Search

Although modulation of claudin-1-based tight junction (TJ) in stratum granulosum is an option for transdermal absorption of drugs, granular permeation enhancers have never been developed. We previously found that homoharringtonine (HHT), a natural alkanoid, weakened intestinal epithelial barrier with changing expression and cellular localization of TJ components such as claudin-1 and claudin-4. In the present study, we investigated whether HHT is an epidermal granular permeation enhancer. Treatment of normal human epidermal keratinocytes (NHEK) cells with HHT decreased claudin-1 and claudin-4 but not zonula occludens-1 and E-cadherin. HHT lowered TJ-integrity in NHEK cells, accompanied by permeation-enhancement of dextran (4 kDa) in a dose-dependent manner. Transdermal treatment of mice with HHT weakened epidermal barrier. HHT treatment enhanced transdermal absorption of dextran with a molecular mass of up to 10 kDa. Together, HHT may be a transdermal absorption enhancer.

Lentinula edodes mycelia solid culture extract (MSCE) is used as a medical food ingredient and provides beneficial effects to patients with cancer and chronic type C hepatitis. Low molecular weight lignin (LM-lignin), which is an active component of MSCE, exhibits hepatoprotective, antitumor, antiviral, and immunomodulatory effects. In this study, we investigated the effect of LM-lignin/lignosulfonic acid on intestinal barrier function. Lignosulfonic acid enhanced transepithelial membrane electrical resistance in human intestinal Caco-2 cell monolayers. In Caco-2 cells treated with lignosulfonic acid, expression of claudin-2, which forms high conductive cation pores in tight junctions (TJs), was decreased. Lignosulfonic acid also attenuated the barrier dysfunction that is caused by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. TNF-α- and IFN-γ-induced activation of NF-κB, such as translocation of NF-κB p65 into the nucleus and induction of gene expression, was inhibited by lignosulfonic acid treatment. Furthermore, lignosulfonic acid decreased the TNF-α- and IFN-γ-induced increase in interleukin (IL)-1β and IL-6 expression in Caco-2 cells. These results suggest that lignosulfonic acid not only enhances TJ barrier function but also restores TJ barrier integrity impaired by inflammatory cytokines. Therefore, lignosulfonic acid may be beneficial for the treatment of inflammation-induced intestinal barrier dysfunction observed in inflammatory bowel disease.

Claudins are key functional and structural components of tight junctions (TJs) in epithelial cell sheets. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to claudin-4 and reversibly modulates intestinal TJ seals, thereby enhancing paracellular transport of solutes. However, the use of C-CPE as an absorption enhancer is limited by the molecule’s immunogenicity and manufacturing cost. Here, we developed a high-throughput screening system based on the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method to identify claudin-4 binders in a library collection of 32,560 compounds. Thiostrepton, identified from the screen, decreased transepithelial electrical resistance and increased flux of 4-kDa fluorescein isothiocyanate–labelled dextran (FD-4) in Caco-2 cell monolayers, a model of intestinal epithelium. Thiostrepton changed the expression, but not the localisation, of TJ components. Treatment of rat jejunum with thiostrepton increased the absorption of FD-4 without tissue toxicity, indicating that thiostrepton is a novel claudin-4 binder that enhances intestinal permeability. The screening system may therefore be a useful tool for identifying claudin-4 binders to enhance drug absorption in mucosa.

Disruption of the gastrointestinal epithelial barrier is a hallmark of chronic inflammatory bowel diseases (IBDs). The transmembrane protein claudin 2 (CLDN2) is a component of epithelial tight junctions (TJs). In the intestines of patients with IBDs, the expression of the pore-forming TJ protein CLDN2 is upregulated. Although CLDN2 is involved in these leaky barriers, whether it can be a target to enhance TJ integrity is unknown because a CLDN2-specific inhibitor has not been developed. Here, we used DNA immunization to generate a monoclonal antibody (mAb) that recognized an extracellular loop of CLDN2. Treatment of epithelial cell monolayers with the mAb increased barrier integrity. In addition, the anti-CLDN2 mAb attenuated the decrease in TJ integrity induced by the proinflammatory cytokine tumor necrosis factor-α (TNF-α), and cotreatment of cells with anti-TNF-α mAb and anti-CLDN2 mAb showed additive attenuating effects. These findings indicate that CLDN2 may be a target for enhancing TJ integrity, and CLDN2 binder may be an enhancer of mucosal barrier integrity and a potential therapeutic option for IBDs.

