Heat-treated Cell Research Articles

Effect of Vacuum Heat Treatment on Larch Earlywood and Latewood Cell Wall Properties

The aim of this study was to evaluate the hygroscopicity and nanomechanics of earlywood (EW) and latewood (LW) larch after thermal modification under vacuum conditions. Wood samples were heat-treated in a vacuum atmosphere at 180–220 °C for 6 h, then their cell wall properties were observed using dynamic water vapor sorption (DVS), imaging Fourier-transform infrared (FTIR) microscopy, and nanoindentation. The results showed that the vacuum heat treatment reduced the hygroscopicity of EW and LW and increased hysteresis between the adsorption and desorption branches of the isotherm. Compared with EW, the treatment temperature had a more pronounced influence on the hygroscopicity of LW. The Hailwood-Horrobin model was found to accurately fit the experimental data. Imaging FTIR microscopy revealed degradation of hemicellulose, cross-linking, condensation reactions, and redistribution of lignin in the cell wall. The elastic modulus for the heat-treated EW and LW cell walls increased at first and then decreased as the treatment temperature increased; the increase in LW was more intense than that in EW. Cell wall hardness also markedly increased after heat treatment. Our analysis suggests that vacuum heat treatment decreases hygroscopicity and alters the chemical composition distribution of cell walls, thus improving wood cell wall mechanics.

Investigation of the relationship between surface colour, contact angle and chemical properties of heat-treated bamboo

ABSTRACT The inherent relationships between the surface phenomena and chemical structures of thermally modified bamboo were investigated to predict the intrinsic properties of bamboo during the thermal modification through the surface phenomena effectively. Moso bamboo was firstly treated by vacuum treatment at 150°C, 170°C, 190°C and 210°C for 1, 2, 3 and 4 h, respectively. And then the surface colour, contact angle and chemical compositions of heat-treated bamboo and in-situ chemical properties of the heat-treated cell wall were analysed by using the colour metre, contact angle metre and confocal scanning microscope. Results showed that the surface colour became darker and the hydrophobicity was increased after heat treatment. As compared to the control, the lightness (L*) value carried out in accordance with CIELAB of heat-treated bamboo decreased by almost 54% and the maximum increase of contact angle was 160%. The surface colour and contact angle of bamboo were closely related to the lignin content. The darkened colour and increased hydrophobicity of the bamboo surface were caused by the crack of C–O–C and the decrease of C=O in lignin. Furthermore, fluorescence analysis indicated that the lignin distribution in the cell wall increased gradually after heat treatment, especially in the thick-walled cells and the corners of cells.

Temperature-Dependent Creep Behavior and Quasi-Static Mechanical Properties of Heat-Treated Wood

In this paper, Berkovich depth-sensing indentation has been used to study the effects of the temperature-dependent quasi-static mechanical properties and creep deformation of heat-treated wood at temperatures from 20 °C to 180 °C. The characteristics of the load–depth curve, creep strain rate, creep compliance, and creep stress exponent of heat-treated wood are evaluated. The results showed that high temperature heat treatment improved the hardness of wood cell walls and reduced the creep rate of wood cell walls. This is mainly due to the improvement of the crystallinity of the cellulose, and the recondensation and crosslinking reaction of the lignocellulose structure. The Burgers model is well fitted to study the creep behavior of heat-treated wood cell walls under different temperatures.

Myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated tumor cell lysates enhance antitumor activity in murine lung cancer.

