Impact of TRPV3 on the development of allergic dermatitis as a dendritic cell modulator.

The transient receptor potential channel vanilloid subfamily V member 3 (TRPV3), which functions as a thermosensor in keratinocytes, plays an important role in the development of allergic and itchy dermatitis in rodents. Although real-time PCR analysis using lesional and non-lesional skin samples from patients with atopic dermatitis showed that TRPV3 was expressed in lesional skin, the role that TRPV3 plays in patients with dermatitis is still relatively obscure. Here, we determined whether TRPV3 was a dendritic cell (DC) modulator using DS-Nh mice with a gain-of-function mutation in TRPV3 (TRPV3Gly573Ser), because increasing skin temperature is associated with the modulation of dermal dendritic cells (DCs). Interestingly, increased responses to haptens by skin and DCs were observed in DS-Nh mice compared with those from DS mice with wild-type TRPV3. Increased thymic stromal lymphopoietin (TSLP) responses were also observed in keratinocytes from DS-Nh mice compared with those from DS mice. Taken together, we propose that the DS-Nh mouse is a good model to use in order to better understand the role of this orphan channel and that TRPV3 may represent a new therapeutic target in certain types of dermatitis through the control of DCs.

Arthritis and pneumonitis produced by the same T cell clones from mice with spontaneous autoimmune arthritis.

SKG mice, a newly established model of rheumatoid arthritis (RA), spontaneously develop autoimmune arthritis accompanying extra-articular manifestations, such as interstitial pneumonitis. To examine possible roles of T cells for mediating this systemic autoimmunity, we generated T cell clones from arthritic joints of SKG mice. Two distinct CD8(+) clones were established and both showed in vitro autoreactivity by killing syngeneic synovial cells and a variety of MHC-matched cell lines. Transfer of each clone to histocompatible athymic nude mice elicited joint swelling and histologically evident synovitis accompanying the destruction of adjacent cartilage and bone. Notably, the transfer also produced diffuse severe interstitial pneumonitis. Clone-specific TCR gene messages in the inflamed joints and lungs of the recipients gradually diminished, becoming hardly detectable in 6-11 months; yet, arthritis and pneumonitis continued to progress. Thus, the same CD8(+) T cell clones from arthritic lesions of SKG mice can elicit both synovitis and pneumonitis, which chronically progress and apparently become less T cell dependent in a later phase. The results provide clues to our understanding of how self-reactive T cells cause both articular and extra-articular lesions in RA as a systemic autoimmune disease.

[New progress in crystallization technology of membrane protein and introduction of pharamaceutical innovation value chain].

We have recently established a Pharamaceutical Innovation Value Chain in collaboration with the SOSHO project (http://www.so-sho.jp) and BioGrid Project (http://www.biogrid.jp/) to accelerate new drug development. The SOSHO project provides novel crystallization technology with laser-irradiation and stirring growth methods, and the BioGrid Project is developing the software necessary for the in silico screening of promising drugs and the simulation of biological responses to proteins. In this paper, we report the recent research work on the crystallization of membrane proteins and the development of a method for in silico drug discovery.

Gene therapy with TRAIL against renal cell carcinoma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer cells. However, TRAIL is not toxic against most normal cells. We have accordingly examined by in vivo electroporation whether TRAIL induces apoptosis in renal cell carcinoma. In addition, combination treatment with TRAIL and 5-fluorouracil (5-FU) against renal cell carcinoma was also investigated. The NC65 renal cell carcinoma line was used as a target. pCAGGS TRAIL was injected into the NC65 tumors in the right flanks of severe combined immunodeficient mice. Tumors were pulsed with the CUY21 electroporator. Electroporation was done once on day 0 or thrice on days 0, 2, and 4. Apoptosis was determined by terminal deoxyribonucleotide transferase-mediated nick-end labeling assay. When TRAIL gene therapy using in vivo i.t. electroporation was done once only, the growth of NC65 tumors was not inhibited. However, when TRAIL gene therapy was done thrice, growth suppression of the NC65 tumors was observed. Transfection of the TRAIL gene by in vivo electroporation induced apoptosis in NC65 tumors. When NC65 cells were treated with TRAIL gene therapy in combination with 5-FU, stronger growth suppression was obtained. TRAIL gene therapy did not induce liver dysfunction in severe combined immunodeficient mice. This study shows that TRAIL gene therapy induced growth suppression and apoptosis in NC65 tumors without severe side effects, and that combination treatment of NC65 cells with TRAIL gene therapy and 5-FU resulted in higher antitumor activity. These findings suggest that TRAIL gene therapy and/or 5-FU may be effective against renal cell carcinoma without harmful toxic effects.

