Novel in vitro effects of bucillamine: inhibitory effects on proinflammatory cytokine production and transendothelial migration of T cells

OBJECTIVE: To investigate the novel antiinflammatory mechanism of a disease-modifying antirheumatic drug, bucillamine, on activated T cells, specifically its effect on T cell proliferation, cytokine production, and migration of T cells. METHODS: T cells were cultured in wells coated with anti-CD3 monoclonal antibodies (mAb) plus anti-CD26 mAb or anti-CD3 plus anti-CD28 mAb, with or without bucillamine. Proliferative responses and the production of interleukin-2 (IL-2), interferon-gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), IL-6, IL-4, and IL-5 were measured under these costimulatory conditions. Phytohemagglutinin (PHA)-activated T cells were cultured on human umbilical vein endothelial cell-coated transwells in the presence or absence of bucillamine, and T cells migrating through the endothelial cell layer were counted. Immunofluorescence analysis was also performed to analyze the effect of bucillamine on the surface expression of adhesion molecules on T cells. RESULTS: Bucillamine (64 microM) significantly inhibited T cell proliferation and the production of IL-2, IFNgamma, TNFalpha, and IL-6, whereas it had no inhibitory effects on the production of IL-4 and IL-5 in the cultures with anti-CD3 plus anti-CD26 mAb. In contrast, bucillamine had no effects on T cell proliferation or any cytokine production in the cultures with anti-CD3 plus anti-CD28 mAb. Furthermore, the same concentration of bucillamine inhibited transendothelial migration of PHA-activated T cells, and reduced the expression level of CD44 on T cells. CONCLUSION: This study demonstrated the novel effects of bucillamine in vitro, showing inhibition of type 1 T helper-type cytokine production and proinflammatory cytokine production induced by certain costimulatory conditions, and inhibition of transendothelial migration of T cells. The inhibition of T cell migration appeared to be mediated partly through the reduced expression of CD44, an adhesion molecule on the T cell surface.     

Microsatellite instability in gallbladder carcinoma: two independent genetic pathways of gallbladder carcinogenesis

Although the genetic basis for gallbladder carcinogenesis has not been clarified, considerable evidence has shown that genetic alterations play an important role in the development and progression of human cancers. In this study, we analyzed 30 gallbladder carcinomas to investigate the role of genetic alterations in their tumorigenesis, and to study correlations with their clinicopathological features. Tissue samples were obtained from 30 patients with gallbladder carcinoma (11 men and 19 women; mean age, 62 years; age range, 38–80 years). Genomic DNAs were extracted from fresh tumor tissue. We examined loss of heterozygosity (LOH) in the p53, APC, DCC, RB, and NM23-H1 gene regions by polymerase chain reaction (PCR)-LOH assay using an automated fluorescent DNA sequencer employing four microsatellite markers (p53, APC, DCC, NM23-H1). Five additional microsatellite markers were used for the determination of microsatellite instability (MSI). LOH was found at p53 in 9 of 15 informative cases (60%), at DCC in 10 of 22 (45%), at APC in 5 of 15 (33%), at RB in 1 of 8 (13%), and at NM23-H1 in 1 of 15 (7%). MSI was observed in 5 of 30 cases (17%) in at least one chromosomal loci of these nine microsatellite markers. None of the patients with MSI-positive tumors showed lymph node metastasis, and there was an inverse correlation between MSI and the presence of LOH in gallbladder carcinoma. These results suggest that there are two independent genetic pathways in gallbladder carcinogenesis; that is, an MSI pathway and an LOH pathway. Received: December 24, 1999 / Accepted: May 26, 2000     

Carboxylic monolayer formation for observation of intracellular structures in HeLa cells with direct electron beam excitation-assisted fluorescence microscopy

Intracellular structures of HeLa cells are observed using a direct electron beam excitation-assisted fluorescence (D-EXA) microscope. In this microscope, a silicon nitride membrane is used as a culture plate, which typically has a low biocompatibility between the sample and the silicon nitride surface to prevent the HeLa cells from adhering strongly to the surface. In this work, the surface of silicon nitride is modified to allow strong cell attachment, which enables high-resolution observation of intracellular structures and an increased signal-to-noise ratio. In addition, the penetration depth of the electron beam is evaluated using Monte Carlo simulations. We can conclude from the results of the observations and simulations that the surface modification technique is promising for the observation of intracellular structures using the D-EXA microscope.OCIS codes: (170.0170) Medical optics and biotechnology, (170.0180) Microscopy     

Study for determination of the optimal cessation period of therapy with anti-platelet agents prior to invasive endoscopic procedures

Background Anti-platelet agents are widely used for the treatment and prevention of thrombotic diseases. On the other hand, continuation of anti-platelet agents increases the risk of hemorrhagic complications in gastrointestinal endoscopy, and cessation of anti-platelet agents exposes the patient to the risk of thromboembolism. Only a few studies have actually studied the whether a cessation period is required prior to endoscopic procedures and if so, the optional duration of the period. The present study assessed the time course of primary hemostasis after the cessation of anti-platelet agents.Methods Eleven healthy men (age range, 19–29 years) were assigned to each of the following regimens: aspirin (ASA; 100 mg/day), ticlopidine (TP; 300 mg/day), and a combination of ASA (100 mg/day) and TP (300 mg/day) for 7 days. There was a washout period of more than 3 weeks between each regimen. A quantitative bleeding time test (QBT test) and platelet aggregation test were performed before the beginning of administration, on the last day of administration, and at 1, 3, and 5 days after cessation, and also at 7 days after cessation for the combination regimen.Results The average bleeding time (BT) and total bleeding loss volume (Tv) of the 11 subjects after administration of the three regimens were significantly increased compared with those before administration. With the administration of ASA, increases of BT and Tv at 3 days after cessation were not significant. The Tv at 5 days after cessation of TP was not significantly increased. With the combination regimen, the BT and Tv at 7 days after cessation were not significantly increased.Conclusions A 3-day cessation period for ASA, a 5-day cessation period for TP, and a 7-day cessation period for ASA + TP administration seem to be sufficient.     

