Minori Kamada

MicroRNA-203 induces apoptosis by upregulating Puma expression in colon and lung cancer cells.

The present study investigated the relationship between microRNA-203 (miR-203) and the p53 upregulated modulator of apoptosis (Puma) in colon (HCT116) and lung cancer (A549) cells. Colon and lung cancer cell lines were selected for this study since a relationship between p53/miR-203 and p53/Puma has been established in both cancers. In the present study, adriamycin and nutlin-3 were used to activate p53, which induced both miR-203 and Puma expression in HCT116 cells. In contrast, HCT 116 cells with downregulated p53 showed decreased miR-203 and Puma expression. Importantly, we found that overexpressed miR-203 in HCT116 cells resulted in significantly increased Puma expression (P<0.05). Based on these findings, we hypothesized that another limb of the p53/Puma axis depends on miR-203 expression. To further validate this relationship, we used lung cancer cells (A549) and found that activated p53 increased both miR-203 and Puma expression. In addition, we found that Puma expression remained elevated in cells with overexpressed miR-203 in the presence of p53 downregulation. Cumulatively, our data purport that p53 not only increased Puma expression directly, but that it may also do so through miR-203. Additionally, functional studies revealed that miR-203 overexpression induced apoptosis and inhibited cell invasiveness.

Expression and distribution of very late antigen-5 in mouse peritoneal macrophages upon ingestion of fibronectin-bound Staphylococcus aureus.

Many pathogens colonize host tissues by binding to the extracellular matrix via their cell surface adhesion molecules, which are called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). Staphylococcus aureus expresses several of these adhesion molecules, some of which bind to filbronectin. Of these adhesion molecules, filbronectin‐binding proteins play a role in the pathogenicity of S. aureus, although it is not yet clear whether they enhance its virulence. We have previously shown that filbronectin‐bound S. aureus is efficiently phagocytosed by thioglycolate‐induced mouse peritoneal macrophages. Bacterial ingestion is mediated by Very Late Antigen‐5 (VLA‐5; α5β1 integrin) and is accompanied by the formation of adhesion complexes. Here we show that the expression of VLA‐5 is restricted to thioglycolate‐induced inflammatory macrophages and is not found in the resident macrophages. When cells were in suspension, α5 integrin was not expressed on the surface of either resident or inflammatory macrophages, whereas in adherent cells, this integrin was distributed on the surface of inflammatory but not resident macrophages. A high level of this integrin was present in the cytoplasmic region only in inflammatory macrophages. In agreement with this, filbronectin‐mediated phagocytosis of S. aureus was observed only in the inflammatory macrophages. In inflammatory macrophages ingesting fibronectin‐bound S. aureus, α5 integrin was concentrated close to the phagocytosed bacteria. This change in distribution was not found in macrophages ingesting untreated bacteria. Together with our previous work, these results indicate that, upon ingestion of fibronectin‐bound S. aureus, VLA‐5 accumulates in the area of phagocytosis in inflammatory macrophages, where it forms adhesion complexes.

Virolysis and In Vitro Neutralization of HIV-1 by Humanized Monoclonal Antibody hNM-01

Antibody humanization by transplanting the complimentarity determining region (CDR) to a human framework aims to reduce the response of the human immune system against a foreign molecule during passive immunization. We transferred the CDR from the murine monoclonal antibody (MAb) NM-01 to a human IgG frame. The humanized NM-01 (hNM-01) recognizes the same epitope on Human Immunodeficiency Virus type 1 (HIV-1) envelope as its murine progenitor, but with greater efficiency, and shows enhanced neutralization of HIV-1. We have shown that this increase in reactivity may be attributed to residue 4 of the humanized kappa chain, where the presence of a methionine residue rather than the murine leucine appears to promote a more advantageous conformation of the antigen-binding site, perhaps via packing interactions with the V(kappa) CDR1. The capacity of humanized NM-01 to neutralize direct clinical isolates was also examined with the expectation that hNM-01 will prove suitable for development as a therapeutic agent. This reshaped antibody reacted with several clinical isolates of HIV-1 tested. Moreover, we proved the ability of this antibody of its activation of complement by flow cytometry and electron microscopy analysis. Although hNM-01 alone was capable of neutralizing HIV-1, the presence of complement enhanced neutralization. The enhancement of complement activation was also observed in hNM-01 than murine progenitor. This finding supports a potential role for antibody-dependent complement-mediated virolysis and more effective neutralization in HIV-1 therapy.