The present study aimed to investigate the efficacy of a myeloid dendritic cell (mDCs) and plasmacytoid (p)DC combined vaccine loaded with heat-treated cancer cell lysates against lung cancer cells. The mDCs and pDCs were selected using magnetic bead sorting. Antigen loading was performed by adding heat-treated Lewis lung cancer cell lysates to mDC, pDC or mDC+pDC (1:1). Surface expression of CD80, CD86, CD40 and major histocompatibility complex (MHC)-II molecules were determined using flow cytometry, and the secretion of cytokines IL-12, IL-6 and TNF-α were assessed using ELISA assays. The effect of the mDC and pDC vaccine on cytotoxic T lymphocytes (CTLs) against tumor cells was investigated. Tumor-bearing nude mice were intravenously injected with the mDC and pDC combined vaccine. Tumor tissues were collected for hematoxylin and eosin and TUNEL staining. Loading with tumor cell lysate significantly upregulated the surface expression of costimulatory molecules MHC-II on DCs and enhanced secretions of IL-6, IL-12 and TNF-α by DCs. In addition, the tumor cell lysate-loaded mDC and pDC combined vaccine significantly promoted lymphocyte proliferation and enhanced CTL-mediated cytotoxicity against Lewis lung cancer cells compared with mDC or pDC treatment alone. Furthermore, intravenous injection of the mDC and pDC combined vaccine into tumor-bearing nude mice significantly inhibited subcutaneous tumor growth and induced necrosis and apoptosis within the tumor tissue. Overall, the pDC and mDC combination vaccine loaded with heat-treated Lewis lung cancer cell lysate had a synergistic effect on the induction of T lymphocyte proliferation and antitumor efficacy, which may be associated with the upregulation of co-stimulatory molecules and cytokine secretions.

Evaluation of Cell-Wall Microstructure and Anti-Swelling Effectiveness of Heat-Treated Larch Wood

In this study, the cell-wall microstructure and anti-swelling effectiveness (ASE) of heat-treated larch wood were evaluated and the correlation between them was analyzed. For this purpose, some larch lumbers were heat-treated for 12, 18, and 24 hours at temperatures of 190°C and 220°C. By observing the scanning electron microscopy cross-sectional image of the heat-treated larch, it was confirmed that the shape of heat-treated wood cell changed, the cut-section of the wood cell wall was rough, and the intercellular space has become wide as the intercellular bonds had broken because of heat-treatment. In addition, the evaluation of the swelling for each treatment condition revealed that, as the heat-treatment temperature and duration increased, the amount of absorbed water and swelling decreased and the ASE increased. The decrease in the amount of absorbed water is thought to be affected by the chemical change in the cell wall by heat-treatment. On the contrary, the decrease in the swelling and the increase in the ASE are thought to be due to a combination of chemical changes and physical changes such as structural changes in the cell wall.

Insufficient radiofrequency ablation promotes epithelial-mesenchymal transition mediated by interleukin-6/signal transducer and activator of transcription 3/Snail pathway in the H22 cells.

Radiofrequency ablation (RFA), an established and minimally invasive therapy for hepatocellular carcinoma, has become an important treatment strategy. However, tumor aggressiveness remains a common problem. The epithelial-mesenchymal transition (EMT) is thought to play an important role in this process. Due to limited sample volumes harvested from patients, we established a heat-treated cell line and a mouse model to investigate the mechanisms of incomplete ablation in EMT. We heat-treated H22 and HepG2 cells using a water bath to determine a suitable temperature for incomplete RFA. Male BALB/c mice were orthotopically transplanted with H22 cells and then subjected to incomplete ablation. Changes in the EMT biomarkers were detected by real-time polymerase chain reaction, western blotting, and immunofluorescence. The experimental results are expressed as means ± standard deviations. Incomplete RFA promoted EMT, downregulated E-cadherin, upregulated vimentin and Snail, and enhanced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) both in vivo and in vitro. Moreover, interleukin (IL)-6 secretion increased after heat treatment in the H22 cells. AG490, an IL-6 inhibitor, inhibited the occurrence of EMT. Insufficient ablation performed at low temperature successfully induces EMT and promotes tumor aggressiveness, which is mediated by the IL-6/STAT3/Snail pathway in both cell and mouse models.

Anti-lung cancer effect of myeloid and plasmacytoid dendritic cell combined vaccines loaded with tumor cell lysates in vitro