Mixed connective tissue disease with interstitial pneumonia in HTLV-1 carrier: case report and review of the literature

We report on a carrier of human T-lymphotropic virus type 1 (HTLV-1) who developed mixed connective tissue disease (MCTD). This patient suddenly manifested clinical symptoms and interstitial pneumonia ascribable to MCTD following long-term infection with HTLV-1. After initiation of oral prednisolone all manifestations quickly improved in parallel with a decrease in inflammatory reactions. In this patient HTLV-1 infection might have played an important role in the pathogenesis of MCTD. Since HTLV-1 can cause adult T-cell leukemia and HTLV-1-associated myelopathy, and also collagen diseases including MCTD, careful observation is necessary even in a carrier, particularly when autoantibodies are detectable in serum.     

Difference in CD22 molecules in human B cells and basophils

OBJECTIVE: CD22 is believed to be restricted to normal and neoplastic B cells. Human basophils were found to express CD22 molecules. Among the antibodies against CD22, Leu14, which recognized the ligand binding domain, reacted to basophils, and B3 and 4KB128, which recognized the amino terminus side and carboxy terminus side of the ligand binding epitope, respectively, did not. To clarify the difference of CD22 antigenicity in human B cells and basophils, we investigated RNA sequence and structures of CD22 molecules. MATERIALS AND METHODS: Purified B cells and basophils were obtained from normal human volunteers by using a MACS magnetic cell sorting system and anti-CD19 and anti-Fc epsilon R1 antibodies, respectively. RT-PCR and sequencing of CD22 mRNA were performed in the exons 3 to 8. Western blotting analysis of CD22 was also performed. RESULTS: The sequence of CD22 mRNA extracted from the basophils was the same as that of B cells in exons 3 to 8 (epitopes recognized by Leu14, B3, and 4KB128 were translated from exons 4 and 5). Reduced CD22 peptide extracted from the basophils reacted to Leu14 as well as B3 and 4KB128, and the molecular size of the reduced and nonreduced products was 130 kDa as expected. CONCLUSION: Disulfide bonds and the resulting 3D conformation of the CD22 molecules may have important roles in the difference of antigenicity of CD22 beta in B cells (CD22 beta 1) and basophils (CD22 beta 2). The difference in molecular structure surrounding the ligand-binding domain of CD22 may imply a specialization of the conformational forms of CD22 according to the ligand isoforms.     

Glycyrrhizin withdrawal followed by human lymphoblastoid interferon in the treatment of chronic hepatitis B

Seventeen patients with chronic hepatitis B were treated with a 4-week administration of glycyrrhizin followed by a 4-week treatment with human lymphoblastoid interferon, then followed for 6 months after the end of treatment. All were positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B virus-associated DNA polymerase (DNA-p) for at least 6 months before entry. All patients were Japanese and none of them were homosexuals. Eleven patients lost DNA-p activity and 10 of them lost HBeAg. Three of these 10 patients had antibody to HBeAg. In 10 patients who became HBeAg-negative, alanine aminotransferase levels after glycyrrhizin administration were higher and initial DNA-p activities relatively lower than the levels found in seven patients who remained HBeAg-positive. The immunomodulater provided by a short course of glycyrrhizin before administration of human lymphoblastoid interferon may be an effective treatment for patients with chronic hepatitis B.     

Helicobacter pylori induces antiapoptosis through buclear factor-kappaB activation

Although Helicobacter pylori is classified as a definite carcinogen, the mechanism underlying gastric carcinogenesis is not yet clear. We previously have shown that H. pylori activates an antiapoptotic gene, the cellular inhibitor of apoptosis protein 2 (c-IAP2), the underlying mechanism of which was investigated in the present study. cDNA array and real-time PCR analyses indicated that H. pylori showed a stimulatory effect on the expression of c-IAP2. Isogenic mutant strains with impaired cag pathogenicity island (cagPAI) expression showed weaker induction. Analyses that used the in situ terminal deoxynucleotide transferase-mediated dUTP nick end-labeling method indicated suppression of antiapoptosis by the antisense c-IAP2 oligonucleotide. Reporter assays with deletion and mutation constructs for the c-IAP2 promoter showed that nuclear factor-kappaB (NF-kappaB) binding sites are indispensable for transactivation. Super-repressor IkappaBalpha or NF-kappaB inhibitor reduced c-IAP2 transactivation by H. pylori, and exogenous expression of c-IAP2 inhibited apoptosis seen with H. pylori. In conclusion, H. pylori induces antiapoptosis through c-IAP2 transactivation following cagPAI-dependent NF-kappaB activation. The interaction of these stimuli may play a role in gastric carcinogenesis.