Mechanistic effect of NMSO3 on replication of human immunodeficiency virus.

NMSO3, a sulphated sialyl lipid, was evaluated for its efficacy against human immunodeficiency virus type 1 (HIV-1). The compound exhibited concentration-dependent inhibition of HIV-1 replication in primary infection cell culture systems. Substantial inhibition was observed at concentrations of NMSO3 that showed little cytotoxicity. NMSO3 also exhibited anti HIV-1 activity in chronically HIV-1 infected cultures. The production of progeny viruses was completely abolished without cytotoxicity by continuous addition of NMSO3 to chronically infected U937 cells. Furthermore, in attempting to define the inhibitory mechanism of NMSO3, we investigated its effect on several steps of the HIV-1 replication cycle. NMSO3 competes with gp120 for binding to CD4 receptors on cells and inhibits the entry of HIV-1. By epitope analysis of the human CD4 molecule, NMSO3 inhibits the binding of antibodies, which recognize the D1 domain of CD4. Moreover, semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR) showed that the integrated provirus is transcriptionally inactive in NMSO3-treated cells, supporting the lack of progeny in the culture supernatant of chronically HIV-1-infected cells treated with NMSO3. These findings indicate that NMSO3 has a unique mechanism of action against HIV-1 in both primary and chronic infection, and may be a valuable compound for the treatment of HIV-1 infection.

Quantification of the CD55 and CD59, Membrane Inhibitors of Complement on HIV-1 Particles as a Function of Complement-Mediated Virolysis

Previous studies have demonstrated that the murine monoclonal antibody (MoAb) NM‐01 activates the human complement classical pathway resulting in lysis of human immunodeficiency virus (HIV). The present study was performed to determine the availability of the V3‐loop of gp120 relative to the complement regulatory proteins, CD55 (DAF) and CD59 (HRF20) molecules on HIV. The results demonstrate that CD55 and CD59 exist on HIV virions, along with gp120 molecules. These findings suggest that activation of human complement on free viral particles is induced by MoAb NM‐01 and that this occurs regardless of the presence of CD55 and CD59 molecules. The destruction of viral particles was demonstrated by a decrease in infectivity. The involvement of human complement in this process was confirmed with an immunoelectron microscopy technique by the presence of a human C9 to prove membrane attack complex (MAC). The results indicate that NM‐01 can induce complement activation because of the ratios of CD55 and CD59 to gp120 molecules on HIV virions. The availability of the gp120 V3 domain on the virion is sufficient for binding of NM‐01 and thereby the formation of MAC that results in virolysis.

Morphological study of the TK cholangiocarcinoma cell line with three-dimensional cell culture

Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well‑characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three‑dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3‑20 days. The morphology of the TK cells was investigated by phase‑contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell‑to‑scaffold attachment in the three‑dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three‑dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19‑9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.

Cholangiocarcinoma cell line TK may be useful for the pharmacokinetic study of the chemotherapeutic agent gemcitabine.

Cholangiocarcinoma is a disease with a poor prognosis. A human cholangiocarcinoma cell line, TK, was previously established to enable further understanding of the disease. We conducted this investigation to determine whether or not the TK line is useful for pharmacokinetic study of the chemotherapeutic agent gemcitabine (GEM). Along with the BXPC3 human pancreatic adenocarcinoma cell line, the sensitivity to and effects on the TK cell line of GEM were compared. The influence of deoxycytidine kinase (dCK) transduction was also comparatively investigated. The effects of GEM in terms of drug sensitivity of the TK cell line, cell cycle and levels of transcripts of key enzymes were comparable to the BXPC3 cell line. Responses to the drug were similar in both cell lines. In contrast to pancreatic carcinoma, cell lines for research on cholangiocarcinoma have been limited. This study suggests the application of the TK cell line to the pharmacokinetic study of the chemosensitization of therapeutic drugs, such as GEM.