Harmine promotes periodontal ligament cell-induced tissue regeneration.

OBJECTIVE: to investigate whether harmine has a promotive effect on human periodontal ligament cells (hPDLCs)-induced tissue regeneration. MATERIALS AND METHODS: Various concentrations of harmine on hPDLCs proliferation were tested. Osteogenic and cementogenic characteristics were examined in hPDLC/rhBMP-2 and hPDLC/harmine by alizarin red S staining, real-time PCR, and Western blotting assay. The activity of harmine was investigated in an ectopic transplantation nude mouse model. RESULTS: We determined that 10 µM of harmine was the threshold concentration. hPDLC/harmine showed similar mineralized nodule formation in alizarin S staining compared to hPDLC/rhBMP-2. In real-time PCR, the highest gene expression level was observed for Runx2 in hPDLC/harmine at all time points. The level of CEMP-1 in hPDLC/harmine was higher at 7 days than hPDLCs alone. Thicker band of Runx2 in hPDLC/harmine was observed than in hPDLC/rhBMP-2 at 7 days by Western blotting. The band for CEMP-1 in hPDLC/harmine was thicker than hPDLCs alone at both 7 and 14 days. In ectopic transplantation, hPDLCs with harmine showed a comparable amount of mineralized tissue formation compared to rhBMP-2. hPDLCs with harmine or rhBMP-2 formed both bone and cementum-like tissue with Sharpey's fiber-like collagen insertion. CONCLUSION: Harmine can be a potential candidate for promoting hPDLCs-induced tissue regeneration.

Intracavernous delivery of clonal mesenchymal stem cells rescues erectile function in the streptozotocin-induced diabetic mouse.

The major hurdle for the clinical application of stem cell therapy is the heterogeneous nature of the isolated cells, which may cause different treatment outcomes. The aim of this study was to examine the effectiveness of mouse clonal bone marrow-derived stem cells (BMSCs) obtained from a single colony by using subfractionation culturing method for erectile function in diabetic animals. Twelve-week-old C57BL/6J mice were divided into four groups: controls, diabetic mice, and diabetic mice treated with a single intracavernous injection of PBS (20 µL) or clonal BMSCs (3 × 10(5) cells/20 µL). Clonal BMSCs were isolated from 5-week-old C3H mice. Two weeks after treatment, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was stained with antibodies to PECAM-1, smooth muscle α-actin, neuronal nitric oxide synthase (nNOS), neurofilament, and phosphorylated endothelial NOS (phospho-eNOS). We also performed Western blot for phospho-eNOS, and eNOS in the corpus cavernosum tissue. Local delivery of clonal BMSCs significantly restored cavernous endothelial and smooth muscle cell contents, and penile nNOS and neurofilament contents, and induced eNOS phosphorylation (Ser1177) in diabetic mice. Intracavernous injection of clonal BMSCs induced significant recovery of erectile function, which reached 80-90% of the control values. Clonal BMSCs successfully restored erectile function through dual angiogenic and neurotrophic effects in diabetic mice. The homogenous nature of clonal mesenchymal stem cells may allow their clinical applications and open a new avenue through which to treat diabetic erectile dysfunction.

Bone marrow-derived clonal mesenchymal stem cells inhibit ovalbumin-induced atopic dermatitis.

Mesenchymal stem cells (MSCs) possess immunomodulatory activities, including suppression of T- and B-cell activation. However, their effects on atopic dermatitis (AD) have not yet been studied. Using an ovalbumin-induced AD mouse model, we investigated whether MSCs can be used as therapeutics in AD. We isolated both allogeneic and syngeneic clonal MSCs (cMSCs) from mouse bone marrow according to the subfractionation culturing method. Our cMSCs suppressed both T- and B-cell activation. T-cell proliferation and cytokine production, including interferon (IFN)-γ and interleukin (IL)-4, were suppressed by inhibition of transcription factors, such as T-bet, GATA-3, and c-Maf. Those transcription factors were nitric oxide dependent. Immunoglobulin E (IgE) suppression occurred through downregulation of AID and BLIMP-1, important regulators for isotype class switch and B-cell differentiation. The cMSCs were injected intravenously into ovalbumin-induced AD mouse model, and the therapeutic effects were analyzed. Injection of both allogeneic and syngeneic cMSCs in an AD mouse model inhibited cell infiltration in skin lesions and decreased the serum level of IgE. IL-4 expression was also suppressed by cMSCs in both the lymph node and skin. The cMSCs migrated to skin lesions and draining lymph nodes. Taken together, these data demonstrated that cMSCs, which suppressed T- and B-cell functions, can be used for the treatment of AD in mice.

