Effects of mechanical stress on cytokine production in mandible-derived osteoblasts.

OBJECTIVE: Mechanical stress is known to be an important factor in the regulation of bone remodeling, and mandibular bone is continuously exposed to mechanical stressors such as occlusal force. Therefore, in this study, we investigated the effects of mechanical stress approaching occlusal force, to which mandible-derived osteoblasts (MDOB) are exposed, on cytokine expression and production using an original hydrostatic pressure apparatus. MATERIALS AND METHODS: The levels of cytokine in MDOB were examined by real-time RT-PCR, ELISA, and western blotting. In addition, mitogen-activated protein kinase inhibitor for ERK1/2, JNK, and p-38 pathways was used to identify the signal transduction pathway. RESULTS: Hydrostatic pressure increased the expression of IL-6 and TNF-α mRNA in a magnitude- and time-dependent manner and also enhanced IL-6 and TNF-α protein production. Furthermore, hydrostatic pressure changed the RANKL/OPG ratio in favor of RANKL for both mRNA and protein levels. Specific inhibitor of p-38 pathway but not that of the ERK1/2 and JNK pathways suppressed the up-regulation of RANKL production induced by hydrostatic pressure loading. CONCLUSION: These results suggest that MDOB play a role in cytokine production in response to mechanical stress and that occlusal force may support the maintenance of mandible bone homeostasis by activating bone remodeling through osteoclastogenesis.

Eradication of the commensal intestinal microflora by oral antimicrobials interferes with the host response to lipopolysaccharide.

The host components and commensal microorganisms of the intestinal microenvironment play roles in the development and maintenance of the host defence. Recent observations have suggested that toll-like receptors (TLRs) are involved in the recognition of innate immunity against intestinal microbes. However, little is known regarding the role of TLR in the maintenance of systemic host defence by intestinal microorganisms. We studied the expression and function of TLR4 and TLR2 on alveolar and peritoneal macrophages in mice after 3 weeks of oral administration of streptomycin and cefotaxime. After active treatment, the intestinal microorganisms were nearly completely eradicated, and the surface expression of TLR4 and TLR2 on the peritoneal macrophages was prominently downregulated. When the actively treated mice were challenged with lipopolysaccharide (LPS), a TLR4 ligand, the host response was markedly impaired. Our results suggest that the oral administration of antimicrobials downregulates the expression of surface TLR on the peritoneal macrophages and modulates the host immune responses against LPS by modifying the intestinal environment.

Induction of chondrogenic phenotype in synovium-derived progenitor cells by intermittent hydrostatic pressure.

OBJECTIVE: The aim of this study was to investigate the effect of intermittent hydrostatic pressure (IHP) on chondrogenic differentiation of synovium-derived progenitor cells (SPCs). METHODS: SPCs, bone marrow-derived progenitor cells and skin fibroblasts from rabbits were subjected to IHP ranging from 1.0 to 5.0 MPa. The mRNA expression of proteoglycan core protein (PG), collagen type II and SOX-9 was examined using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The production of SOX-9 protein and glycosaminoglycan (GAG) by SPCs was analyzed by Western blot and the dimethylmethylene blue assay. In addition, mitogen-activated protein (MAP) kinase inhibitors for c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and the p38 pathway were used to identify the signal transduction pathways. RESULTS: Real-time RT-PCR showed that mRNA expression of PG, collagen type II and SOX-9 was significantly enhanced only in SPCs receiving 5.0 MPa of IHP. The production of SOX-9 protein and GAG by SPCs was also increased by exposure to 5.0 MPa of IHP. These up-regulated expressions were suppressed by pretreatment with an inhibitor of JNK, but not with inhibitors of ERK or p38. CONCLUSION: Our results demonstrated that the exposure of SPCs to 5.0 MPa of IHP could facilitate induction of the chondrogenic phenotype by the MAP kinase/JNK pathway. This finding suggests the potential for IHP utilization in regenerative treatments for cartilage injuries or osteoarthritis.

Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf.