Objective: To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated Lewis lung cancer cell lysates for treatment of lung cancer in mice. Methods: Bone marrow cells were induced by the recombinant mouse fms-like tyrosine kinase receptor 3 ligand (rmFlt3-L) in vitro, myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC∶pDC=1∶1 were stimulated with heat-treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ (MHC-Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin-12 (IL-12), interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Results: The lymphocyte proliferation in mice stimulated with mDC+ pDC group loaded with heat-treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively (P<0.05). When the ratio of effector cells: target cells (E∶T) was 10∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P<0.05). When the ratio of E∶T was 20∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 54.77%±3.28%, significantly higher than 35.25%±1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P<0.05). When the ratio of E∶T was 40∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 73.01%±0.91%, significantly higher than 51.36%±0.58% of mDC group and 22.65%±1.28% of pDC group, respectively (P<0.05). With the rate of E∶T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC-Ⅱ of mDC: pDC=1 group pulsed with heat-treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL-6 cytokine concentrations of mDC+ pDC group, mDC group and pDC group loaded with heat-treated Lewis lung cancer cell lysates were (586.67±52.52) pg/ml, (323.33±67.14) pg/ml and (166.67±16.07) pg/ml, respectively. The concentrations of IL-12 in each group were (2 568.75±119.24) pg/ml, (2 156.25±120.55) pg/ml and (672.92±31.46) pg/ml, respectively. The concentrations of TNF-α in each group were (789.33±48.08) pg/ml, (584.89±116.49) pg/ml and (291.56±40.73) pg/ml, respectively. The concentrations of IL-6, IL-12 and TNF-α secreted by mDC+ pDC group were much higher than those of mDC group and pDC group under the same culture conditions (P<0.05). Conclusions: The mDCs and pDCs combined vaccines pulsed with heat-treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti-tumor effect is related with up-regulation of co-stimulatory molecules and increased secretion of cytokines.

The Interleukin-6/Signal Transducer and Activator of Transcription 3/Snail Pathway Mediates Epithelial-Mesenchymal Transition in Recurrent Liver Cancer after Radiofrequency Ablation

Background: Radiofrequency ablation (RFA), as a relatively mature and minimally invasive therapy for hepatocellular carcinoma (HCC), has become an important treatment strategy. However, tumor recurrence remains common and epithelial-mesenchymal transition (EMT) is thought to play an important role in this process. Based on limited sample volumes harvested from patients, we established a heat-treated cell line and mouse model, in addition to collecting patient samples, to investigate the mechanism of recurrence after RFA due to incomplete ablation. Methods: We heat-treated H22 cells using a water bath to determine a suitable temperature for incomplete RFA. Male BALB/c mice were orthotopically transplanted with H22 cells and then subjected to incomplete ablation. Clinical specimens collected from patients with HCC who had undergone RFA were also collected. Changes in EMT biomarkers were detected by real-time polymerase chain reaction, western blotting, and immunofluorescence. Findings: Incomplete RFA promoted EMT, downregulated E-cadherin, upregulated vimentin and Snail, and enhanced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) both in vivo and in vitro. Moreover, interleukin (IL)-6 secretion increased after heat treatment in H22 cells. AG490, an IL-6 inhibitor, was found to block the occurrence of the EMT. Interpretation: Insufficient ablation performed at low temperatures can successfully induce EMT, which is mediated by the IL-6/STAT3/Snail pathway in cell and mouse models. Funding: Supported by National Natural Science Foundation of China (61671276) and the Natural Science Foundation of Shandong Province (2014ZRE27479, ZR2018PH032, ZR201709250376). Declaration of Interest: All authors declare no conflicts-of-interest related to this article. Ethical Approval: The study design was approved by the appropriate ethics review board.

Prorocentrum lima from the South Atlantic: Morphological, molecular and toxicological characterization

Morphological descriptions using light and scanning electron microscopy and molecular characterization of two Prorocentrum lima strains (UNR-01 and UNR-09) isolated from Armação dos Búzios, Rio de Janeiro, Brazil are provided. Okadaic acid (OA), dinophysistoxin-1 (DTX1) and DTX2 production by strain UNR-01 was investigated using liquid chromatography with mass spectrometry. Toxins were extracted from heat-treated (boiled) and non-boiled cell pellets to obtain respective quantities of free and total OA and DTX1. Growth parameters (growth rate and mean generation time) were determined for strain UNR-01. Prorocentrum lima cells were oblong-to-ovate in shape, broad in the middle region, and narrow at the anterior end. The periflagellar area was triangular, set into a V-shaped depression and was composed of eight periflagellar platelets of different sizes. The morphology fits well the characterization of the species isolated from elsewhere. Phylogenetic analysis based on internal transcribed spacer – ITS – and D1–D3 large subunit – LSU – of ribosomal RNA gene sequences revealed that both strains were identical and closely related to P. lima isolates from the Caribbean Sea and USA. The growth rate of strain UNR-01 was 0.24divday−1. OA concentrations were on average 15.2 and 38.5pg[OA]cell−1 for heat-treated and non-treated cells respectively, while DTX1 mean concentration was 0.5pg[DTX1]cell−1 for both heat-treated and non-treated cells. DTX2 was not detected. To date, these are the first strains of P. lima from the south Atlantic that have been characterized.