Application of in vivo electroporation to cancer gene therapy.

Much intensive research has gone into the development of safe and efficient methods for the delivery of therapeutic genes. In vivo electroporation is a non-viral delivery protocol in which plasmid DNA solutions are injected into targeted tissues, followed by electric pulses (typically 100 V, 50 ms). In general, in vivo electroporation enhances gene expression in targeted tissues by 2-3 orders of magnitude, as compared to the injection of plasmid DNA solutions without electric pulses, and the tissue damage appears to be minimal. Among the other advantages of this technique are that it can safely be administered repeatedly, and it is simpler and more economical to use than viral vectors, especially in clinical cases. Using this approach, highly efficient gene transfer has already been achieved in muscle and liver as well as in tumors. In fact, gene therapies for cancer utilizing in vivo electroporation have been proved effective in a number of experimental murine tumor models. The therapeutic genes delivered in those cases were diverse including, for example, cytokine genes (IL-12) and cytotoxic genes (TRAIL), making possible a wide range of therapeutic strategies. Moreover, systemic antitumor effects were also observed, suggesting that this approach may be effective for the treatment of metastatic as well as primary tumors.

Protective effect against ischemia and light damage of iris pigment epithelial cells transfected with the BDNF gene.

PURPOSE: Brain-derived neurotrophic factor (BDNF) has been reported to protect retinal neurons against ischemia and light-induced damage. In the current study, the BDNF gene was transfected into iris pigment epithelial (IPE) cells of Long-Evans rats, and the neuroprotective ability of the IPE cells transfected with the BDNF gene against N-methyl-D-aspartate (NMDA)-induced neuroretinal cell death and against phototoxic damage was examined. METHODS: The level of BDNF mRNA and protein expressed in the transfected cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) and by sandwich enzyme-linked immunosorbent assay (ELISA). The neuroprotective effects were determined by culturing BDNF gene-transfected IPE cells or nontransfected cells with neuroretinal cells in the presence of NMDA. The neuroprotective activity was also evaluated for the damage induced by constant exposure to light on the photoreceptors by transplanting BDNF gene-transfected IPE cells into the subretinal region of the superior half of the eye. RESULTS: BDNF gene-transfected IPE cells expressed higher levels of BDNF mRNA and protein than did nontransfected IPE cells. A significant increase in the protection against NMDA was observed in the neuroretinal cells cultured with BDNF-transfected IPE cells than in those cultured with nontransfected IPE cells (P = 0.0029) or with nontreated cells (P = 0.0010). The effect was partially attenuated by the addition of anti-BDNF antibody. Significant photoreceptor cell protection against injury from constant light was also observed by the subretinal transplantation of BDNF-transfected IPE cells when compared with those receiving transplants of nontransfected cells or vehicle injection. CONCLUSIONS: BDNF-transfected IPE cells demonstrated a neuronal rescue effect in both in vitro and in vivo experiments. IPE cells may be a potential source for autologous cell transplantation for some retinal diseases, and the transfection of the genes of neurotrophic factors into the transplanted cell may be a useful tool for delivering these factors to the retina.

Combination of IL-12 and IL-18 of electro-gene therapy synergistically inhibits tumor growth.