A limiting barrier for mucosal absorption of drugs is the tight junction (TJ). TJs exist between two adjacent cells (bicellular TJ, bTJ) and at the sites where three cells meet (tricellular TJ, tTJ). We present a novel approach which employs a physiologically regulated pathway for the passage of large molecules through the tTJ. Main barrier-relevant tTJ proteins are tricellulin and angulin-1 to -3. We developed an angulin binder from Clostridium perfringens iota-toxin (Ib) whose receptor is angulin-1. An Ib fragment corresponding to amino acids 421-664 (Ib421-664) of iota-toxin proved to bind in cells expressing angulin-1 and -3, but not angulin-2. This binding led to removal of angulin-1 and tricellulin from the tTJ which enhanced the permeation of macromolecular solutes. Ib421-664 enhanced intestinal absorption in rats and mice. Our findings indicate that Ib421-664, which we designate angubindin-1, is a modulator of the tTJ barrier and that modulation of that barrier qualifies for a new strategy of developing a mucosal absorption enhancer.

Background/Aims: Although proinflammatory cytokine–induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α. Methods: To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER) and paracellular permeability were measured. Production levels and localization of tight junction (TJ) proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC) and MLC kinase (MLCK) mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively. Results: Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition. Conclusion: Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation.

Tight junctions (TJs) are complex biochemical structures that seal the intercellular space and prevent the free movement of solutes across epithelial cell sheets. Modulating the TJ seal is a promising option for increasing the transdermal absorption of drugs. Within TJs, the binding of the claudin (CLDN) family of tetratransmembrane proteins through cis- and trans-interactions is an integral part of seal formation. Because epidermal TJs contain CLDN-1 and CLDN-4, a binder for these CLDNs may be a useful modulator of the permeability of the epidermal barrier. Here, we investigated whether m19, which can bind to CLDN-1/-4 (also CLDN-2/-5), modulates the integrity of epidermal TJs and the permeability of cell sheets to solutes. Treatment of normal human epidermal keratinocytes (NHEKs) with the CLDN binder reduced the integrity of TJs. A CLDN-1-specific binder (a monoclonal antibody, clone 7A5) also weakened the TJ seal in NHEKs. Although m19 attenuated the TJ barrier in human intestinal epithelial cells (Caco-2), 7A5 did not. Treatment of NHEKs with 7A5 enhanced permeation of a paracellular permeation marker. These findings indicate that CLDN-1 is a potential target for modulating the permeability of the epidermis, and that our CLDN-1 binder is a promising candidate molecule for development as a dermal absorption enhancer.

Lentinula edodes mycelia solid culture extract (MSCE) contains several bioactive molecules, including some polyphenolic compounds, which exert immunomodulatory, antitumor, and hepatoprotective effects. In this study, we examined the anti-hepatitis C virus (HCV) activity of MSCE and low-molecular-weight lignin (LM-lignin), which is the active component responsible for the hepatoprotective effect of MSCE. Both MSCE and LM-lignin inhibited the entry of two HCV pseudovirus (HCVpv) types into Huh7.5.1 cells. LM-lignin inhibited HCVpv entry at a lower concentration than MSCE and inhibited the entry of HCV particles in cell culture (HCVcc). MSCE also inhibited HCV subgenome replication. LM-lignin had no effect on HCV replication, suggesting that MSCE contains additional active substances. We demonstrate here for the first time the anti-HCV effects of plant-derived LM-lignin and MSCE. The hepatoprotective effect of LM-lignin suggests that lignin derivatives, which can be produced in abundance from existing plant resources, may be effective in the treatment of HCV-related diseases.