In situ measurement of heat-treated wood cell wall at elevated temperature by nanoindentation

Nanoindentation equipped with hot stage to investigate the temperature-dependent real-time mechanical behavior of wood cell wall is introduced. In this study, wood samples were heat-treated at 180°C and 210°C. The effects of heat treatment on Larix gmelinii (Rupr.) Kuzen, a species of larch native to eastern Siberia, were studied at the cell-wall level. Nanoindentation at high temperature was used to track changes in the micromechanical properties in the longitudinal direction and to obtain measurements for both the temperature-dependent reduced modulus and hardness. The average reduced modulus of the cell wall slightly increased, while hardness tended to increase after heat treatment under room temperature conditions. The nanoindentation tests showed that both the reduced modulus and hardness of the wood cell walls tended to decrease as the temperature increased from 20°C to 180°C. Moreover, it was shown that heat treatment resulted in better heat resistance compared to that of untreated wood, especially in the samples indented at 180°C.

Post-annealing of thin-film yttria stabilized zirconia electrolytes for anode-supported low-temperature solid oxide fuel cells.

The effects of a post-annealing treatment on the performance of low-temperature solid oxide fuel cells (LT-SOFCs) were investigated. Nickel oxide-samarium doped ceria (NiO-SDC) anodes and yttria stabilized zirconia (YSZ) electrolytes were deposited on anodized aluminum oxide (AAO) membranes by RF sputtering and DC reactive sputtering, respectively. The half-cell of YSZ/NiO-SDC was then heat-treated at 600 degrees C for 10 h, and a porous platinum (Pt) cathode was deposited on the annealed YSZ/NiO-SDC structure by DC magnetron sputtering. Electrochemical impedance spectroscopy (EIS) analysis revealed a significant decrease in the ohmic resistance and a slight increase in the cathodic impedance. Such a result may be attributed to the increased grain size and enhanced crystallinity of the YSZ electrolyte after the heat treatment. The maximum power density observed for the heat-treated cell was 35 mW/cm2 at 450 degrees C, more than three times higher than the 10 mW/cm2 value obtained for the as-deposited cell.

Hyperthermia increases natural killer cell cytotoxicity against SW-872 liposarcoma cell line.

Although there is convincing data in support of the effectiveness of hyperthermia in tumor therapy, the molecular mechanisms underlying the clinical effects of hyperthermia are still poorly understood. To investigate natural killer (NK) cell cytotoxicity against heat-treated SW-872 and HeLa tumor cell lines. NKG2D ligands and HLA class I transcription were examined using quantitative real-time PCR in treated tumor cell lines at 0, 2, 4, 6 and 12 h following thermal treatment at 39C and 42C for 1 h. The expression of MICA/B, ULBP1 and ULBP2 were also determined by flow cytometry. NK92-MI cytotoxic activity against heat-treated target cell lines was assessed by LDH release as well as annexin-V and 7-AAD assays. Our results showed that heat treatment at 39C improved the cytolytic activity of NK cells against SW-872 cells without increasing NKG2D ligand concentration or decreasing HLA class I levels. The observed increase in the cytotoxicity of NK cells against SW-872 cells after hyperthermia does not coincide with changes in MICA/B, ULBP1 and ULBP2 ligands of NKG2, however, the expression of other ligands in target cells may have made the cells susceptible to the cytotoxic effect of NK cells.

Binding mechanism of patulin to heat-treated yeast cell

This study aims to assess the removal mechanism of patulin using heat-treated Saccharomyces cerevisiae cells and identify the role of different cell wall components in the binding process. In order to understand the binding mechanism, viable cells, heat-treated cells, cell wall and intracellular extract were performed to assess their ability to remove patulin. Additionally, the effects of chemical and enzymatic treatments of yeast on the binding ability were tested. The results showed that there was no significant difference between viable (53·28%) and heat-treated yeast cells (51·71%) in patulin binding. In addition, the cell wall fraction decreased patulin by 35·05%, and the cell extract nearly failed to bind patulin. Treatments with protease E, methanol, formaldehyde, periodate or urea significantly decreased (P<0·05) the ability of heat-treated cells to remove patulin. Fourier transform infrared (FTIR) analysis indicated that more functional groups were involved in the binding process of heat-treated cells. Polysaccharides and protein are important components of yeast cell wall involved in patulin removal. In addition, hydrophobic interactions play a major role in binding processes. Heat-treated S.cerevisiae cells could be used to control patulin contamination in the apple juice industry. Also, our results proof that the patulin removal process is based mainly on the adsorption not degradation.