Mesenchymal stem cells infected with Mycoplasma arginini secrete complement C3 to regulate immunoglobulin production in B lymphocytes.

Mesenchymal stem cells (MSCs) have immunomodulatory functions such as the suppression of T and B cells. MSCs suppress immunoglobulin (Ig) production by B cells via cell-cell contact as well as via secretion of soluble factors. Our study showed that the conditioned medium (CM) of MSCs infected with a mycoplasma strain, Mycoplasma arginini, has marked inhibitory effects on Ig production by lipopolysaccharide/interleukin-4-induced B cells compared with mycoplasma-free MSC-CM. We analyzed mycoplasma-infected MSC-CM by fast protein liquid chromatography and liquid chromatography to screen the molecules responsible for Ig inhibition. Complement C3 (C3) was the most critical molecule among the candidates identified. C3 was shown to be involved in the suppression of the Ig production of B cells. C3 was secreted by mycoplasma-infected MSCs, but not by mycoplasma-free MSCs or B cells. It was able to directly inhibit Ig production by B cells. In the presence of a C3 inhibitor, Ig inhibition by MSC-CM was abrogated. This inhibitory effect was concomitant with the downregulation of B-cell-induced maturation protein-1, which is a regulator of the differentiation of antibody-secreting plasma cells. These results suggest that C3 secreted from mycoplasma-infected MSCs has an important role in the immunomodulatory functions of MSCs. However, its role in vivo needs to be explored.

Variations of clonal marrow stem cell lines established from human bone marrow in surface epitopes, differentiation potential, gene expression, and cytokine secretion.

Bone marrow has been considered to contain many different types of progenitor or stem cells. This study aims to establish a new strategy that provides for the rapid establishment of human clonal marrow stem cell (hcMSC) lines with a relatively small amount of bone marrow aspirate and to characterize newly generated hcMSC lines for their cell phenotype, differentiation potential, lineage-specific gene expression, and cytokine secretion. Human cMSC lines were generated with human bone marrow aspirates using a new protocol, called the subfractionation culturing method. The newly established hcMSC lines were analyzed for their cell surface epitopes by fluorescence-activated cell sorting (FACS), differentiation potential by in vitro differentiation assays, lineage-specific gene expression by RT-PCR, and cytokine secretion by enzyme-linked immunoassay (ELISA). The overall profile of the cell-surface epitopes of the newly established hcMSC lines was similar to those of the known MSCs. These hcMSC lines were capable of differentiating into multilineages with some differences in differentiation capability. In addition, these hcMSC lines secrete high levels of transforming growth factor-beta1 (TGF-beta1), leukemia inhibitory factor (LIF), TGF-alpha, and interleukein-10 (IL-10), again with some variation in each cell line. The newly designed protocol may be an efficient method to establish hcMSC lines rapidly with a relatively small amount of bone marrow sample, and these newly established hcMSC lines possess stem cell characteristics and exhibit some differences in cell-surface epitopes, differentiation potential, lineage-specific gene expression, and cytokine secretion.

Combined treatment with intratumoral injection of dendritic cells and topical application of imiquimod for murine melanoma.

BACKGROUND: The maturation state of dendritic cells is one of the factors that affect their capacity to induce antigen-specific cytotoxic T lymphocytes. Topical cutaneous application of imiquimod can induce the maturation and migration of cutaneous dendritic cells. OBJECTIVES: To evaluate the synergistic effect of topical application of imiquimod plus intratumoral injection of syngeneic bone marrow-derived dendritic cells in the treatment of melanoma. METHODS: For the B16F10 melanoma model, naive C57BL/6 mice were inoculated intradermally with 2x10(3) B16F10 melanoma cells in the right upper flank. Four groups (untreated control, dendritic cells alone, imiquimod alone and imiquimod plus dendritic cells) were included in the animal study, with five mice in each group. Tumour size was measured every 2 weeks, and histochemical and immunohistochemical staining carried out. ELISpot and PKH assays were performed to assess immune activity. RESULTS: Combined treatment of topical application of imiquimod and intratumoral injection of dendritic cells led to significant tumour regression, in contrast to partial eradication of the tumours with imiquimod or dendritic cells alone. CONCLUSION: These findings suggest that combination therapy with topical application of imiquimod and intratumoral administration of dendritic cells is a potent strategy for the treatment of melanoma.

