Gene therapy improves dental manifestations in hypophosphatasia model mice.

BACKGROUND AND OBJECTIVE: Hypophosphatasia is a rare inherited skeletal disorder characterized by defective bone mineralization and deficiency of tissue non-specific alkaline phosphatase (TNSALP) activity. The disease is caused by mutations in the liver/bone/kidney alkaline phosphatase gene (ALPL) encoding TNSALP. Early exfoliation of primary teeth owing to disturbed cementum formation, periodontal ligament weakness and alveolar bone resorption are major complications encountered in oral findings, and discovery of early loss of primary teeth in a dental examination often leads to early diagnosis of hypophosphatasia. Although there are no known fundamental treatments or effective dental approaches to prevent early exfoliation of primary teeth in affected patients, several possible treatments have recently been described, including gene therapy. Gene therapy has also been applied to TNSALP knockout mice (Alpl-/- ), which phenocopy the infantile form of hypophosphatasia, and improved their systemic condition. In the present study, we investigated whether gene therapy improved the dental condition of Alpl-/- mice. MATERIAL AND METHODS: Following sublethal irradiation (4 Gy) at the age of 2 d, Alpl-/- mice underwent gene therapy using bone marrow cells transduced with a lentiviral vector expressing a bone-targeted form of TNSALP injected into the jugular vein (n = 3). Wild-type (Alpl+/+ ), heterozygous mice (Alpl+/- ) and Alpl-/- mice were analyzed at 9 d of age (n = 3 of each), while Alpl+/+ mice and treated or untreated Alpl-/- mice were analyzed at 1 mo of age (n = 3 of each), and Alpl+/- mice and Alpl-/- mice with gene therapy were analyzed at 3 mo of age (n = 3 of each). A single mandibular hemi-section obtained at 1 mo of age was analyzed using a small animal computed tomography machine to assess alveolar bone formation. Other mandibular hemi-sections obtained at 9 d, 1 mo and 3 mo of age were subjected to hematoxylin and eosin staining and immunohistochemical analysis of osteopontin, a marker of cementum. RESULTS: Immunohistochemical analysis of osteopontin, a marker of acellular cementum, revealed that Alpl-/- mice displayed impaired formation of cementum and alveolar bone, similar to the human dental phenotype. Cementum formation was clearly present in Alpl-/- mice that underwent gene therapy, but did not recover to the same level as that in wild-type (Alpl+/+ ) mice. Micro-computed tomography examination showed that gene therapy improved alveolar bone mineral density in Alpl-/- mice to a similar level to that in Alpl+/+ mice. CONCLUSIONS: Our results suggest that gene therapy can improve the general condition of Alpl-/- mice, and induce significant alveolar bone formation and moderate improvement of cementum formation, which may contribute to inhibition of early spontaneous tooth exfoliation.

Antiproliferative activity of REIC/Dkk-3 and its significant down-regulation in non-small-cell lung carcinomas.

We recently reported the cloning of the REIC/Dkk-3 gene, whose expression was shown to be down-regulated in many human immortalized and tumor-derived cell lines [T. Tsuji et al. (2000) Biochem. Biophys. Res. Commun. 268, 20-24]. In the present study, we demonstrated that expression of the exogenous REIC/Dkk-3 gene in tumor cells inhibited cell growth. Furthermore, the level of REIC/Dkk-3 mRNA in normal human cells was lowest in the late G(1) phase during the cell cycle. Then we found that the expression of REIC/Dkk-3 was significantly down-regulated in surgically resected non-small-cell lung carcinomas. We determined the REIC/Dkk-3 locus on chromosome 11p15, where loss of heterozygosity has frequently been observed in human tumors. These findings indicate that REIC/Dkk-3 may function as a tumor suppressor.

Reduced expression of REIC/Dkk-3 gene in non-small cell lung cancer.

Dickkopfs (Dkks) are secretory glycoproteins and are important for inducing amphibian head formation. Some Dkks have the ability to antagonize the Wnt proto-oncoprotein, which can promote cell proliferation and transformation when it is mutated or overexpressed. We recently cloned the REIC/Dkk-3 gene, one of the Dkks, and found that expression of the gene was markedly decreased in many human immortalized and tumor-derived cell lines. To further investigate the function of the REIC/Dkk-3 gene in vivo, the gene expression was quantified in 57 surgically-resected non-small cell lung cancer (NSCLC) tissues and their adjacent normal lung tissues by real-time quantitative PCR analysis based on TaqMan methodology. The REIC/Dkk-3 mRNA level was significantly lower in NSCLC tissues than in normal lung tissues. Reduced expression of REIC/Dkk-3 in NSCLC was observed in 36 (63%) of the 57 tumors. Reduced expression of REIC/Dkk-3 was frequently observed in poorly differentiated adenocarcinoma and squamous cell carcinoma. These results indicate that reduced expression of REIC/Dkk-3 may be correlated with rapid cell proliferation in human NSCLC.