BACKGROUND: We previously showed that IL-12 electro-gene therapy (EGT) induces significant antitumor immunity. However, because interleukin (IL)-18 acts synergistically with IL-12 to augment Th1 responses and interferon (IFN)-gamma, we designed an EGT protocol using IL-12 + IL-18 with the aim of enhancing the antitumor effects obtained with IL-12 alone. MATERIALS AND METHODS: Expression plasmids harboring the gene for IL-12 or IL-18 were cotransferred to subcutaneous murine CT26 tumors. Subsequent expression of IFN-gamma and the presence of CD8+ T cells within treated tumors were analyzed by semiquantitative RT-PCR. RESULTS: IL-12 + IL-18 EGT inhibited tumor growth significantly better than IL-12 EGT, which was consistent with significantly higher intratumoral levels of IFN-gamma. Enhanced infiltration of tumor tissues by CD8+ T cells was confirmed with both IL-12 and IL-12 + IL-18 EGT. Finally, IL-12 + IL-18 EGT, but not IL-12 EGT, significantly suppressed untreated contralateral tumors. CONCLUSION: EGT with IL-12 and IL-18 is potentially an effective antitumor gene therapy.

The in silico screening and X-ray structure analysis of the inhibitor complex of Plasmodium falciparum orotidine 5'-monophosphate decarboxylase.

Orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) catalyses the final step in the de novo synthesis of uridine 5'-monophosphate (UMP) from orotidine 5'-monophosphate (OMP). A defective PfOMPDC enzyme is lethal to the parasite. Novel in silico screening methods were performed to select 14 inhibitors against PfOMPDC, with a high hit rate of 9%. X-ray structure analysis of PfOMPDC in complex with one of the inhibitors, 4-(2-hydroxy-4-methoxyphenyl)-4-oxobutanoic acid, was carried out to at 2.1 Å resolution. The crystal structure revealed that the inhibitor molecule occupied a part of the active site that overlaps with the phosphate-binding region in the OMP- or UMP-bound complexes. Space occupied by the pyrimidine and ribose rings of OMP or UMP was not occupied by this inhibitor. The carboxyl group of the inhibitor caused a dramatic movement of the L1 and L2 loops that play a role in the recognition of the substrate and product molecules. Combining part of the inhibitor molecule with moieties of the pyrimidine and ribose rings of OMP and UMP represents a suitable avenue for further development of anti-malarial drugs.

LAT1 expression in non-small-cell lung carcinomas: analyses by semiquantitative reverse transcription-PCR (237 cases) and immunohistochemistry (295 cases).

OBJECTIVE: System l-amino acid transport mediates the uptake of aromatic neutral amino acids and nutritionally essential amino acids from extracellular fluids. Little is known about the role of l-amino acid transporter 1 (LAT1), a member of the system l-amino acid transporter family, in non-small-cell lung carcinomas (NSCLCs). PATIENTS AND METHODS: We examined (i) LAT1 mRNA levels in 40 normal lung tissues (NLTs) and 237 NSCLCs using semiquantitative RT-PCR, (ii) LAT1 protein expression in 295 NSCLCs using immunohistochemistry, and (iii) whether LAT1 mRNA and protein expressions were related to clinicopathologic findings and outcome. RESULTS: The LAT1 mRNA level was significantly higher in all NSCLCs (6.81+/-1.13) than in NLT (1.00+/-0.18). The LAT1 mRNA level showed no association with clinicopathologic findings or outcome. LAT1 protein was detected with a diffuse or granular appearance within the cytoplasm and/or on the plasma membrane of tumor cells. When tumors were graded as positive if staining indicating a plasma membrane expression of LAT1 protein made up more than 10% of the tumor, the frequency of this membrane expression was found to be associated with tumor histology, differentiation grade, pathologic stage, T classification, pleural invasion, lymph-vessel invasion, and overall survival rate. CONCLUSION: Detection of a plasma membrane expression of LAT1 protein would appear to be of value in informing the prognosis in NSCLC cases.

Role of TRPV3 in immune response to development of dermatitis.