Microphthalmia-associated transcription factor (Mitf) is critically involved in melanin synthesis as well as differentiation of cells of the melanocytic lineage. Some earlier studies suggested that Mitf is also essential in the survival of melanoma cells, but this notion remains controversial. We synthesized short interfering RNA (siRNA) duplexes corresponding to the mitf sequence and transfected them into B16 melanoma. Lipid-mediated transfection in vitro of Mitf-specific siRNA resulted in specific downregulation of Mitf and of the tyrosinase that is a transcriptional target of Mitf. This treatment also remarkably reduced the viability of melanoma cells by inducing apoptosis. To examine the potential feasibility of RNAi therapy against melanoma, B16 cells were subcutaneously injected into syngenic mice and siRNA was transfected into the pre-established tumor by means of electroporation. The Mitf-specific siRNA drastically reduced outgrowth of subcutaneous melanoma, while nonspecific siRNA failed to affect tumor progression. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-based analysis of tumor specimens demonstrated that the tumor cells transfected with Mitf-siRNA effectively underwent apoptosis in vivo. The present results indicate that Mitf plays important roles in melanoma survival. Intratumor electrotransfer of Mitf-specific siRNA may provide a powerful strategy for therapeutic intervention of malignant melanoma.

Cytokine production in human periodontal ligament cells stimulated with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Although some functions and characterizations of human periodontal ligament (hPDL) cells have been reported, the role of hPDL cells in periodontal disease is poorly understood. We have previously reported that hPDL cells produce many kinds of inflammatory cytokines by stimulation with Prevotella intermedia. In this study, we examined the production of cytokines in hPDL cells stimulated with Porphyromonas gingivalis as compared with P. intermedia. MATERIAL AND METHODS: hPDL cells cultured in Dulbecco's modified Eagles's medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics. After three to four passages, hPDL cells were stimulated with P. intermedia (ATCC25601) or P. gingivalis (ATCC33277) for 24 h. Total RNA was extracted by ISOGEN and the expression of cytokine mRNA was determined using reverse transcription-polymerase chain reaction. Cytokines in the culture supernatants were assessed by enzyme-linked immunosorbent assay. RESULTS: The expression of interleukin-1beta, interleukin-6, interleukin-8, tumor necrosis factor-alpha (TNF-alpha), receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) mRNA was detected in hPDL cells after stimulation with P. gingivalis as well as P. intermedia. There were no significant differences in the kind of cytokines expressed in hPDL cells between P. gingivalis and P. intermedia. However, P. gingivalis induced a significantly higher production of cytokines in hPDL cells than P. intermedia (p < 0.05). CONCLUSION: This study demonstrated that hPDL cells produce many kinds of cytokines as a result of bacterial stimulation, including stimulation with P. gingivalis and P. intermedia. These results suggest that hPDL cells may play a role in cytokine production in periodontal disease.

Interferon-gamma is causatively involved in experimental inflammatory bowel disease in mice.

Cytokines may be crucially involved in the pathogenesis of inflammatory bowel diseases (IBD), but it remains controversial whether interferon (IFN)-gamma, a typical proinflammatory cytokine, is an essential mediator to cause the disorders. In the present study, IFN-gamma(-/-) and wild-type (WT) C57BL/6 mice were fed 2.5% dextran sodium sulphate (DSS) in drinking water for 7 days, in order to investigate DSS-induced intestinal inflammation. The DSS-treated WT mice exhibited a robust production of IFN-gamma in the gut, a remarkable loss of body weight, as well as high rate of mortality (60%). In striking contrast, IFN-gamma deficient mice did not develop DSS-induced colitis, as indicated by the maintenance of body weight and survival rate of 100%. Severe intestinal inflammation was demonstrated exclusively in WT animals in terms of the shortening of the bowel as well as the elevation of the disease activity index, myeloperoxidase (MPO) activity and serum haptoglobin level. Histological study of DSS-treated WT intestine revealed disruption of mucosal epithelium and massive infiltration of inflammatory cells, while the organ from IFN-gamma(-/-) mice remained virtually normal in appearance. Enzyme-linked immunosorbent assay (ELISA) analyses indicated abundant production of three chemokines, i.e. monokine induced by interferon-gamma (MIG), interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1), in the DSS-irritated intestine of WT but not of IFN-gamma(-/-) mice. The present results demonstrate clearly that IFN-gamma plays indispensable roles in the initiation of DSS colitis, and some chemokines are produced in an IFN-gamma-dependent fashion.

