Kohta Kurohane

Anti-neovascular therapy using novel peptides homing to angiogenic vessels.

Cancer chemotherapy targeted to angiogenic vessels is expected to cause indirect tumor regression through the damage of the neovasculature without the induction of drug resistance. To develop a tool for neovasculature-specific drug delivery, we isolated novel peptides homing to angiogenic vessels formed by a dorsal air sac method from a phage-displayed peptide library. Three distinct phage clones that markedly accumulated in murine tumor xenografts presented PRPGAPLAGSWPGTS-, DRWRPALPVVLFPLH- or ASSSYPLIHWRPWAR-peptide respectively. After the determination of the epitope sequences of these peptides, we modified liposomes with epitope penta-peptides. Liposome modified with APRPG-peptide showed high accumulation in murine tumor xenografts, and APRPG-modified liposome encapsulating adriamycin effectively suppressed experimental tumor growth. Finally, specific binding of APRPG-modified liposome to human umbilical endothelial cells, and that of PRP-containing peptide to angiogenic vessels in human tumors, i.e., islet cell tumor and glioblastoma, were demonstrated. The present study indicates the usefulness of APRPG-peptide as a tool for anti-neovascular therapy, a novel modality of cancer treatment.

Photodynamic therapy targeted to tumor-induced angiogenic vessels

Cancer photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) may be effective not only by being directly cytotoxic to tumor cells, but also by being cytotoxic to the endothelium of tumor neovasculature. In the present study, we investigated the effect of PDT with an experimental liposomal formulation of BPD-MA on tumor-induced angiogenic vessels using a murine dorsal air sac model. First, hemostasis of neovasculature was examined by varying the regimen of PDT. Laser irradiation at 15 min after injection of 2 mg/kg liposomal BPD-MA (15 min PDT) caused complete blocking of blood flow in neovasculature. In contrast, PDT did not inhibit blood flow when the irradiation occurred 3 h after the injection of liposomal BPD-MA (3 h PDT). Next, the antitumor effect of PDT on Meth A sarcoma-bearing mice was investigated by using the hemostasis-inducing regimen. Tumor growth was strongly inhibited after the 15 min PDT with BPD-MA at a dose of 0.5-2 mg/kg. In contrast, 3 h PDT with BPD-MA at a dose of 2 mg/kg suppressed tumor growth only partially. The current study indicates that 15 min PDT causes strong suppression of tumor growth, perhaps through damaging endothelial cells in the tumor neovasculature rather than through a direct cytotoxic effect on tumor cells.

Effects of phthalate esters on the sensitization phase of contact hypersensitivity induced by fluorescein isothiocyanate.

Background Many different types of phthalate ester are used as plasticizers and are thus found in the air. There have been several studies that suggest an association between allergies and phthalate esters. We previously found that di‐butyl phthalate (DBP) has an adjuvant effect in a mouse contact hypersensitivity model, in which fluorescein isothiocyanate (FITC) is involved as an immunogenic hapten.

Induction of Intensive Tumor Suppression by Antiangiogenic Photodynamic Therapy Using Polycation-Modified Liposomal Photosensitizer

The authors previously observed that antiangiogenic scheduling of photodynamic therapy (PDT) was effective in causing tumor regression through hemostasis. It would thus be expected that photosensitizer entrapped in polycation liposomes (PCLs) would be efficiently taken up in tumor‐derived angiogenic vascular endothelial cells due to the strong electrostatic adhesion between the polycation and the plasma membrane, thus resulting in enhanced phototherapeutic efficacy.

Suppression of tumor growth by novel peptides homing to tumor-derived new blood vessels.

Novel peptides homing to angiogenic vessels were recently isolated from a phage‐displayed random pentadecapeptide library. One of the isolated peptides, ASSSYPLIHWRPWAR, significantly suppressed the migration of VEGF‐stimulated human umbilical vein endothelial cells. Dendoric ASSSYPLIHWRPWAR‐peptide suppressed the formation of new blood vessels in dorsal air sac model mice. Furthermore, ASSSYPLIHWRPWAR‐peptide and the fragment peptides containing WRP, which is revealed to be an epitope sequence, significantly suppressed the tumor growth, although 15‐mer shuffled peptide derived from ASSSYPLIHWRPWAR and pentapeptides with alanine substitution of each residue of WRP did not. Taken together, ASSSYPLIHWRPWAR‐peptide may cause tumor dormancy through inhibition of angiogenesis, and the WRP sequence may be the minimal and essential sequence for this activity.

Induction of preferential chemotaxis of unstimulated B-lymphocytes by 2-arachidonoylglycerol in immunized mice

2‐Arachidonoylglycerol (2‐AG) is an endogenous ligand for cannabinoid receptors. There are two types of cannabinoid receptors, CB1 and CB2. We investigated the chemotactic activity of 2‐AG using mouse lymphocytes because cells in the immune system are known to express CB2. Spleen cell migration toward 2‐AG was observed, which was completely inhibited by SR144528, a CB2‐specific antagonist 2‐AG has been reported to induce a preferential B cell chemotaxis. We examined whether there is any difference in responsiveness during the activation of B cells. When spleen cells from immunized mice were tested, naive B cells but not germinal center B cells (GL7‐positive) were increased in the fraction attracted by 2‐AG. Furthermore, when Peyers patch lymphocytes were tested after oral administration of cholera toxin, the number of IgA– B cells was increased in the fraction attracted by 2‐AG. These results suggested that 2‐AG preferentially attracts unstimulated naive B cells rather than activated and/or class‐switched B cells. This property may influence the structure of B cell compartments in secondary lymphoid tissues.