Claudins constitute a family of at least 27 proteins with four transmembrane domains, and play a pivotal role in maintaining tight-junctions seals in diverse epithelial tissues. The expression of claudin-4 often changes in intestinal tissues of inflammatory bowel disease and various human cancers. Therefore, claudin-4 is a promising target for treatment of these diseases. In our previous study, we established a reporter cell line to monitor claudin-4 expression on the basis of a functional claudin-4 promoter. Using this cell line, we have performed a cell-based screen of a library containing 2642 biologically active small-molecule compounds to identify modulators of claudin-4 expression. The screen identified 24 potential modulators of the claudin-4 promoter activity. Fourteen of these compounds (12 of them novel) induced endogenous claudin-4 expression. The identified compounds might serve as lead compounds targeting aberrant gene expression in inflammatory bowel disease.

Homoharringtonine (HHT), a natural alkaloid produced by various Cephalotaxus species, has antileukemic activity in acute and chronic myelogenous leukemia. However, HHT can also induce unanticipated effects in the gastrointestinal tract, such as diarrhea and nausea/vomiting, but the mechanism behind these adverse effects has not been clarified. In the present study, we show that HHT affects the epithelial permeability of intestinal Caco-2 cell monolayers. HHT reduced the transepithelial electrical resistance (TER) of Caco-2 cells in a dose- and time-dependent manner. The HHT effect was reversible and no cytotoxicity was observed at the concentrations used. HHT simultaneously increased the paracellular flux of the 4 kDa and 40 kDa FITC-dextrans associated with the TER reduction. Immunoblotting analysis revealed that HHT decreased the protein expression of TJ components such as claudin-3, -5, and -7. However, the transcription levels of these claudins were not repressed by HHT treatment. HHT also disturbed the cellular localization of claudin-1 and -4. These changes coincided with the reduced barrier function. Our findings suggest that HHT enhances the paracellular permeability of Caco-2 cell monolayers by modulating the protein expression and localization of claudin isoforms; these actions might be responsible for the gastrointestinal effects of HHT.

Claudin-2 (CLDN-2), a pore-forming tight junction protein with a tetra-transmembrane domain, is involved in carcinogenesis and the metastasis of some cancers. Although CLDN-2 is highly expressed in the tight junctions of the liver and kidney, whether CLDN-2 is a safe target for cancer therapy remains unknown. We recently generated a rat monoclonal antibody (mAb, clone 1A2) that recognizes the extracellular domains of human and mouse CLDN-2. Here, we investigated the safety of CLDN-2-targeted cancer therapy by using 1A2 as a model therapeutic antibody. Because most human therapeutic mAbs are IgG1 subtype that can induce antibody-dependent cellular cytotoxicity, we generated a human–rat chimeric IgG1 form of 1A2 (xi-1A2). xi-1A2 activated Fcγ receptor IIIa in the presence of CLDN-2-expressing cells, indicating that xi-1A2 likely exerts antibody-dependent cellular cytotoxicity. At 24 h after its intravenous injection, xi-1A2 was distributed into the liver, kidney, and tumor tissues of mice bearing CLDN-2-expressing fibrosarcoma cells. Treatment of the xenografted mice with xi-1A2 attenuated tumor growth without apparent adverse effects, such as changes in body weight and biochemical markers of liver and kidney injury. These results support xi-1A2 as the lead candidate mAb for safe CLDN-2-targeted cancer therapy.

Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood-brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood-brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood-brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood-brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders.