Transthoracic direct current shock facilitates intramyocardial transfection of naked plasmid DNA infused via coronary vessels in canines.

Catheter-mediated, percutaneous, transluminal delivery of naked plasmid DNA (pDNA) into myocardium may offer a valuable strategy to heart diseases. Here, we examined whether clinically available transthoracic direct current (DC) shock improves intracoronary naked DNA transfection into myocardium. Plasmid vector encoding the GL3 luciferase was infused retrogradely into the coronary veins of beagle dogs, whereas another pDNA solution was infused into the left coronary artery. During and after these procedures, the coronary venous sinus was occluded by balloon, and transthoracic DC shock of 200 J was applied immediately after the infusions. Without DC shock, no remarkable increase in luciferase activity was demonstrated in any part of the left ventricular myocardium. In the presence of DC pulsation, significant luciferase expression was detected in the regions that were supplied by left anterior descending coronary artery (LAD), whereas the gene expression in the right coronary artery (RCA) regions was much less drastic. X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) staining of cardiac cross-sections also revealed regional expression of beta-galactosidase. Immunohistochemical examinations of heart cryosections revealed that cardiomyocytes in LAD regions successfully expressed transgene product. The present system may enable a new strategy for myocardial gene therapy, without any special device or technique other than cardiac catheterization and DC cardioversion that are generally performed in ordinary hospitals.

Glutamine protects articular chondrocytes from heat stress and NO-induced apoptosis with HSP70 expression.

OBJECTIVE: To investigate the effect of l-glutamine (Gln) on stress responses of chondrocytes exposed to heat stress or nitric oxide (NO). METHODS: Cultures of articular chondrocytes were established from rabbit joints, and treated for 12h with various concentrations of Gln (0-20 mM). In some experiments, cells were also treated with quercetin (Que), a heat shock protein 70 (HSP70) inhibitor. Heat stress (43 degrees C) was applied to the cells for 0-120 min. Apoptosis was induced by 0.5mM sodium nitroprusside (SNP) dihydrate that produces NO. After stress loading, HSP70 expression was detected by Western blot analysis. Cell viability was assessed by lactate dehydrogenase (LDH) release and tetrazolium salt-based assays, while apoptosis was evaluated by Hoechst 33342 staining, TUNEL methods and active caspase-3 determination. RESULTS: Gln demonstrated dose-dependent enhancing effect on stress-mediated induction of HSP70, while in the absence of any stress HSP70 was not induced by Gln alone. After heating or SNP loading, chondrocytes showed severe reduction in viability, while the cytotoxic outcome was almost completely abrogated by conditioning with Gln. The protective effect of Gln was significantly blocked by Que that effectively suppressed stress-induced HSP70 expression in chondrocytes. The Gln also rendered chondrocytes unsusceptible to NO-induced apoptosis that was frequently seen in SNP-treated culture. CONCLUSION: This study demonstrated that the treatment of chondrocytes with Gln protected the cells from heat stress and NO-induced apoptosis. These chondroprotective effects of Gln may be mediated by HSP70.

Cytokine genetic adjuvant facilitates prophylactic intravascular DNA vaccine against acute and latent herpes simplex virus infection in mice.

Intravascular plasmid DNA (pDNA) vaccine encoding herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) effectively induces prophylactic immunity against lethal HSV-1 infection in mice. We investigated whether the vaccine potency is further improved by coadministration of cytokine genes together with a low dose of genetic vaccine. pDNA encoding IL-12, IL-15, IL-18 or IL-21 was capable of elevating survival rates of HSV-1-infected mice when coinjected with 1 microg of gB pDNA, while IL-10 gene delivery failed to affect the effectiveness of the genetic immunization. Although only 17% of mice survived acute HSV infection after the gB pDNA vaccination at a dose of 1 microg, all mice coadministered with 1 microg each of gB and IL-12 pDNAs not only survived the acute infection but also escaped latent infection. In these animals, the neutralizing antibody against HSV-1 was abundantly produced, and CTL activity against the gB antigen was augmented. Coadministration of the gB and IL-12 genes also elevated the serum level of interferon-gamma. Adaptive transfer experiments indicated that soluble factors contributed to preventive immunity, while cell components alone were not capable of protecting mice from fatal viral infection. These results strongly suggest potential usefulness of Th1 cytokine genes as effective molecular adjuvants that facilitate specific humoral as well as cellular immune responses elicited by intravascular molecular vaccination.