TRPA1 and TRPV1 activation is a novel adjuvant effect mechanism in contact hypersensitivity

We have revealed that local stimulation of sensory neurons is involved in the adjuvant effect of dibutyl phthalate (DBP) in a fluorescein isothiocyanate-induced mouse contact hypersensitivity model. Transient receptor potential (TRP) A1 and TRPV1 seemed to be candidate DBP targets. Here we directly demonstrated that DBP activates a subset of neurons in mouse dorsal root ganglia responsive to TRPA1 and TRPV1 agonists. TRPA1 and TRPV1 activation was further demonstrated using cultured cells expressing TRP channels. Among structurally different phthalate esters, there is a positive relationship between the activation of TRPA1- or TRPV1-expressing cells and the adjuvant effect.

Effects of Phthalate Esters on Dendritic Cell Subsets and Interleukin‐4 Production in Fluorescein Isothiocyanate‐Induced Contact Hypersensitivity

Phthalate esters with short alkyl chains, such as di‐ethyl (DEP), di‐n‐propyl (DPP), and di‐butyl phthalate (DBP), have adjuvant effects on an FITC‐induced contact hypersensitivity mouse model. The adjuvant effects of DPP and DBP are associated with enhanced trafficking of FITC‐presenting CD11b+ dendritic cells (DC). DEP has relatively weak activity as to FITC‐positive cell migration. Here we demonstrated that DBP and DPP also increased the number of FITC‐positive CD8alpha+ DC in draining lymph nodes. We also found enhanced production of interleukin‐4 in draining lymph nodes after FITC sensitization with DEP, DPP, or DBP, suggesting an additional adjuvant mechanism of phthalate esters.

Influence of Local Treatments with Capsaicin or Allyl Isothiocyanate in the Sensitization Phase of a Fluorescein-Isothiocyanate-Induced Contact Sensitivity Model

Background: In fluorescein isothiocyanate (FITC)-induced contact hypersensitivity models, dibutyl phthalate has been empirically used as a solvent ingredient. We have demonstrated that dibutyl phthalate has an adjuvant effect through the facilitation of trafficking FITC-presenting dendritic cells (DC) from the skin to draining lymph nodes. Here we investigated the effects of local pretreatment with substances that are capable of desensitizing sensory neurons in the sensitization phase. Methods: Local pretreatment of BALB/c mice with capsaicin (epicutaneous), allyl isothiocyanate (epicutaneous) or a truncated form of calcitonin gene-related peptide (CGRP8–37; intradermal) was performed before contact sensitization to FITC. The ear swelling test was employed to monitor sensitization. The appearance of FITC-presenting CD11c-positive cells in the draining lymph nodes was detected by flow cytometry. Cytokine production in local lymph node cell cultures was determined by ELISA. Results: The ear swelling response was reduced in mice pretreated with capsaicin or allyl isothiocyanate. DC trafficking and maturation (based on the levels of costimulators CD80 and CD86) were inhibited. Interleukin-4 production by local lymph nodes was suppressed with allyl isothiocyanate but not with capsaicin. Pretreatment with CGRP8–37 suppressed sensitization to FITC. Conclusions: Local pretreatment with substances that are capable of desensitizing sensory neurons through the respective transient receptor potential channels suppressed skin sensitization to FITC in a mouse model. This was associated with reduced trafficking and maturation of FITC-presenting DC. A CGRP antagonist also suppressed the sensitization to FITC, suggesting the possible involvement of sensory neurons in sensitization.

Production of Secretory Immunoglobulin A against Shiga Toxin-Binding Subunits in Mice by Mucosal Immunization

ABSTRACT The toxicity of Shiga toxins (Stx) depends on the binding of their B subunits to carbohydrate ligands on host cells. The production of antibodies against B subunits, especially immunoglobulin A (IgA) secreted on the mucosal surface, should contribute to host defense. One of the major problems in attempts to produce IgA against Stx was the poor immunogenicity of B subunits. We were able to produce serum IgA as well as IgG against Stx1B in mice of the H-2d haplotype by means of intranasal immunization with recombinant B subunits of Stx (Stx1B) together with cholera toxin as a mucosal adjuvant. Secretory IgA (S-IgA) was detected in nasal washes but not in feces. We prepared chemically cross-linked Stx1B for use as an immunogen, and the formation of stable oligomers was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. When the cross-linked Stx1B was used together with cholera toxin for the intranasal immunization of BALB/c mice, strong enhancement of the immune response was observed. The S-IgA titers in nasal washes were 16- to more than 64-fold higher than those in mice immunized with native Stx1B plus cholera toxin. Furthermore, fecal IgA was detectable when the cross-linked Stx1B was used. The use of cholera toxin was necessary for the induction of high titers of S-IgA in the nasal washes. However, the effect of cross-linking was dependent on the major histocompatibility complex haplotype; that is, no enhancement of IgA production was observed in C57BL/6 mice. The present results provide a practical means of producing IgA against Stx1B in BALB/c mice.