Intestinal permeation enhancers are a crucial component of many oral formulations, without which many drugs would show an insufficient absorption in the gut. The present study sought to provide a better understanding of the molecular interaction of such absorption enhancers with the intestine, by investigating the effect of the surfactant-like permeation enhancer dodecylmaltoside (DDM) on Caco-2 cells. The extent to which the action of DDM is apportioned between the para- and transcellular routes was addressed by examining the transport of relevant marker compounds ([3H]-mannitol and [3H]-propranolol, respectively). In the case of [3H]-mannitol, a robust permeation enhancement was achieved with 0.5 mM DDM (∼6-fold), whereas little effect was seen on the permeation of [3H]-propranolol. Concomitantly measured TEER values revealed a rapid onset of action of DDM with a swift recovery and complete restitution (>90%) within 4 h after washout. To localize the site(s) of action of DDM at the absorptive surface of Caco-2 cells, sulfo-NHS-SS-biotin, a membrane-impermeable compound, was applied apically. In the presence of 0.5 mM DDM, translocated biotin was found to be accumulated toward bicellular contacts, whereas no biotin permeation was observed in untreated control cells. Western blot analysis of DDM-treated and untreated Caco-2 cells revealed an interaction of DDM with specific tight junction associated proteins, resulting in a reduction of claudin-3 and -4 and also occludin, as well as a depletion of claudin-2 from lipid rafts. Collectively, the results presented provide a more in depth understanding of the molecular mechanism(s) underlying the permeation-enhancing actions of DDM.

A current bottleneck in the development of central nervous system (CNS) drugs is the lack of drug delivery systems targeting the CNS. The intercellular space between endothelial cells of the blood-brain barrier (BBB) is sealed by complex protein-based structures called tight junctions (TJs). Claudin-5 (CLDN-5), a tetra-transmembrane protein is a key component of the TJ seal that prevents the paracellular diffusion of drugs into the CNS. In the present study, to investigate whether CLDN-5 binders can be used for delivery of drugs to the CNS, we generated monoclonal antibodies (mAbs) specific to the extracellular domains of CLDN-5. In an in vitro model of the BBB, the anti-CLDN-5 mAbs attenuated trans-epithelial/endothelial electrical resistance and enhanced solute permeation. These anti-CLDN-5 mAbs are potential leads for the development of novel drug delivery systems targeting the CNS.

We previously found that claudin (CL) is a potent target for cancer therapy using a CL-3 and -4-targeting molecule, namely the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Although CL-3 and -4 are expressed in various normal tissues, the safety of this CL-targeting strategy has never been investigated. Here, we evaluated the tissue distribution of C-CPE in mice. Ten minutes after intravenous injection into mice, C-CPE was distributed to the liver and kidney (24.0% and 9.5% of the injected dose, respectively). The hepatic level gradually fell to 3.2% of the injected dose by 3 h post-injection, whereas the renal C-CPE level gradually rose to 46.5% of the injected dose by 6 h post-injection and then decreased. A C-CPE mutant protein lacking the ability to bind CL accumulated in the liver to a much lesser extent (2.0% of the dose at 10 min post-injection) than did C-CPE, but its renal profile was similar to that of C-CPE. To investigate the acute toxicity of CL-targeted toxin, we intravenously administered C-CPE-fused protein synthesis inhibitory factor to mice. The CL-targeted toxin dose-dependently increased the levels of serum biomarkers of liver injury, but not of kidney injury. Histological examination confirmed that injection of CL-targeted toxin injured the liver but not the kidney. These results indicate that potential adverse hepatic effects should be considered in C-CPE-based cancer therapy.

Claudin‐4 (CLDN‐4), a tight‐junction protein, is overexpressed in various malignant tumors, including gastric, colorectal, pancreatic, and breast cancers. However, CLDN‐4 is also expressed in normal tissues, including the liver, pancreas, kidney, and small intestine. Whether CLDN‐4 is an effective and safe target for cancer therapy has been unclear owing to the lack of a binder with both CLDN‐4 specificity and cross‐reactivity to human and murine cells. In this study, we successfully generated a rat anti‐CLDN‐4 monoclonal antibody (5D12) that was specific to, and cross‐reactive with, human and mouse CLDN‐4. 5D12 recognized the second extracellular domain of human CLDN‐4 in a conformation‐dependent manner. A human–rat chimeric IgG1 of 5D12 (xi‐5D12) activated the Fcγ IIIa receptor, indicating the activation of antibody‐dependent cellular cytotoxicity in CLDN‐4‐expressing cells. Moreover, xi‐5D12 significantly suppressed tumor growth in mice bearing human colorectal and gastric tumors without apparent adverse effects, such as weight loss or liver and kidney damage. These results suggest that CLDN‐4 is a potent target for cancer therapy and that an anti‐CLDN‐4 antibody is a promising candidate anticancer agent.

