 0.01). Thus, the major route of HCV transmission in haemodialysis patients without blood transfusion may be via the haemodialysis treatment. 2) The prevalence of anti-HCV increased significantly with the ALT level and abnormal ALT activity of the anti-HCV positive group were significantly greater than that of the negative group. Thus, it is suggested that HCV infection may be an etiologic factor of liver dysfunction in haemodialysis patients.

[HBV carriers and HDV superinfection--studies on the cases of HBsAg clearance].

We studied the annual clearance rates of hepatitis B surface antigen (HBsAg) and the annual seroconversion rates of HBsAg (HBs seroconversion rates), and the correlation between HBsAg clearance and hepatitis delta virus (HDV) superinfection in hepatitis B virus (HBV) carriers in Japan. Out of 1,029 HBV carriers followed for more than 36 months, 56 cases were cleared of HBsAg from the sera, and 24 of these cases developed hepatitis B surface antibody. The annual clearance rate of HBsAg was 0.94% and the annual HBs seroconversion rate was 0.27%. These rates increased with aging, especially above 30 years of age. Antibody to HDV was detected in three cases with increased serum alanine aminotransferase activity preceding HBsAg clearance. These data indicate that HDV superinfection may play a role in induction of the HBsAg clearance in HBV carriers in Japan.

[Combination therapy with ofloxacin and chloramphenicol against typhoid fever].

Recently we experienced two male typhoid patients who required additional treatment with CP sodium succinate (CP succinate) to OFLX therapy. Although both of the cases were administered 900 mg/day of OFLX orally for three and four days, respectively, the clinical efficacies were not sufficient. We added 2.0 g/day of CP succinate intravenously for six days, although a lasting high fever returned to normal level in a few days. The administration of OFLX was continued up to 14 days. The daily doses of OFLX during the co-medication with CP succinate were 600 mg in one case and 900 mg in the other case. In both cases no recurrence was observed clinically and bacteriologically for six months after their discharge. No adverse reaction was observed throughout the therapy.

[Studies on the clinical and bacteriological effects of ofloxacin on the typhoid patients and carriers].

Fundamental and clinical studies of OFLX were performed against the patients with typhoid fever and typhoid carriers. 1) Clinical and bacteriological effects: Eight patients with typhoid fever and 3 typhoid carriers were treated with OFLX. Daily doses of the agent were 900 mg in 5 adult patients, 600 mg in a child patient and 3 adult carriers. In one case of the remaining 2 adult patients, daily doses of the agent changed from 800 mg to 1200 mg and from 900 mg to 1200 mg in the other one. The duration of the treatment was 9, 14 or 21 days. Clinical efficacies of OFLX against the patients proved 4 cases were "excellent", 3 cases were "good" and one case was "poor". The eradication of Salmonella typhi recognized in all cases containing 3 carriers with the exception of the "poor" case. Adverse reactions were observed transiently in 3 patients, such a slight decrease of RBC count, decrease of granulocyte count and elevation of GPT value respectively. 2) Antimicrobial activity: MICs of OFLX against 40 strains of S. typhi were 0.05 micrograms/ml and 0.1 micrograms/ml. The MICs of NFLX, CPFX and T-3262 were almost the same as that of OFLX, and those of ENX, NY-198 and NA were higher than that of OFLX. The peaks of MIC of CP and ABPC, first choice drug against typhoid fever, were 1.56 micrograms/ml and 0.38 micrograms/ml respectively. 3) Serum concentration; Serum concentrations of OFLX were serially measured on 5 patients through the day. The concentrations of the drug were distributed from 0.82 micrograms/ml to 6.34 micrograms/ml at 6.30 a.m. and from 2.52 micrograms/ml to 11.2 micrograms/ml at 9:00 p.m. Those of the day time showed considerable individual differences.

Waldenström's macroglobulinemia associated with amyloidosis and membranous nephropathy.

A 61-year-old man with massive proteinuria and hyper gamma-globulinemia was admitted to hospital because of massive edema and pulmonary infection. He showed significantly high level of serum IgM (3244 mg/dl) with lambda-type M-protein and Bence Jones protein detected by immunoelectrophoresis. Renal biopsy specimen showed not only the diffuse amorphous amyloid deposition in mesangial area but global thickening of capillary wall with spike formation by silver staining which was similar to the spicular formation. Immunofluorescence disclosed find granular deposition of IgG and C3 along the capillary wall and the electromicroscopic findings clearly showed both massive amyloid fibril at mesangial area and diffuse epimembranous electron dense deposits. lambda-type Bence Jones protein in macroglobulinemia was suggested not only the cause of renal amyloidosis but also the antigenic origin of membranous nephropathy in this case.

[Diagnosis of hepatocellular carcinoma using chicken anti-monoclonal antibody--as tumor marker on the heterophilic hanganutziu-deicher antigen].

The Hanganutziu-Deicher (HD) antigen is a heterophilic antigen with N-glycolylneuraminic acid as the terminal carbohydrate, and has attracted much attention as one of tumor-associated antigens. This antigen is absent from normal tissues of human and chicken but expressed in human neoplasms. Extensive research on HD antigen has not be made yet to show the intimate correlation with human neoplasm, but it is attracting an attention as a diagnostic marker for the definition of cancer. We made two different chicken monoclonal antibodies against HD antigen as a pathological tool for hepatocellular carcinoma (HCC). The hybrid cell, which produced antibodies, were prepared by means of fusion technology with HD3 antigen-immunized chicken spleen cells and HAT-sensitive chicken B cell line termed R27H4. The two antibodies had a different characteristic, one recognized only glycolipid, and the other recognized glycoprotein as well as glycolipid. Glycolipid type HD antigen and glycoprotein type HD antigen were identified by using two chicken anti-HD monoclonal antibodies. Glycolipid type HD antigen was detected by flow cytometry in 4 out of 24 HCC samples (87.5%). In our study, increased serum levels of IgG type and/or IgM type HD antibodies were observed in 21 out of 24 patients with HCC (87.5%). The presence of these antibodies is attributable to the expression of the HD antigen in cells of HCC. Eleven out of 24 patients with HCC were negative for AFP and PIVKA-II, and 10 out of these 11 patients (90.9%) were positive for anti-HD antibodies. We consider that patients with HCC should be screened for HD antigen and antibody for an early diagnosis of hepatocellular carcinoma; the same as for AFP and PIVKA-II both of which are tumor-associated antigens that have been associated with for hepatocellular carcinoma.

IFPA Meeting 2012 Workshop Report II: epigenetics and imprinting in the placenta, growth factors and villous trophoblast differentiation, role of the placenta in regulating fetal exposure to xenobiotics during pregnancy, infection and the placenta.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology: 1) epigenetics and imprinting in the placenta; 2) growth factors and villous trophoblast differentiation; 3) role of the placenta in regulating fetal exposure to xenobiotics during pregnancy; 4) infection and the placenta.