Augmentation of antigen-specific cytokine responses in the early phase of vaccination with a live-attenuated simian/human immunodeficiency chimeric virus expressing IFN-gamma.

A nef-deleted SHIV-NM-3rN (SHIV-NI) was previously shown to be nonpathogenic and to induce protective immunity. In the present study, a SHIV-NI expressing human interferon-gamma (SHIV-IFN-gamma) was constructed and the effect of co-expression of IFN-gamma on virus replication and immunopotentiation was investigated in macaques that were vaccinated with both viruses, by comparing cytokine responses during the first 4 weeks after vaccination. Peripheral blood mononuclear cells (PBMC) isolated from vaccinated macaques were stimulated with inactivated viral particles for 24 h, and the production of IL-2, IL-4, IL-6, IL-10, IL-12, TNF-alpha and IFN-gamma was determined by ELISA and flow cytometry. All of the vaccinated macaques showed increases in cytokine production. However, the production of IFN-gamma (Th1-type cytokine) was more rapidly induced by SHIV-IFN-gamma vaccination, and IFN-gamma-producing cells appeared to be still increasing at 4 weeks after vaccination, although the difference of virus replication during the time was not significant in contrast to in vitro replication in cultured PBMC. These results suggest that co-expression of IFN-gamma with SHIV can modulate the antiviral immune responses into the Th1 type response, which would probably provide more protective immunity.

Intravascular naked DNA vaccine encoding glycoprotein B induces protective humoral and cellular immunity against herpes simplex virus type 1 infection in mice.

Naked plasmid DNA (pDNA) vaccine expressing herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) was tested for protective activity against acute HSV-1 infection in mice. The pDNA was intravenously injected into Balb/c mice via their tail vein under high pressure, and the vaccination was performed two times at an interval of 7 days. The gB gene vaccination significantly protected the mice from subsequent intraperitoneal challenge with a lethal dose of HSV-1, which killed all the animals given control plasmid or saline. The protective activity was correlated with the dose of the plasmid inoculated, the survival rate reaching 83% in mice vaccinated with 5 microg of pDNA. The vaccinated mice were also protected from latent HSV infection. The immunized mice showed significant elevation in neutralizing antibody against HSV-1 as well as serum levels of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma). When mice were immunized with 5 microg of an Epstein-Barr virus (EBV)-based plasmid vector harboring the gB, the cytotoxic T lymphocytes (CTLs) activity and proliferative response for HSV-1 were also induced. The results strongly suggest that intravenous immunization of naked pDNA may induce humoral and cellular immune responses against the virus, leading to a significant prophylactic outcome against HSV-1 infection in mice.

Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo.

To accomplish efficient nonviral gene therapy against prostate cancer (PC), Epstein-Barr virus (EBV)-based plasmid vectors containing EBNA1 gene and oriP were employed and combined with a cationic polymer or cationic lipid. When EBV-plasmid/poly-amidoamine dendrimer complex was injected into PC-3-derived tumors established in severe combined immunodeficiency mice, a considerable expression of marker gene was obtained in the tumors, and the expression level was more than eight-fold higher than that achieved by conventional plasmid vector/dendrimer. Since most PC cells express the apoptotic signal molecule Fas (Apo-1/CD95) on their surface, Fas ligand (FasL) gene was transferred into PC cells to kill the tumor cells. In vitro transfection with pGEG.FasL (an EBV-plasmid with the FasL gene) significantly reduced the viability of PC cells, which subsequently underwent apoptosis. Intratumoral injections of pGEG.FasL into PC induced significant growth suppression of the xenograft tumors, in which typical characteristics of apoptosis were demonstrated by TUNEL staining and electron microscopic observations. When pGEG.FasL transfer was accompanied by systemic administrations of cisplatin, the tumors were inhibited even more remarkably, leading to prolonged survival of the animals. FasL gene transfection by means of EBV-based plasmid/cationic macromolecule complexes may provide a practical therapeutic strategy against PC.