Claudin-1 (CLDN-1), an integral transmembrane protein, is an attractive target for drug absorption, prevention of infection, and cancer therapy. Previously, we generated mouse anti-CLDN-1 monoclonal antibodies (mAbs) and found that they enhanced epidermal absorption of a drug and prevented hepatitis C virus infection in human hepatocytes. Here, we investigated anti-tumor activity of a human-mouse chimeric IgG1, xi-3A2, from one of the anti-CLDN-1 mAbs, clone 3A2. Xi-3A2 accumulated in the tumor tissues in mice bearing with human CLDN-1-expressing tumor cells. Xi-3A2 activated Fcγ receptor IIIa-expressing reporter cells in the presence of human CLDN-1-expressing cells, suggesting xi-3A2 has a potential to exhibit antibody-dependent cellular cytotoxicity against CLDN-1 expressing tumor cells. We also constructed a mutant xi-3A2 antibody with Gly, Ser, and Ile substituted with Ala, Asp, and Arg at positions 236, 239, and 332 of the Fc domain. This mutant antibody showed greater activation of Fcγ receptor IIIa and in vivo anti-tumor activity in mice bearing human CLDN-1-expressing tumors than xi-3A2 did. These findings indicate that the G236A/S239D/I332E mutant of xi-3A2 might be a promising lead for tumor therapy.

Claudin-4, a member of a tetra-transmembrane protein family that comprises 27 members, is a key functional and structural component of the tight junction-seal in mucosal epithelium. Modulation of the claudin-4-barrier for drug absorption is now of research interest. Disruption of the claudin-4-seal occurs during inflammation. Therefore, claudin-4 modulators (repressors and inducers) are promising candidates for drug development. However, claudin-4 modulators have never been fully developed. Here, we attempted to design a screening system for claudin-4 modulators by using a reporter assay. We prepared a plasmid vector coding a claudin-4 promoter-driven luciferase gene and established stable reporter gene-expressing cells. We identified thiabendazole, carotene and curcumin as claudin-4 inducers, and potassium carbonate as a claudin-4 repressor by using the reporter cells. They also increased or decreased, respectively, the integrity of the tight junction-seal in Caco-2 cells. This simple reporter system will be a powerful tool for the development of claudin-4 modulators.

The protein p14ARF is a known tumor suppressor protein controlling cell proliferation and survival, which mainly localizes in nucleoli. However, the regulatory mechanisms that govern its activity or expression remain unclear. Here, we report that a novel proapoptotic nucleolar protein, PANO, modulates the expression and activity of p14ARF in HeLa cells. Overexpression of PANO enhances the stability of p14ARF protein by protecting it from degradation, resulting in an increase in p14ARF expression levels. Overexpression of PANO also induces apoptosis under low serum conditions. This effect is dependent on the nucleolar localization of PANO and inhibited by knocking-down p14ARF. Alternatively, PANO siRNA treated cells exhibit a reduction in p14ARF protein levels. In addition, ectopic expression of PANO suppresses the tumorigenicity of HeLa cells in nude mice. These results indicate that PANO is a new apoptosis-inducing gene by modulating the tumor suppressor protein, p14ARF, and may itself be a new candidate tumor suppressor gene.

Tumor metastasis of epithelium-derived tumors is the major cause of death from malignant tumors. Overexpression of claudin is observed frequently in malignant tumors. However, claudin-targeting antimetastasis therapy has never been investigated. We previously prepared a claudin-4-targeting antitumor molecule that consisted of the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) fused to protein synthesis inhibitory factor (PSIF) derived from Pseudomonas exotoxin. In the present study, we investigated whether claudin CPE receptors can be a target for tumor metastasis by using the C-CPE-fused PSIF as a claudin-targeting agent. One of the most popular murine metastasis models is the lung metastasis of intravenously injected B16 cells. Therefore, we first investigated the effects of the C-CPE-fused PSIF on lung metastasis of claudin-4-expressing B16 (CL4-B16) cells. Intravenous administration of the C-CPE-fused PSIF suppressed lung metastasis of CL4-B16 cells but not B16 cells. Injection of C-CPE-fused PSIF also inhibited tumor growth and spontaneous lung metastasis of murine breast cancer 4T1 cells inoculated into the subcutis. Treatment with C-CPE-fused PSIF did not show apparent side effects in mice. These findings indicate that claudin targeting may be a novel strategy for inhibiting some tumor metastases.