Biomineralization of manganese by Bacillus spp isolated from a marine biofilm

Biomineralization of manganese on titanium condenser material exposed to seawater has been illustrated. Biomineralization occurs when the fouling components, namely, the microbes, are able to oxidize minerals present in water and deposit them as insoluble oxides on biofilm surfaces. Extensive biofilm characterization studies Showed that an alarmingly large number of bacteria in these biofilms are capable of oxidizing manganese and are, thereby, capable of causing biomineralization on the condenser material exposed to seawater. This paper addresses studies on understanding the exact role of the microbes in bringing about oxidation of manganese. The kinetics of manganese oxidation by marine Gram-positive manganese oxidizing bacterium Bacillus spp. that was isolated front the titanium surface was studied in detail. Manganese oxidation in the presence of Bacillus cells, by cell free extract (CFE) and heat-treated cell free extract was also studied. The study confirmed that bacteria mediate manganese oxidation and lead to the formation of biogenic oxides of MnO2 eventually leading to biomineralization on titanium surface exposed to seawater.

Screening of cytokines to enhance vaccine effects of heat shock protein 70-rich tumor cell lysate

Heat shock proteins (HSPs) have been recognized as significant participants in immune reactions. We have previously reported that heat-treated cells expressing HSP70 can mediate potent antitumor immune responses. As successful immunotherapy is dependent on the host immune system, the present study evaluated whether systemic administration of immunocyte stimulatory and growth promoting cytokines could enhance heat-treated cell lysate vaccine (HCLV) immunization to further promote the antitumor immunity. After heating mouse melanoma B16 cells (43 degrees C, 30 min) to elicit increased HSP70 expression, cells were lysed by freeze thawing to prepare HCLV. In approaches using a poorly immunogenic melanoma B16, the effects of various cytokines (IL-1beta, -2, -4, -6 and -12, IFN-beta and -gamma, GM-CSF and TNF-alpha) were assessed in combination with HCLV. Syngenic C57BL/6 mice were immunized subcutaneously with HCLV twice, on days -14 and -7, while cytokines were injected intraperitoneally on day -7. Subcutaneous B16 cell challenge was performed on day 0. IL-12 significantly enhanced the efficacy of HCLV, compared to non-heated cell lysate vaccine (CLV) and non-vaccination. Systemic administration of recombinant IL-12 augmented the efficacy of HCLV, inducing protective immunity against tumor challenge and enhancing cytotoxicity assessed in primed splenocytes against B16 cells in treated mice. These results suggest that IL-12 represents an important modulator of antitumor immune responses induced by HCLV, and may facilitate further efforts to develop novel cancer immunotherapies based on HSP70-mediated vaccination.

Effect of pertussis toxin on the induction of nitric oxide synthesis in murine macrophages and on protection in vivo

Macrophages from mice immunised with whole cell pertussis vaccine (WCV) responded in vitro to selected antigens by nitric oxide (NO) synthesis. This process was closely associated with macrophage activation. Because of the postulated role of traces of pertussis toxin (PT) in the protective effects of WCV, native PT and a genetically detoxified PT (g-PT) in combination with either a heat-treated whole cell pertussis vaccine (dWCV) or a three component acellular vaccine (ACV), were examined for their effects on NO induction in murine macrophages. The protective effects of these two forms of PT were examined in parallel using the intracerebral (ic) and aerosol challenge routes. Cultures of macrophages from mice immunised with dWCV and ACV, PT or g-PT produced less NO than comparable cultures from mice vaccinated with WCV. However, vaccination with either dWCV or ACV in combination with PT but not with g-PT, induced a significant increase (126–157%) in NO production by cultured cells and was associated with increased protection against challenge by both the ic and aerosol routes. These data indicate that a low concentration of PT acting as a co-factor in combination with other Bordetella pertussis antigens, can potentiate the activation of macrophages and that this process plays a key role in protection against infection.