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein-Barr virus (EBV)-based plasmid vectors.

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 microg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 microg of DNA. More than 70% of liver cells stained with X-gal when beta-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver.

Apoptosis induced by in vitro infection with simian-human immunodeficiency chimeric virus in macaque and human peripheral blood mononuclear cells.

We investigated apoptosis induced by in vitro infection with the chimeric virus of simian immunodeficiency virus and human immunodeficiency virus (SHIV). Macaque and human peripheral blood mononuclear cells (PBMCs) were infected with pathogenic SHIV-89.6p (89.6p) or nonpathogenic SHIV-NM-3rN (NM-3rN). In macaque PBMCs, the extent of virus production and apoptosis induction in CD4(+) cells was much greater in 89.6p infection than in NM-3rN infection. The result was consistent with our previous study of in vivo SHIV infection. In human PBMCs, 89.6p replicated and induced apoptosis more extensively than did NM-3rN, when the cells were infected with the same infectious doses of the viruses. However, in cells infected with a high dose of NM-3rN, the levels of virus production and apoptosis induction were comparable to those in 89.6p infection. There was no significant difference in the extent of apoptosis induction between 89.6p and NM-3rN infection when growth curves of the two viruses matched. Thus, apoptosis induction by SHIV might depend quantitatively on the amount of virus production rather than on the strains of the virus. Moreover, the correlation between the extent of apoptosis induction and virus pathogenicity in macaque PBMCs has also been found in SHIV-infected macaques. This suggests that the profiles of SHIV infection in vitro reflect the in vivo phenomena. Therefore, the in vitro evaluation of apoptosis induction by SHIV could be useful as a safety test for the development of live-attenuated vaccines.

Unique place of Kampo (Japanese traditional medicine) in complementary and alternative medicine: a survey of doctors belonging to the regional medical association in Japan.

Although there are various kinds of complementary and alternative medicine (CAM) therapies, it is conjectured that medical doctors consider individual CAM therapies to be heterogeneous in nature. Therefore, to investigate the relationship among Kampo (Japanese traditional medicine) and other CAM, a survey using a structured, self-administered questionnaire was performed for 540 randomly selected doctors of the Kyoto Medical Association (KMA). The results showed that some form of CAM was practiced by 73% of the KMA doctors. The most common CAM practice was Kampo, which corresponded to 96.1% of CAM-practicing doctors. A smaller percentage of doctors practiced other forms of alternative medicine. Kampo was best known by doctors among other CAM therapies. Almost all doctors believed in the effectiveness of Kampo. Doctors who believed in the effectiveness of Kampo tended to believe that other CAM therapies were also effective. Cluster analysis revealed that Kampo was distant from the other CAM. It was concluded that Kampo was most frequently practiced and most believed by doctors in Japan among CAM therapies. Since Kampo was independent of other CAM therapies, Kampo's place in CAM therapies was very unique in Japan.

In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice.

Direct intratumoral transfection of cytokine genes was performed by means of the in vivo electroporation as a novel therapeutic strategy for cancer. Plasmid vectors carrying the firefly luciferase, interleukin (IL)-12 and IL-18 genes were injected into established subcutaneous B16-derived melanomas followed by electric pulsation. When plasmid vectors with Epstein--Barr virus (EBV) nuclear antigen 1 (EBNA1) gene were employed, the expression levels of the transgenes were significantly higher in comparison with those obtained with conventional plasmid vectors. In consequence of the transfection with IL-12 and IL-18 genes, serum concentrations of the cytokines were significantly elevated, while interferon (IFN)-gamma also increased in the sera of the animals. The IL-12 gene transfection resulted in significant suppression of tumor growth, while the therapeutic effect was further improved by co-transfection with IL-12 and IL-18 genes. Repetitive co-transfection with IL-12 and IL-18 genes resulted in significant prolongation of survival of the animals. Natural killer (NK) and cytotoxic T lymphocyte (CTL) activities were markedly enhanced in the mice transfected with the cytokine genes. The present data suggest that the cytokine gene transfer can be successfully achieved by in vivo electroporation, leading to both specific and nonspecific antitumoral immune responses and significant therapeutic outcome.