The mycelia of the edible mushroom Lentinula edodes can be cultured in solid medium containing lignin, and the hot-water extracts (L.E.M.) is commercially available as a nutritional supplement. During the cultivation, phenolic compounds, such as syringic acid and vanillic acid, were produced by lignin-degrading peroxidase secreted from L. edodes mycelia. Since these compounds have radical scavenging activity, we examined their protective effect on oxidative stress in mice with CCl(4)-induced liver injury. We examined the hepatoprotective effect of syringic acid and vanillic acid on CCl(4)-induced chronic liver injury in mice. The injection of CCl(4) into the peritoneal cavity caused an increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The intravenous administration of syringic acid and vanillic acid significantly decreased the levels of the transaminases. Four weeks of CCl(4) treatment caused a sufficiently excessive deposition of collagen fibrils. An examination of Azan-stained liver sections revealed that syringic acid and vanillic acid obviously suppressed collagen accumulation and significantly decreased the hepatic hydroxyproline content, which is the quantitative marker of fibrosis. Both of these compounds inhibited the activation of cultured hepatic stellate cells, which play a central role in liver fibrogenesis, and maintained hepatocyte viability. These data suggest that the administration of syringic acid and vanillic acid could suppress hepatic fibrosis in chronic liver injury.

We developed a unique method for converting atmospheric aldehyde into alcohol using formaldehyde dehydrogenase from Pseudomonas putida (PFDH) doped in a polymer film. A film of poly(2-methacryloyloxyethylphosphorylcholine-co-n-butyl methacrylate) (PMB), which has a chemical structure similar to that of a biological membrane, was employed for its biocompatibility. A water-incorporated polymer film entrapping PFDH and its cofactor NAD(+) was obtained by drying a buffered solution of PMB, PFDH, and NAD(+). The aldehydes in the air were absorbed into the polymer film and then enzymatically oxidized by PFDH doped in the PMB film. Interestingly, alcohol and carboxylic acid were produced by the enzymatic reaction, indicating that PFDH catalyzes dismutation of aldehyde in the PMB film. Importantly, a PFDH-PMB film catalyzes aldehyde degradation without consuming the nucleotide cofactor, thereby allowing repeated use of the film. The activity of PFDH in the PMB film was higher than that in other common water-soluble polymers, suggesting that the hydrational state in a phospholipid polymer matrix is suitable for enzymatic activity.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive form of human leukemia/lymphoma. Although this disease is initiated by infection with human T-lymphotropic virus type 1 (HTLV-1), many HTLV-1 carriers survive for a long period without aggressive illness, suggesting that other factors may play roles in the progression of ATLL to an aggressive state. However, the mechanism involved in this progression still remains unclear. Previously, we have reported that ASY/Nogo mRNA was markedly down-regulated in human small-cell lung carcinomas, whereas it was expressed in normal tissues and other lung carcinomas, such as adenocarcinoma and squamous cell carcinoma. To understand whether or not ASY/Nogo gene is involved in the progression of ATLL, we examined the expression of ASY/Nogo mRNA in smoldering, chronic and aggressive ATLL, and found that the expression level of ASY/Nogo mRNA was markedly reduced in clinically aggressive ATLL. HTLV-1 Tax expression was not affected by the down-regulation of ASY/Nogo mRNA. These results indicate that the ASY/Nogo gene may act as a suppressor against ATLL progression, independent of Tax expression.