Water-soluble lipopolymer as a gene carrier to corpus cavernosum.

Adenovirus or naked plasmid DNA (pDNA) has been used to deliver the therapeutic gene into corpus cavernosum. However, the potential risks of viral vector and inefficiency of naked pDNA have limited their clinical application. In this study, water-soluble lipopolymer (WSLP) was evaluated as a gene carrier to corpus cavernosum. The WSLP/pDNA complex was transfected to smooth muscle cells in vitro. WSLP had high transfection efficiency, which was comparable to poly(ethylenimine) (PEI). In addition, WSLP had much less cytotoxicity than PEI, suggesting that WSLP is a safer carrier than PEI. To evaluate the transfection efficiency to corpus cavernosum, the WSLP/pDNA complex was injected into the rat corpus cavernosum. As a result, the WSLP/pDNA complex showed higher transfection efficiency than naked pDNA. In addition, the gene expression was dependent upon the dose of the complex. The results suggest that WSLP may be useful for gene therapy of erectile dysfunction.

Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1-producing fibroblasts.

Transforming growth factor beta (TGF-beta) has been considered as a candidate for gene therapy of orthopedic diseases. The possible application of cell-mediated TGF-beta gene therapy as a new treatment regimen for degenerative arthritis was investigated. In this study, fibroblasts expressing active TGF-beta 1 were injected into the knee joints of rabbits with artificially made cartilage defects to evaluate the feasibility of this therapy for orthopedic diseases. Two to 3 weeks after the injection there was evidence of cartilage regeneration, and at 4 to 6 weeks the cartilage defect was completely filled with newly grown hyaline cartilage. Histological analyses of the regenerated cartilage suggested that it was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline cartilage. Our findings suggest that cell-mediated TGF-beta 1 gene therapy may be a novel treatment for orthopedic diseases in which hyaline cartilage damage has occurred.

A possible relation of the Helicobacter pylori pfr gene to iron deficiency anemia?

BACKGROUND: H. pylori infection is thought to contribute to iron-deficiency anemia, especially during puberty. The ferritin protein Pfr of H. pylori is homologous to eukaryotic and prokaryotic ferritins. The purpose of this study was to analyze the H. pylori pfr status in gastric biopsy specimens according to clinical data, including antral gastritis with or without iron-deficiency anemia. METHODS: A total of 26 H. pylori-positive patients aged from 10-18 years were categorized into subgroups based on the presence or absence of iron-deficiency anemia. All of them had antral gastritis. Sixteen patients were proved to have iron-deficiency anemia by hematological study, two of which had a duodenal ulcer. The other 10 patients showed normal hematological findings. DNA isolation was performed from each of the gastric biopsy specimens. PCR amplification of the pfr gene coding was done using two sets of primers. The pfr region, 501 bp, was generated by linking the sequences of the two PCR products. The nucleotide and protein sequences were compared between the pfr regions from Korean H. pylori strains and the NCTC 11638 strain, which was obtained from the Genbank. Sequence comparisons were also performed for the pfr regions between the iron-deficiency anemia (+) and (-) groups. RESULTS: Analysis of the complete coding region of the pfr gene revealed three sites of mutation. The Ser39Ala mutation was found in 100% (26/26), Gly111Asn in 26.9% (7/26), and Gly82Ser in 11.5% (3/26). There were no significant differences in the mutations of the pfr regions between the iron deficiency anemia (+) and (-) groups. CONCLUSION: The mutation in the pfr gene did not relate with the clinical phenotype, iron deficiency anemia. Further studies are needed on the aspects of host side or other complex factors to elucidate the mechanisms by which the H. pylori infection might lead to iron deficiency anemia.

Combination treatment for osteosarcoma with baculoviral vector mediated gene therapy (p53) and chemotherapy (adriamycin).