Relation between cytokines and Helicobacter pylori in gastric cancer.

BACKGROUND: Helicobacter pylori is etiologically involved in the development of gastric cancer and infected gastric mucosa has been shown to possess elevated levels of cytokines [for example interleukin (IL)-1beta, IL-6 and IL-8]. Because specific cytokines have also been shown to enhance the development of certain cancers, we examined the relationship between the levels of cytokines, the type and stage of gastric cancers, and the H. pylori infection. MATERIALS AND METHODS: Cytokines were measured from gastric cancer tissues, adjacent normal appearing mucosa, and the serum in 66 patients with early or advanced gastric cancer and from controls using semiquantitative RT-PCR and ELISA. RESULTS: IL-6 and IL-8 levels were more than 10-fold increased in cancer tissues as compared with normal gastric tissues. IL-8 levels in cancer tissues were more than 2-fold higher in advanced gastric cancer as compared with early gastric cancer irrespective of H. pylori status. IL-6 levels were significantly higher in early gastric cancer with active H. pylori infection as compared with early cancer without H. pylori infection (8.7 + 1.4 vs. 1.2 + 0.3 pg/mg protein, p <.001) and decreased significantly after the cure of H. pylori (11.1 + 2.9-8.2 + 2.3 pg/mg protein, p <.05). CONCLUSIONS: IL-8 levels in gastric cancer tissue are largely independent of H. pylori infection. In contrast, tissue IL-6 levels were high in H. pylori infected early gastric cancer and fell significantly after the cure of H. pylori suggesting a relationship between H. pylori infection and early gastric cancer.

Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma-derived HCS-2/8 cells.

The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent; Oiagen), linear polyethylenimine (PEI; ExGen 500; Euromedex), and branched PEI in gene delivery into chondrocytes was examined comparatively. A plasmid vector containing the Escherichia coli LacZ (pSES.beta) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8. Gene expression was evaluated by an O-nitrophenyl beta-D-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal; Nacalai Tesque). The ONPG assay showed that the highest delivery rate was achieved when 2microg of pSES.beta was combined with either 21 microg of dendrimer, 1.7microg of linear PEI, or 2.0microg of branched PEI. At the same DNA/polymer ratios, the proportions of X-gal-stained cells were also the highest (31.3 +/- 7.5%, 30.3 +/- 9.0%, and 8.3 +/- 3.1%, respectively). LacZ expression reached the highest level 3 days after the dendrimer-mediated transduction, and gradually declined, returning to the background level on day 14. Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations. Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer. The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful nonviral vector for gene delivery to cells of chondrocytes.

Expression of transduced HSP70 gene protects chondrocytes from stress.

OBJECTIVE: To investigate the efficacy of adenovirus vector mediated transduction of heat shock protein 70 (HSP70) gene to human chondrocyte-like cell (HCS-2/8) against heat stress. METHODS: Two adenovirus vectors that contain wild-type (AxSHEwt) or mutant-type (AxSHEmt) HSP70 gene, and that are regulated by SRalpha promoter, were constructed. The mutant-type lacks the area that expresses stress durability. One of the 2 adenovirus vectors was added to the cultures of human chondrocyte-like cells (HCS-2/8). Heat stress (48 degrees C) was applied to the transduced cells for 2 h, and the efficacy of adenovirus vector mediated transduction of HSP70 gene against heat stress in the chondrocytes was investigated using alamar blue assay and MTT assay. RESULTS: Absorbance levels at 48 degrees C were 300.3 +/- 51.9 and 1.173 +/- 0.011 in the controls, 278.5 + 33.8 and 1.217 +/- 0.018 in the AxSHEmt transduced cells, and 349 +/- 14.7 and 1.371 +/- 0.033 in the AxSHEwt transduced cells. The level in the AxSHEwt transduced cells was significantly higher than in the other 2 groups (p < 0.05). With 37 degrees C treatment, no significant difference was observed. CONCLUSION: Chondrocytes to which HSP70 gene was transduced had a significantly higher metabolic activity and viability under heat stress.