BACKGROUND: Recently, it has been reported that the Gly573Ser substitution of transient receptor potential V3 (TRPV3) leads to increased ion-channel activity in keratinocytes. Our previous studies have indicated that the spontaneous hairless and dermatitis phenotypes of DS-Nh mice, which were newly established as an animal model of atopic dermatitis (AD), are caused by TRPV3Gly573Ser. Although this substitution causes hairlessness in several kinds of rodents, in our investigations, dermatitis developed in only a few animals. Here, we generated NC/Nga-Nh mice to elucidate the role of TRPV3Gly573Ser in NC/Nga mice, which is one of the most studied animal models of AD. METHODS: To establish and validate the new AD animal model, NC/Nga-Nh mice were generated using NC/Nga and DS-Nh mice, and their clinical features were compared. Next, T-cell receptor (TCR) Vbeta usage in splenocytes, evaluation of bacterial colonization, and serological and histological analyses were carried out. Finally, repeated-hapten-application dermatitis was induced in these mice. RESULTS: NC/Nga-Nh mice did not develop spontaneous dermatitis, whereas DS-Nh mice displayed this phenotype when maintained under the same conditions. Serological analysis indicated that there really was a phenotypic difference between these mice, and TCR repertoire analysis indicated that TCRVbeta haplotypes played an important role in the development of dermatitis. Artificial dermatitis developed in DS and NC/Nga-Nh mice, but not in DS-Nh and NC/Nga mice. Histological and serological analyses indicated that mouse strains were listed in descending order of number of skin mast cells: DS-Nh > DS approximately NC/Nga-Nh > NC/Nga, and serum IgE levels were increased after 2,4,6 trinitrochlorobenzene application in these mice. Serum IgE level in DS-Nh mice was lower than that mesured in other strains. CONCLUSION: Our results confirm the contribution of the TRPV3Gly573Ser gene to the development of repeated hapten dermatitis, but not spontaneous dermatitis in NC/Nga mice.

Impact of the Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatitis in mice.

We reported that the Gly573Ser substitution in transient receptor potential vanilloid 3 (TRPV3) led to increased ion channel activity in keratinocytes and caused spontaneous hairlessness in DS-Nh mice. DS-Nh mice also develop allergic and pruritic dermatitis. As the hairless and dermatitis phenotypes were both inherited in an autosomal dominant fashion and could not be segregated from each other, we speculated that TRPV3(Gly573Ser) might be responsible for the dermatitis. Here, we constructed TRPV3(Gly573Ser) transgenic mice, with a putative promoter sequence in the 5' region of TRPV3, to investigate the involvement of TRPV3 in the development of specific types of dermatitis. These transgenic mice spontaneously developed dermatitis, whereas wild-type mice did not display this phenotype when maintained under the same conditions. Histological and serological analyses were carried out to better understand the clinical features of TRPV3(Gly573Ser) transgenic mice. A physiological study revealed that TRPV3(Gly573Ser) induced a higher nerve growth factor response to heat. Finally, C57BL-Nh mice were used to investigate the penetrance of the TRPV3(Gly573Ser) gene for dermatitis. Interestingly, C57BL-Nh mice developed spontaneous scratching behavior, separately from the development of dermatitis. We propose that TRPV3(Gly573Ser) is a cause of pruritus and/or dermatitis associated with scratching, and suggest that TRPV3 may represent a therapeutic target in pruritic dermatitis.

Impact of T-cell receptor Vbeta haplotypes on the development of dermatitis in DS-Nh mice: synergistic production of interleukin-13 caused by staphylococcal enterotoxin C and peptide glycans from Staphylococcus aureus.

Although the pathogenic role of interleukin-13 (IL-13) is a key for atopic dermatitis (AD), the mechanism of IL-13 production in AD remains unclear. To investigate the role of the T-cell receptor Vbeta (TCR Vbeta) haplotype in the development of dermatitis and the production of IL-13 in the naturally occurring dermatitis model by staphylococcal enterotoxin C (SEC)-producing Staphylococcus aureus, we raised DS-Nh mice harbouring the TCR Vbeta(a) haplotype with a central deletion in the TCRBV gene segments, including TCR Vbeta8S2. Observation and histopathological analysis of the two mouse substrains with spontaneous dermatitis indicated that later onset and weaker severity of AD-like dermatitis were identified in mice with TCR Vbeta(a) compared to those with TCR Vbeta(b). Immunohistochemical examination revealed the infiltration of a large number of CD4-bearing T cells in the skin lesions in mice with TCR Vbeta(b) but not in those with TCR Vbeta(a). Interestingly, much lower levels of serum IL-13 were detected in mice with the TCR Vbeta(a) than in those with the TCR Vbeta(b) haplotype. In vitro, synthetic ligands (Pam(2)CSK4) of toll-like receptor 2 (TLR2) synergistically produced IL-13 with SEC in splenocytes of mice with TCR Vbeta(b) but not of those with TCR Vbeta(a), and natural killer T cells were essential for this synergism. Our findings suggested that this TCR Vbeta-haplotype-dependent synergism with TLR2 plays an important role in the development of AD-like dermatitis in DS-Nh mice.