Claudin-1 (CLDN1), a known host factor for hepatitis C virus (HCV) entry and cell-to-cell transmission, is a target molecule for inhibiting HCV infection. We previously developed four clones of mouse anti-CLDN1 monoclonal antibody (mAb) that prevented HCV infection in vitro. Two of these mAbs showed the highest antiviral activity. Here, we optimized the anti-CLDN1 mAbs as candidates for therapeutics by protein engineering. Although Fab fragments of the mAbs prevented in vitro HCV infection, their inhibitory effects were much weaker than those of the whole mAbs. In contrast, human chimeric IgG1 mAbs generated by grafting the variable domains of the mouse mAb light and heavy chains inhibited in vitro HCV infection as efficiently as the parental mouse mAbs. However, the chimeric IgG1 mAbs activated Fcγ receptor, suggesting that cytotoxicity against mAb-bound CLDN1-expressing cells occurred through the induction of antibody-dependent cellular cytotoxicity (ADCC). To avoid ADCC-induced side effects, we prepared human chimeric IgG4 mAbs. The chimeric IgG4 mAbs did not activate Fcγ receptor or induce ADCC, but they prevented in vitro HCV infection as efficiently as did the parental mouse mAbs. These findings indicate that the IgG4 form of human chimeric anti-CLDN1 mAb may be a candidate molecule for clinically applicable HCV therapy.

Efficient vaccine delivery to mucosal tissues including mucosa-associated lymphoid tissues is essential for the development of mucosal vaccine. We previously reported that claudin-4 was highly expressed on the epithelium of nasopharynx-associated lymphoid tissue (NALT) and thus claudin-4-targeting using C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) effectively delivered fused antigen to NALT and consequently induced antigen-specific immune responses. In this study, we applied the C-CPE-based vaccine delivery system to develop a nasal pneumococcal vaccine. We fused C-CPE with pneumococcal surface protein A (PspA), an important antigen for the induction of protective immunity against Streptococcus pneumoniae infection, (PspA-C-CPE). PspA-C-CPE binds to claudin-4 and thus efficiently attaches to NALT epithelium, including antigen-sampling M cells. Nasal immunization with PspA-C-CPE induced PspA-specific IgG in the serum and bronchoalveolar lavage fluid (BALF) as well as IgA in the nasal wash and BALF. These immune responses were sufficient to protect against pneumococcal infection. These results suggest that C-CPE is an efficient vaccine delivery system for the development of nasal vaccines against pneumococcal infection.

Epithelium plays pivotal roles in barriers that separate the inside of the body from the outside environment. Passage across epithelial barriers is the first step in drug absorption. Epidermal absorption is an ideal route for drug administration because it is non-invasive and easily controlled. Claudin-1 (CLDN1) seals tight junctions in the stratum granulosum, but whether modulation of CLDN1 enhances epidermal permeability is unclear. We investigated the effect of an anti-CLDN1 antibody on the epidermal epithelial barrier by using human epidermal keratinocytes (NHEK), a popular in vitro model of human epithelial cell sheets. Immunoblot and FACS analyses showed that CLDN1 was expressed on NHEK cell membranes. Treatment of NHEK monolayers with anti-CLDN1 antibody decreased the integrity of tight junctions between adjacent cells and enhanced the permeation of solutes across the cell sheets. Furthermore, treatment of Caco-2 cell monolayers, a popular model of the human intestinal barrier that expresses CLDN1 ...

Epithelial cell sheets have two types of intercellular tight junctions (TJs): bicellular tight junctions (bTJs) and tricellular tight junctions (tTJs). Modulation of bTJs has been investigated in n...

Claudin (CLDN) is a key component of the epithelial tight junction. Expression of the various CLDNs differs among tissues and is deregulated in malignant tumors. For example, CLDN4 is frequently overexpressed in pancreatic, breast, and ovarian cancers and therefore may be an attractive target for anticancer drugs. However, the low antigenicity of CLDN4 has hampered the development of antibody-based CLDN4-targeted cancer therapy. We successfully generated monoclonal antibodies (mAbs) to CLDN4 and evaluated their anti-tumor activity. Briefly, we immunized rats with plasmids containing the CLDN4 coding sequence. We fused the resulting B cells with myeloma cells and screened 8 hybridoma clones that produced anti-CLDN4 mAbs. All 8 mAbs bound to CLDN4-expressing cells but not to those expressing CLDN1, 2, 5, 6, 7or 9. All 8 mAbs showed CLDN4 binding-dependent activation of the Fcγ receptor. A clone with high affinity to CLDN4 suppressed tumor growth in vitro and in vivo. Interestingly, treatment of cancer cells...