Selective killing of human immunodeficiency virus-infected cells by targeted gene transfer and inducible gene expression using a recombinant human immunodeficiency virus vector.

A human immunodeficiency virus type 1 (HIV-1)-based retroviral vector pseudotyped with HIV envelope containing the herpes simplex virus-thymidine kinase (HSV-TK) gene under the control of the HIV LTR promoter (pHXTKN) was constructed and stably transferred into human CD4(+) H9, CEM, and U937 cells. RNase protection assays did not initially detect expression of the HSV-TK gene in HXTKN-transduced CD4(+) cells (HXTKN/CD4), but expression was then efficiently induced by infection with HIV-1. MTT assays showed that after HIV-1 infection, the susceptibility of HXTKN/CD4 cells to ganciclovir (GCV) was 1000-fold higher than prior to infection. This enabled HIV-1-infected cells to be selectively killed by transduction with HXTKN followed by exposure to GCV. Because the HSV-TK gene is specifically transferred into HIV-1-permissive cells and expressed only after HIV-1 infection, the frequency of unwanted cell death should be low. Elimination of the HIV-1-infected cells effectively inhibited further spread of infectious virus. In addition, the integrated HIV vector sequences were repackaged on infection with HIV-1 and transferred to surrounding untransduced cells. These results are indicative of the potential benefits of using HIV vectors in gene therapies for the treatment of HIV-1 infection.

Suppressive effect of the herbal medicine Oren-gedoku-to on cyclooxygenase-2 activity and azoxymethane-induced aberrant crypt foci development in rats.

The present study is part of a program to obtain effective chemopreventive agents with low toxicity from medicinal herbs and traditional herbal medicines. We previously reported that Oren (Coptidis rhizoma) and Ogon (Scutellariae radix) inhibit azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation. In the present investigation, we found Sanshishi (Gardeniae fructus) and the traditional herbal medicine Oren-gedoku-to (OGT), composed of Ogon, Oren, Sanshishi and Obaku, also have preventive potential. Sanshishi and OGT decreased the numbers of ACF to 25.2 and 59.4% of the control value at 2% in the diet, respectively. Adverse effects, evidenced by body weight loss, were weaker with OGT than component herbs. To investigate their mechanisms of action, the influence on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) activities was studied. Both OGT and Sanshishi inhibited COX-2 but not COX-1, this presumably contributing to their suppressive effects on ACF development. The results suggest that OGT may be useful for colon cancer chemoprevention in terms of efficacy and toxicity.

Antioxidative effects of Choi-oki-to and its ability to inhibit the progression of atheroma in KHC rabbits.

Agents which inhibit the oxidative modification of low density lipoprotein (LDL) have been thought to be helpful in preventing the formation of atherosclerotic lesions; the so called "oxidation hypothesis". To test this hypothesis, we examined the antioxidative activities of 127 Kampo medicines in vitro and their inhibitory effects on the development of atheromatous plaque formation in KHC rabbits, a model of spontaneous familial hypercholesterolemia. Some of the 127 Kampo medicines showed scavenging or antioxidative effects equal to or stronger than those of probucol in vitro. Choi joki to, which had the strongest antioxidative effects on LDL in vitro, was chosen for a study in vivo. After 24 weeks, 1 g/kg of Choi joki to successfully inhibited the progression of atherosclerotic lesions in KHC rabbits (P < 0.01). Further investigations regarding the antioxidative effects of Kampo medicines are expected.

Effects of San'o-shashin-to on the antioxidative mechanism in spontaneous familial hypercholesterolaemic rabbits.

We investigated the effects of the Kampo medicine San'o-shashin-to which showed strong antioxidative effects in vitro on antioxidative mechanism in Kurosawa and Kusanagi- hypercholesterolaemic (KHC) rabbits, which are a good model of spontaneous familial hypercholesterolaemia. A 1-g kg(-1)dose of San'o-shashin-to was administered orally for 24 weeks and changes in plasma lipids, low-density lipoprotein (LDL) oxidation and redox dynamics of alpha-tocopherol in plasma and erythrocyte membrane were measured in a control group and San'o-shashin-to-treated group. A significant prolongation of the lag time was found in the 12th and 24th week of the experiment in San'o-shashin-to-treated group, indicating antioxidative effects on LDL. At the end of the administration period, the alpha-tocopherolquinone/alpha-tocopherol ratio in erythrocyte membrane, which reflects the utilization rate of alpha-tocopherol was significantly higher in San'o-shashin-to-treated group. There were no significant differences in the two groups with regards to plasma lipids. These results indicate that San'o-shashin-to had antioxidative effects on LDL in vivo and beneficial effects on redox dynamics of alpha-tocopherol, which is the strongest endogenous antioxidant.