Influence of TRPV3 mutation on hair growth cycle in mice.

We recently reported that Gly573Ser substitution of the transient receptor potential cation channel, subfamily V member 3 (TRPV3) caused hair loss in DS-Nh mice. To further elucidate the effects of this mutation on the development of the spontaneous hairless phenotype, we examined the temperature-response to epidermal sheets from DS-Nh and DS mice. It was indicated that the mutation was gain-of-function. We also performed genetic and histological analyses with both strain skins. DNA microarray data revealed that the levels of keratin-associated protein 16-1, 16-3, and 16-9 genes related to the anagen phase were decreased in the skins of DS-Nh mice compared with those of three days old DS mice. Histological analysis revealed that the anagen phase persisted in DS-Nh mice, and that the telogen phase was seen in DS but not DS-Nh mice at 21 days of age. Regulation of TRPV3 appears to be important for appropriate hair development in rodents.

Association of a mutation in TRPV3 with defective hair growth in rodents.

DS-Nh mice and WBN/Kob-Ht rats are spontaneous hairless mutant rodent strains. These animals develop spontaneous dermatitis under normal conditions. The non-hair Nh and Ht phenotypes are inherited in an autosomal dominant fashion, and the Nh mutation possesses a high potency for penetration. We previously reported that genes involved in dermatitis and hairlessness did not segregate from each other. Here, we carried out genetic analysis to identify the genes responsible for these hairless mutations. An amino-acid substitution at the same position in one gene was detected in DS-Nh mice and WBN/Kob-Ht rats: Gly573 to Ser (Nh mutation) or Gly573 to Cys (Ht mutation), located in the transient receptor potential (TRP) cation channel subfamily V member 3 (TRPV3) gene. Mutated TRPV3 was expressed in skin keratinocytes of DS-Nh mice. Histopathological analyses revealed that mast cells in skin lesions were increased in both rodents compared to their age-matched parent strains, and that this may partially be due to hairlessness and dermatitis. We concluded that TRPV3 was the gene responsible for Nh and Ht mutations, and that mutation in TRPV3 possibly correlated with increased mast cell numbers.

WBN/Kob-Ht rats spontaneously develop dermatitis under conventional conditions: another possible model for atopic dermatitis.

WBN/Kob-Ht rats (Ht-rats) raised under conventional conditions spontaneously developed dermatitis. In this study, we carried out histopathological analysis to elucidate the pathological features of the dermatitis in Ht-rats. We then tried to detect Staphylococcus species recovered from the skin lesions of Ht-rats. We also measured the serum levels of total IgE, IL-4 and IFN-gamma in these rats. The histopathological data indicated that inflammatory cells had infiltrated the skin lesions. Staphylococcus aureus was recovered from the skin lesions, and the serum levels of total IgE and IL-4 were elevated in Ht-rats with dermatitis. These results suggest that dermatitis in Ht-rats is similar to that in the DS-Nh mice, which has recently been proposed as an animal model for human atopic dermatitis.

Augmented antitumor activity of a secondary lymphoid-tissue chemokine (SLC)-interleukin (IL) 2 fusion protein in mouse.