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been evaluated as a vector for gene delivery to human tumor cells. A human osteogenic sarcoma cell line, Saos-2, was found to be highly susceptible to infection with a baculoviral vector, with nearly 100% of Saos-2 cells being able to express a lacZ reporter gene after a brief exposure to the virus at a m.o.i. of 30 pfu/cell. The production of beta-galactosidase protein was 18-times greater than that in HepG2 cells which were previously thought to be the mammalian cells most susceptible to the baculovirus. The possibility of developing a baculovirus as a cytotoxic vector for p53-defective cancer was tested by destruction of Saos-2 cells (p53-/-) with a recombinant baculovirus containing the wild type p53 gene (BV-p53) in vitro. The p53 baculovirus induced apoptotic cell death in tumor cells in a dose-dependent manner with approximately 60% killing at an m.o.i. of 160 pfu/cell. Combined treatments of gene therapy (p53) and chemotherapy (adriamycin) resulted in synergistic and potent killing of the osteogenic sarcoma cells. For example, greater than 95% of Saos-2 cells were killed by the combination of BV-p53 (m.o.i. of 100) and adriamycin (35 ng/ml), whereas approximately 50% and approximately 55% cells were killed by BV-p53 and adriamycin alone, respectively. These results indicate that a baculoviral gene delivery vector can be used to efficiently target certain types of mammalian cells and the combination treatment of gene-therapy mediated by a baculovirus and chemotherapy may enhance induction of apoptosis in cancer cells.

Identification of a negative Cis-regulatory element and multiple DNA binding proteins that inhibit transcription of the transforming growth factor-beta type II receptor gene.

Expression of the transforming growth factor-beta type II receptor (TGF-beta RII) is highly regulated and is a critical determinant of the cellular response to TGF-beta. Previous analysis of the promoter region for the TGF-beta RII gene introduced the possible existence of a negative regulatory element (NRE) upstream adjacent to the core promoter region (Bae et al., 1995. J. Biol. Chem. 270, 29460-29468). We have confirmed the presence of a strong NRE located between base pairs -100 and -67 relative to the transcription start site. Utilizing DNA transfection techniques and a series of synthesized oligonucleotide promoter fragments, we have shown that this NRE is active in a variety of cell lines. Electrophoretic mobility shift assays have revealed the presence of multiple DNA binding proteins specifically interacting with the NRE. At least three distinct protein complexes are variably present depending on the specific cell line examined, and mutational analysis of the NRE has identified a ten-base pair recognition sequence which is shared by all three complexes. This palindromic sequence has not been previously reported and does not share homology with any known transcription factor consensus sequences. When inserted into an E4Delta heterologous promoter construct, the NRE binding sequence failed to inhibit either basal or activated transcription of the target gene, indicating that the NRE does not act as a general repressor but may specifically operate within the context of the TGF-beta RII core promoter.

Mouse endostatin inhibits the formation of lung and liver metastases.

Angiogenesis is required for tumor formation. Several studies have demonstrated that tumor angiogenesis is regulated by a balance between proangiogenesis and antiangiogenesis factors and that this balance varies in different organ environments. To investigate whether expression of an angiogenesis inhibitor by cancer cells could alter this balance and prevent tumor formation in different organ environments, we engineered stable transfectants from RenCa mouse renal carcinoma cells and SW620 human colon carcinoma cells to constitutively secrete a mouse endostatin protein with c-myc and polyhistidine (His) tags. Production and secretion of the endostatin-c-myc-His fusion protein by endostatin-transfected cells were confirmed by immunofluorescence staining and Western blot analysis. The endostatin transfectants and control transfectants, stably transfected with a control plasmid, had similar in vitro growth rates compared with their parental cell lines. Conditioned medium from endostatin-transfected cells inhibited human umbilical vein endothelial cell proliferation by 36-51% compared with conditioned medium from control cells. After inoculation into mice, flank tumors from endostatin-transfected cells were 73-91% smaller than flank tumors from control cells after 3 weeks. Inoculation of a cell mixture containing 25% endostatin-transfected cells and 75% control cells resulted in inhibition of flank tumor formation as effective as after inoculation of 100% endostatin-transfected cells. Formation of lung metastases by RenCa endostatin-transfected cells and formation of liver metastases by SW620 endostatin-transfected cells were dramatically inhibited compared with formation of metastases by control cells. These findings demonstrate that endostatin can inhibit tumor formation in different organ environments and that gene delivery of endostatin into even a minority of tumor cells may be an effective strategy to prevent progression of micrometastases to macroscopic disease.