In situ gene transfer and suicide gene therapy of gastric cancer induced by N-ethyl-N'-nitro-N-nitrosoguanidine in dogs.

Gene therapy could potentially revolutionize the treatment of gastrointestinal (GI) tract cancer. The aim of this study was to establish a practical method of gene transfer which would be applicable to human gastric cancer. Retrovirus or/and adenovirus vectors carrying the lacZ marker gene were transferred in situ by needle through an endoscopic biopsy channel into primary gastric cancer in six male beagle dogs that had been treated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). In addition, an adenovirus vector carrying the herpes simplex virus thymidine kinase (Ad.CAGHSV-TK) gene was introduced in situ into cancer tissues in the stomach of three dogs, and the animals were treated with intravenous ganciclovir (GCV). Retrovirus-producing cells which expressed the lacZ gene were specifically localized to the injection site in the stomach. The lacZ gene was more widely transferred into the tumor by the adenovirus vector than by retrovirus-producing cells. Improvement of the needle used for gene transfer and the use of multiple injections per tumor led to more diffuse transfer of the vector into the tumor. The Ad.CAGlacZ gene was also transferred into regional lymph nodes of the stomach. Moderate to diffuse degeneration of the primary cancer tissues of the stomach was found after Ad.CAGHSV-TK/GCV gene therapy. Moreover, almost complete tissue degeneration was observed in the regional lymph nodes of the stomach. An adverse effect of HSV-TK/GCV gene therapy was acute hepatotoxicity, which was not found after Ad.CAGlacZ gene transfer, but was found after high-titer Ad.CAGHSV-TK gene transfer followed by GCV. These findings suggest that in situ gene transfer of a suicide gene followed by prodrug treatment may be applicable not only to primary tumors, but also to lymph node metastases of gastric cancer, though further study of both beneficial and adverse effects is required before clinical usage.

Anti-inflammatory effects of mao-bushi-saishin-to in mice and rats.

Effects of Mao-Bushi-Saishin-to (MBS) on anti-inflammatory activities were examined in mice and rats. MBS significantly inhibited the increase in vascular permeability induced by acetic acid, the ear edema induced by arachidonic acid and phorbol ester, and the cutaneous extravasation induced by bradykinin and histamine. MBS, however, was not effective against the serotonin-induced cutaneous permeability increase in mice. MBS significantly inhibited carrageenin-induced hind foot edema and cotton pellet-induced granulation tissue growth in rats. These results show that MBS may exert anti-inflammatory effects through the underlying mechanism(s) of preventing mediator release from mast cells and macrophages.

Inhibitory effects of Dai-saiko-to (Da-Chai-Hu-Tang) on the progression of atherosclerotic lesions in Kurosawa and Kusanagi-hypercholesterolemic rabbits.

The inhibitory effects of the traditional herbal medicine Dai-saiko-to (Da-Chai-Hu-Tang) on the progression of the atherosclerotic lesions were studied using the spontaneous familial hypercholesterolemia (FH) model, Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits. Changes in blood chemistry, pathology and low-density lipoprotein (LDL) oxidation were measured in a control group and a Dai-saiko-to-treated group. In the control group, the area of atheromatous plaques of the aorta progressed between week 12 (29.1%) and 26 (51.5%). This progression of atherosclerotic lesions did not happen in the Dai-saiko-to-treated group between week 12 (26%) and 26 (27.4%). Antioxidative effects on LDL were seen in the Dai-saiko-to-treated group in weeks 16 and 18. Dai-saiko-to did not improve the hypercholesterolemia in the KHC rabbits. These results suggest that Dai-saiko-to has inhibitory effects on the development of atheromatous plaque formation in spontaneous FH model rabbits. It is possible that the antioxidative effects of Dai-saiko-to on LDL led to the beneficial effects observed in this study.

Regulation of hepatic macrophage function by oral administration of xiao-chai-hu-tang (sho-saiko-to, TJ-9) in rats.