BACKGROUND: To enhance the antitumor efficacy of IL2 gene therapy, combinations of several other genes, such as p53, a tumor suppressor gene, or lymphotactin, a C-chemokine, and the IL2 gene are attempted, and synergistic effects are observed. We report here on the enhanced antitumor activity of a fusion protein (mSLC-IL2) comprised of a newly identified member of the CC-chemokine family, mouse SLC (mSLC), and mouse IL2 (mIL2). METHODS: We constructed mSLC-IL2 by connecting the N-terminus of mIL-2 to the C-terminus of mSLC using a two-amino-acid linker. The resultant fusion protein retained both mIL2 activity, as measured in a standard proliferation assay using a mouse IL-2 dependent cell line, and chemokine activity, as measured in a chemotaxis assay using a preB cell line expressing mSLC-specific receptor, CCR7. The gene encoding mSLC-IL2 was retrovirally transduced into fibroblast CL.7 cells, derived from Balb/c mice. RESULTS: Intradermal transplantation of fibroblasts expressing mSLC-IL2 into syngenic mice induced a dense accumulation of CD4(+) and CD8(+) cells at the sites of transplantation. Moreover, when CT-26 cells, derived from colon adenocarcinoma cells, were co-transplanted with mSLC-IL2-transduced fibroblasts, the CT-26 cell exhibited significantly lower tumorigenicity than CT-26 cells co-transplanted with mIL2-transduced fibroblasts. CONCLUSIONS: These findings, obtained from both in vitro and in vivo data, suggest that the gene encoding mSLC-IL2 may be a good candidate for inclusion as part of an anticancer gene therapy protocol.

Large-scale production of functional human adrenomedullin: expression, cleavage, amidation, and purification.

Human adrenomedullin (hAM) is a 52-amino-acid regulatory peptide containing a six-membered ring structure and an amidated C-terminus, features that are essential for its biological activity. Here, we describe a simple and effective protocol for producing large quantities of highly pure, functional recombinant hAM. A peptide precursor (hAM-Gly) was expressed in Escherichia coli as a fusion protein with thioredoxin and collected as inclusion bodies. The fusion protein was then digested with BLase, a glutamate-specific endopeptidase, to prepare hAM-Gly. The essential ring structure formed spontaneously, while the terminal amide was generated by conversion of the added glycine residue using peptidylglycine alpha-amidating enzyme. The low solubility of hAM-Gly enabled the use of a selective precipitation/extraction method to generate a product that was 80-90% pure, which was sufficient to proceed with the alpha-amidating enzyme reaction. The resultant hAM was then purified further by column chromatography. The final yield was about 82 mg/L of bacterial culture, and the purity, determined by reverse phase HPLC, was >99.5%. The recombinant hAM was biologically active, eliciting concentration-dependent increases in cAMP in CHO-K1 cells expressing a specific hAM receptor and hypotensive responses when intravenously injected into rats. This new approach to the synthesis of hAM is simpler and more cost-effective for large-scale production than chemical synthesis. It therefore represents a new powerful tool that has the potential to facilitate analysis of the structure and function of hAM, as well as the development of new therapeutic protocols for the treatment of ailments such as hypertension.

Combination electro-gene therapy using herpes virus thymidine kinase and interleukin-12 expression plasmids is highly efficient against murine carcinomas in vivo.

We report the use of plasmid DNA-mediated combination gene therapy for tumor-bearing mice using in vivo electroporation, also called electro-gene therapy (EGT), that resulted in uncomplicated and complete cures in more than 90% of the mice. Subcutaneously inoculated CT26 tumors in syngeneic BALB/c mice were subjected to repeated EGT treatments consisting of intratumoral co-injection of naked plasmids encoding the cytokine interleukin-12 (IL-12) (p35 and p40 subunits) and the suicide gene herpes simplex virus thymidine kinase (HSV-tk), followed by in vivo electroporation. The early anti-tumor effect was always stronger, and the rate of cure, as seen in the long-term follow-up, was always greater in the groups treated with combination EGT than in those treated with IL-12 or HSV-tk EGT alone. Systemic levels of IL-12 and IFN-gamma increased in both combination and IL-12-alone EGT-treated groups. Moreover, combination EGT for established subcutaneous tumors strongly reduced hematogenous lung metastases and increased survival time when live CT26 tumor cells were injected through the tail vein. Limited experiments on C57/B16 mice with murine melanoma also showed very similar trends. These results suggest that this simple and safe method of plasmid-mediated combination EGT may provide a potentially effective gene therapy for cancer.