Mechanisms of instantaneous inactivation of SARS-CoV-2 by silicon nitride bioceramic.

The hydrolytic processes occurring at the surface of silicon nitride (Si3N4) bioceramic have been indicated as a powerful pathway to instantaneous inactivation of SARS-CoV-2 virus. However, the virus inactivation mechanisms promoted by Si3N4 remain yet to be elucidated. In this study, we provide evidence of the instantaneous damage incurred on the SARS-CoV-2 virus upon contact with Si3N4. We also emphasize the safety characteristics of Si3N4 for mammalian cells. Contact between the virions and micrometric Si3N4 particles immediately targeted a variety of viral molecules by inducing post-translational oxidative modifications of S-containing amino acids, nitration of the tyrosine residue in the spike receptor binding domain, and oxidation of RNA purines to form formamidopyrimidine. This structural damage in turn led to a reshuffling of the protein secondary structure. These clear fingerprints of viral structure modifications were linked to inhibition of viral functionality and infectivity. This study validates the notion that Si3N4 bioceramic is a safe and effective antiviral compound; and a primary antiviral candidate to replace the toxic and allergenic compounds presently used in contact with the human body and in long-term environmental sanitation.

Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts.

INTRODUCTION: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. MATERIALS AND METHODS: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. RESULTS: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. DISCUSSION: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases.

High pressure treatment under subfreezing temperature results in drastic inactivation of enveloped and non-enveloped viruses.

Some viruses are sensitive to high pressure. The freeze-pressure generation method (FPGM) applies pressure as high as 250 MPa on a substance, simply by freezing a pressure-resistant reservoir in which the substance is immersed in water. Here we examined whether the FPGM successfully inactivates herpes simplex virus type 1 (HSV-1), an enveloped DNA virus belonging to the human Herpesviridae, and encephalomyocarditis virus (EMCV), an envelope-free RNA virus belonging to the Picornaviridae. After the treatment, HSV-1 drastically reduced the ability to form plaque in Vero cells in vitro as well as to kill mice in vivo. EMCV that had been pressurized failed to proliferate in HeLa cells and induce interferon response. The results suggest that the FPGM provides a feasible procedure to inactivate a broad spectrum of viruses.

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.

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.

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.

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.

Prolonged gene expression in mouse lung endothelial cells following transfection with Epstein-Barr virus-based episomal plasmid.

The development of a strategy to deliver a gene to pulmonary endothelium will be useful for gene function study and for pulmonary gene therapy. Cationic lipidic vectors are efficient in gene transfer to pulmonary endothelium via the vascular route; however, gene expression is transient and lasts for only a few days. In this study, we show that pulmonary gene transfer via cationic lipidic vectors can be significantly improved using an Epstein-Barr virus (EBV)-based expression plasmid. Systemic administration of cationic liposomes followed by the EBV-based plasmid led to gene expression in the lung that lasted for more than 3 weeks. Prolonged and high levels of gene expression can also be obtained in primary mouse lung endothelial cells (MLEC) following lipofection with an EBV-based plasmid. These results suggest the utility of this gene transfer protocol in studying the expression of cloned genes in lung endothelial cells and in pulmonary gene therapy.

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.

Effect of hot water extract from Agaricus blazei Murill on antibody-producing cells in mice.

We investigated the immunopotentiating activities of boiled water-soluble extracts from desiccated Agaricus blazei Murill (ABM). Effect of ABM extract on antibody production was investigated by method of hemolytic plaque-forming cells (PFC) against sheep red blood cells (SRBC) antigen. ABM extracts significantly (p<0.01) increased the number of PFC in spleen with intraperitoneal administration at doses of 25 mg/kg as compared with control group. The populations of Mac-1- or CD25-positive cells significantly (p<0.01, p<0.001) increased, but in CD19-positive cells, there were no differences in ABM-treated mice as compared with control mice. The expressions of IL-6 and IL-1beta mRNA were augmented by ABM extract in both peritoneal macrophages and spleen cells. These results suggested that ABM extract might be an effective stimulator for T cell and macrophage to IL-1beta and IL-6 release, resulting in augmentation of antibody production against SRBC antigen.

Improvement of nonviral gene therapy by Epstein-Barr virus (EBV)-based plasmid vectors.

The nonviral gene transfer technologies include naked DNA administration, electrical or particle-mediated transfer of naked DNA, and administration of DNA-synthetic macromolecule complex vectors. Each method has its advantage, such as low immunogenicity, inexpensiveness, ease in handling, etc., but the common disadvantage is that the transfection efficiency has been relatively poor as far as conventional plasmid vectors are involved. To improve the nonviral gene transfer systems, Epstein-Barr virus (EBV)-based plasmid vectors (also referred to EBV-based episomal vectors) have been employed. These vectors contain the EBNA1 gene and oriP element that enable high transfer efficiency, strong transgene expression and long term maintenance of the expression. In the current article, I review recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, drastic tumor suppression was achieved by gancyclovir administrations following an intratumoral injection with an EBV plasmid vector encoding the HSV1-TK suicide gene. Equiping the plasmid with carcinoembryonic antigen (CEA) promoter sequences enabled targeted killing of CEA-positive tumor cells, which was not accomplished by conventional plasmid vectors without the EBV genetic elements. Transfection with an apoptosis-inducing gene was also effective in inhibiting tumors. Interleukin (IL)-12 and IL-18 gene transfer, either local or systemic, induced therapeutic antitumoral immune responses including augmentation of the cytotoxic T lymphocyte (CTL) and natural killer (NK) activities, while an autologous tumor vaccine engineered to secrete Th1 cytokines via the EBV system also induced growth retardation of tumors. Non-EBV conventional plasmids were much less effective in eliciting these therapeutic outcomes. Intracardiomuscular transfer of the beta-adrenergic receptor gene induced a significant elevation in cardiac output in cardiomyopathic animals, suggesting the usefulness of the EBV system in treating heart failure. The EBV-based nonviral delivery also worked as genetic vaccine that triggered prophylactic cellular and humoral immunity against acute lethal viral infection. All the nonviral delivery vehicles so far tested showed an improved transfection rate when combined with the EBV-plasmids. Collectively, the EBV-based plasmid vectors may greatly contribute to nonviral gene therapy against a variety of disorders, including malignant, congenital, chronic and infectious diseases.

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.

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.

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.