The effect of Xiao-Chai-Hu-Tang (Sho-saiko-to, TJ-9), the extract of a mixture of 7 herbs, on hepatic macrophage function was studied using rats. Hepatic macrophages were activated by injection of Corynebacterium parvum or 70% partial hepatectomy. Oral administration of TJ-9 for 3 weeks did not affect the ability of these macrophages to produce superoxide anions evaluated in situ by liver perfusion with nitro blue tetrazolium (NBT) and phorbol myristate acetate (PMA). However, the similar administration of TJ-9 attenuated the blocking of the activation after partial hepatectomy produced by pretreatment with gum arabic, a polysaccharide of high molecular weight. When gum arabic was added to the medium of rat hepatic macrophages cultured with normal rat sera, their ability to produce superoxide anions was reduced in a dose-related manner. This reduction was attenuated by changing the sera to the sera obtained from rats given oral doses of TJ-9 for 3 weeks. These results suggest that TJ-9 may improve the blocked function of hepatic macrophages in activation.

[Basal studies on combination of Chinese medicine in cancer chemotherapy: protective effects on the toxic side effects of CDDP and antitumor effects with CDDP on murine bladder tumor (MBT-2)].

Effects of oral administration of Juzentaihoto, a herb drug, on the toxic side effects of cis-diammine dichloroplatinum (CDDP) and combination effects with CDDP on murine bladder tumor (MBT 2) were studied using C3H/He male mice. The following results were obtained; 1. When the mice were treated with the mixture diet of 1% or 0.5% Juzentaihoto before 2 weeks, adverse effects of high dose CDDP (15 or 17.5 mg/kg) which were included with lethal toxicity, renal and hepatic toxicity, and myelosuppression were protected significantly. The administration of 0.5% Juzentaihoto mixture diet markedly shifted the LD50 to the right. Furthermore, the effects of Juzentaihoto were clearly revealed on the histological findings of testis and kidney. 2. On murine bladder tumor, when the mice were treated with the mixture diet of 1% or 0.5% Juzentaihoto and injected CDDP (2.5 mg/kg/week) for 8 weeks, significantly greater inhibition of the tumor growth and prolongation of the survival rate were observed than those in the group of CDDP alone. These results indicates that Juzentaihoto lessens the toxic side effects of CDDP and that it enhances the effects of CDDP experimentally. Juzentaihoto, a kind of Chinese herb medicine, might come under the category of biological response modifiers.

[Protective effects of the Chinese medicine Juzentaiho-to from the adverse effects of mitomycin C and cisplatin].

Some Chinese medicines in Japan have been reported to have not only antitumour effects, but also to offer protection from the adverse effects of anti-tumour agents. However, there is controversy regarding the protective effects of such Chinese medicines against the adverse effects of anti-tumour agents, in this study, we examined the effects of Tsumura Juzentaiho-to (TJ-48) on the toxicity of mitomycin C (MMC) and cisplatin (CDDP). Both the pre-administration of TJ-48 a single time and for seven days shifted the dose response curve and LD50S of MMC and CDDP to the right. Seven days of treatment using TJ-48 delayed deaths due to lethal dose of MMC or CDDP and markedly changed their survival curves. Also, TJ-48 reduced the atrophy of the testis, thymus and spleen caused by MMC. TJ-48 also had beneficial effects on leukopenia, anemia and body weight loss caused by MMC, and increase of BUN and creatinine caused by CDDP. These results indicate that the combined use of TJ-48 may be a new way to in prevent or minimize the toxicity of MMC or CDDP.

Epizootiological studies of Salmonella typhimurium infection in guinea pigs.

In two natural outbreaks of S. typhimurium infection in guinea pigs, frequent isolations of the organism from the conjunctiva and cervical lymph nodes and high incidences of the conjunctivitis and abscess formation in the cervical lymph nodes were shown, suggesting more importance of the conjunctiva as infection route than oral route. These features in salmonellosis of guinea pigs were tried to reproduce in experimental infections by conjunctival and oral inoculations of 10(2) and 10(6) cells of 4 different strains of S. typhimurium and also by contact infection simulating natural conditions. As the results, it was demonstrated that guinea pigs were more susceptible to the conjunctival infection than the oral infection, because higher infection rates and more frequent incidence of abscess formation in the cervical lymph nodes as well as conjunctivitis were produced by the conjunctival inoculation than the oral inoculation of the organism. Main localization sites of the organism were the cervical lymph nodes, conjunctiva and upper respiratory tract in conjunctivally inoculated guinea pigs but more widely distributed in orally infected ones. These findings were common in animals infected with 4 different strains of S. typhimurium and also in contact infection. Thus the conjunctiva was regarded as an important route of S. typhimurium in guinea pigs.