Natural transformation of Acinetobacter sp. strain BD413 with cell lysates of Acinetobacter sp., Pseudomonas fluorescens, and Burkholderia cepacia in soil microcosms.

To elucidate the biological significance of dead bacterial cells in soil to the intra- and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacter spp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transform Acinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80 degrees C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus provides Acinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.

Heat sensitivity of double-stranded DNA-dependent protein kinase (DNA-PK) activity

Purpose: The heat sensitivity of DNA-PK activity in hybrid cells and the possible restoration of this activity with extracts from scid cells (defective in DNA-PKcs), sxi-3 cells (defective in Ku80) and V79-4 (sxi-3 parental wild-type cells) was analysed. Materials and methods: Heat treatment of cells was performed in a water bath at 44 C. The cell extract from scid cells or sxi-3 cells was added to heat-treated hybrid cell extracts, and the DNA-PK activity was assayed. Results: When hybrid cells were heated at 44 C for 15min, DNA-PK activity was reduced to undetectable levels. The decreased DNA-PK activity could be restored in a concentration-dependent manner with the addition of scid cell extract. The sxi-3 cell extract could not restore heat-inactivated DNA-PK activity. Conclusions: DNA-PK was inactivated by heat treatment at 44 C. Ku70/Ku80, but not Ku70 alone, could restore heat-inactivated DNA-PK.

Detection of isotype-specific autoantibodies to calpastatin in sera from patients with rheumatic diseases using heat-treated HeLa cell extract as an antigen source for immunoblotting

To detect immunoglobulin isotype-specific autoantibodies to native human calpastatin in patients with rheumatic diseases, we performed immunoblot analysis using the heated HeLa cell extracts to enrich heat-resistant calpastatin. The calpastatin molecule that was apparently migrated to 110 kD by SDS-PAGE was confirmed to react with monoclonal anti-human calpastatin antibody in immunoblotting. IgG antibodies to calpastatin were detected in 22 of 48 sera (46%) from patients with RA, whereas only 20% (5/25), 11% (2/19) and 13% (2/15) of sera from SLE, SSc and PM/DM had IgG anti-calpastatin antibodies, respectively. IgM antibodies were also found in 40% (19/48) of RA and 12% (3/25) of SLE patients but not detected in sera from patients with other rheumatic diseases. IgA antibodies were found in only one RA and one SLE serum. In RA, 7 of 48 sera (15%) had IgM antibodies alone, but all SLE sera with IgM antibodies had IgG antibodies. Thus, anti-calpastatin autoantibodies were detected by using the native human calpastatin. Although these autoantibodies were found in patients with various rheumatic diseases, they were present in RA patients at the highest frequency. In particular, the presence of IgM antibodies appeared to be more specific in RA patients.

Heat-treated osteoblastic cell (ROS17/2.8)-conditioned medium induces the formation of osteoclast-like cells

To study the effects of osteoblast products on osteoclast formation, we added the conditioned medium (CM) of rat osteoblastic cell line ROS 17/2.8 to rat bone marrow cultures, in which tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleate cells (MNCs) formed in the presence of 10 −8 M 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3). The formation of 1,25(OH) 2D 3-dependent TRAP-positive MNC at day 7 of culture was strongly inhibited by the >10 kDa fraction of ROS 17/2.8 cell-CM (ROSCM), but heat treated ROSCM (htROSCM) expressed marked stimulation in the formation of the MNCs. The expression of several osteoclastic phenotypes of the MNCs induced by htROSCM and 1,25(OH) 2D 3 was more enhanced compared with that of the MNCs induced by 1,25(OH) 2D 3 alone. The MNCs induced by htROSCM and 1,25(OH) 2D 3 were highly motile, were sensitive to calcitonin (CT), and had high bone resorbing activity. These data suggest that htROSCM promotes the osteoclast differentiation in the presence of 1,25(OH) 2D 3 in a rat bone marrow culture system. The stimulatory activity of TRAP-positive MNC formation in htROSCM is derived from heat-stable protein(s) that is (are) thought to be different from colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF) or granulocyte-macrophage-CSF (GM-CSF).