Claudins (CLDNs) comprise a tetra-transmembrane protein family with ~20 members, which are located in the lateral membrane between adjacent epithelial cells. Because of loss of cellular polarity during malignant transformation, CLDN often migrates from the lateral membrane and becomes exposed on epithelial cell surfaces. CLDN3 and CLDN4 are targets for tumor therapy because they are overexpressed in various malignant tumors. We previously optimized a protocol for immunization with CLDN and developed 8 rat anti-CLDN4 monoclonal antibodies, one of which recognized both CLDN3 and CLDN4. In this study, we focused on the CLDN3/4 antibody. The CLDN3/4 antibody showed high affinity for CLDN4 (KD value: 4.3 nM). We fluorescently labeled the CLDN3/4 antibody and intravenously injected it into mice bearing tumors (human colon cancer Lovo cells, or human gastric cancer MKN74 cells). The CLDN3/4 antibody accumulated in tumor tissues and attenuated tumor growth without apparent adverse effects in the Lovo- and MKN74-b...

In this study, we evaluated a baculoviral display system for analysis of viral entry by using a recombinant adenovirus (Ad) carrying a luciferase gene and budded baculovirus (BV) that displays the adenoviral receptor, coxsackievirus and adenovirus receptor (CAR). CAR-expressing B16 cells (B16-CAR cells) were infected with luciferase-expressing Ad vector in the presence of BV that expressed or lacked CAR (CAR-BV and mock-BV, respectively). Treatment with mock-BV even at doses as high as 5 µg/mL failed to attenuate the luciferase activity of B16-CAR cells. In contrast, treatment with CAR-BV with doses as low as 0.5 µg/mL significantly decreased the luciferase activity of infected cells, which reached 65% reduction at 5 µg/mL. These findings suggest that a receptor-displaying BV system could be used to evaluate viral infection.

The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is the most well-characterized claudin-3 and -4 (CL-3 and -4) binder. The mucosal absorption-enhancing activity of C-CPE is over 400-fold more potent than a clinically used enhancer. C-CPE has potential in cancer therapy and for mucosal vaccination. Since CL-3 and -4 are ubiquitously expressed in heart, lung, liver, kidney, thyroid gland, stomach, and intestine, safety evaluations are important for their future therapeutic use. Here, we investigated the tissue distribution of C-CPE in mice after intravenous administration. We prepared CF750-labeled wild-type C-CPE and C-CPE harboring mutant CL-3/-4 binding regions. FACS analysis showed that labeled wild-type C-CPE bound to CL-4 but that labeled mutant C-CPE did not, indicating that labeling did not alter the CL-binding properties of C-CPE. Most wild-type and mutant C-CPE promptly accumulated in the kidney, and C-CPE were more distributed in the liver, thyroid gland and intestine than m...

Previously, we isolated cell lines that display various degrees of transformed phenotypes from a single-cell population of human diploid fibroblasts (RB) containing a large deletion (13q14-22) in one copy of chromosome 13. They included a cell line transfected with SV40 early genes (RBSV), an immortalized cell line (RBI), an anchorage-independent cell line (RBS), and a tumorigenic cell line (RBT). Here, we analyzed gene expression profiles in these cell lines and showed that expression of some fibroblast-specified or mesenchyme-specified genes were downregulated, and those of stem cell-specified genes, including NANOG, were upregulated during malignant progression. When NANOG expression was knocked down with a short hairpin NANOG expression vector (shNANOG vector) in the RBS and RBT cells, the anchorage independency and tumorigenicity were repressed. We next examined various cancer cell lines for NANOG expression and showed that some cancer cell lines expressed a high level of normal and/or variant NANOG proteins. Overexpression of NANOG mRNA in lung adenocarcinoma was also shown by in situ hybridization. All these data indicate the involvement of NANOG in tumorigenesis.