Infection of the germ line by retroviral particles produced in the follicle cells: a possible mechanism for the mobilization of the gypsy retroelement of Drosophila.

The gypsy retroelement of Drosophila moves at high frequency in the germ line of the progeny of females carrying a mutation in the flamenco (flam) gene. This high rate of de novo insertion correlates with elevated accumulation of full-length gypsy RNA in the ovaries of these females, as well as the presence of an env-specific RNA. We have prepared monoclonal antibodies against the gypsy Pol and Env products and found that these proteins are expressed in the ovaries of flam females and processed in the manner characteristic of vertebrate retroviruses. The Pol proteins are expressed in both follicle and nurse cells, but they do not accumulate at detectable levels in the oocyte. The Env proteins are expressed exclusively in the follicle cells starting at stage 9 of oogenesis, where they accumulate in the secretory apparatus of the endoplasmic reticulum. They then migrate to the inner side of the cytoplasmic membrane where they assemble into viral particles. These particles can be observed in the perivitelline space starting at stage 10 by immunoelectron microscopy using anti-Env antibodies. We propose a model to explain flamenco-mediated induction of gypsy mobilization that involves the synthesis of gypsy viral particles in the follicle cells, from where they leave and infect the oocyte, thus explaining gypsy insertion into the germ line of the subsequent generation.

An env-like protein encoded by a Drosophila retroelement: evidence that gypsy is an infectious retrovirus.

The gypsy element of Drosophila differs from most LTR retrotransposons in containing a third open reading frame that resembles retroviral env genes. The protein encoded by ORF3 is glycosylated and processed, like all retroviral envelope proteins. The protein is expressed at high levels in fly strains in which gypsy elements are active. In these strains the protein is found primarily in viral particles. When larvae of fly strains in which gypsy is normally inactive are exposed to sucrose gradient fractions containing these particles, a high level of gypsy insertion activity is observed in their progeny. Thus, gypsy has the expected properties of an insect retrovirus.

Gypsy transposition correlates with the production of a retroviral envelope-like protein under the tissue-specific control of the Drosophila flamenco gene.

Gypsy displays striking similarities to vertebrate retroviruses, including the presence of a yet uncharacterized additional open reading frame (ORF3) and the recent evidence for infectivity. It is mobilized with high frequency in the germline of the progeny of females homozygous for the flamenco permissive mutation. We report the characterization of a gypsy subgenomic ORF3 RNA encoding typical retroviral envelope proteins. In females, env expression is strongly repressed by one copy of the non-permissive allele of flamenco. A less dramatic reduction in the accumulation of other transcripts and retrotranscripts is also observed. These effects correlate well with the inhibition of gypsy transposition in the progeny of these females, and are therefore likely to be responsible for this phenomenon. The effects of flamenco on gypsy expression are apparently restricted to the somatic follicle cells that surround the maternal germline. Moreover, permissive follicle cells display a typically polarized distribution of gypsy RNAs and envelope proteins, both being mainly accumulated at the apical pole, close to the oocyte. We propose a model suggesting that gypsy germinal transposition might occur only in individuals that have maternally inherited enveloped gypsy particles due to infection of the maternal germline by the soma.

Therapy of an animal model of human gastric cancer using a combination of anti-erbB-2 monoclonal antibodies.

Amplification and/or overexpression of the erbB-2 gene have been demonstrated in 20-30% of adenocarcinomas of the breast, ovary, lung, and stomach and are associated with aggressive clinical course and poor prognosis. Interference with erbB-2 function by the use of monoclonal antibodies is a promising approach to the treatment of these diseases. In this study we demonstrate that a combination of two anti-erbB-2-specific antibodies inhibited the growth of human gastric tumor cells in vitro. This combination antibody therapy also inhibited the growth of human tumor cell lines growing as xenografts in nude mice and was able to dramatically reduce established tumors. This is the first reported observation of tumor regression induced by anti-erbB-2 monoclonal antibodies. Treatment was not curative in that tumors regrew after 6 weeks. Treatment with either single antibody alone did not inhibit cell growth or tumor formation. Pulse chase and tyrosine kinase activity experiments were used to investigate the activity of the erbB-2 gene product (gp185erbB-2). The formation of complexes by two antibodies was found to interfere with receptor function and mimic some properties of a typical receptor ligand. Selective interference of the erbB-2 receptor by combination antibody therapy may be advantageous for the treatment of human cancers.