Vector-averaged gravity regulates gene expression of receptor activator of NF-kappaB (RANK) ligand and osteoprotegerin in bone marrow stromal cells via cyclic AMP/protein kinase A pathway.

Bone loss due to unloading of the skeleton may be caused by an acceleration of osteoclastic bone resorption as well as a decline of osteoblastic bone formation. Recently, two molecular species that play important roles in osteoclastogenesis were discovered: (i) the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG) ligand/osteoclast differentiation factor induces osteoclastogenesis; and (ii) the OPG/osteoclastogenesis inhibitory factor potently inhibits osteoclastogenesis. To investigate the effects of gravity on gene expression of RANKL and OPG, a mouse bone marrow-derived stromal cell line, ST2, was cultured on a single axis clinostat, which generates a vector-averaged gravity environment. Northern blot analysis revealed that RANKL mRNA was increased, whereas that of OPG decreased. The clinostat culture also caused an increase in intracellular cyclic (cAMP) level. Both forskolin and dibutyryl-cAMP mimicked the regulation of RANKL and OPG transcription in clinostat culture. These modulations of gene expression in clinostat culture were blocked by a protein kinase A (PKA) inhibitor, H89, but not by a cyclooxygenase inhibitor, indomethacin. The enhancement of RANKL gene expression under clinostat culture and its inhibition by H89 were confirmed by a reporter assay with the murine RANKL 5'-flanking region. These results suggest that modulations of RANKL and OPG expression in stromal cells might be one of the causes of bone loss during skeletal unloading. An elevation of intracellular cAMP level caused through an as yet undetermined pathway is involved in modulation of RANKL and OPG expression during clinostat culture.

Effects of microgravity on c-fos gene expression in osteoblast-like MC3T3-E1 cells.

The paper summarizes the data on proliferation and gravity-related gene expression of osteoblasts that were obtained from an experiment conducted under simulated and real microgravity conditions. Simulated microgravity conditions obtained in a clinostat depress proliferation of both osteoblast-like MC3T3-E1 and HeLa carcinoma cells. This depression of proliferation occurs in a collagen gel culture in which the flow of culture medium by rotation may be reduced. Interestingly, MC3T3-E1 cells which are probably one of target cells to microgravity are more sensitive than the HeLa cells. Simulated microgravity inhibited the epidermal growth factor (EGF)-induced c-fos gene expression in the MC3T3-El cells. To examine in detail the effect of real microgravity on the EGF signal transduction cascade in osteoblasts, MC3T3-E1 cells were cultured in the Cell Culture Experiment Module of the sounding rocket TR-1A6. The EGF-induced c-fos expression in cells was depressed under short-term microgravity conditions in the sounding rocket, while the phosphorylation of mitogen-activated protein kinase (MAPK) was not affected compared with the controls grown on the ground. These results suggest that an action site of microgravity in the signal transduction pathway may be downstream of MAPK.

Water purification system by using the biofilter for long-term experiment equipment with aquatic animals for the space station.

We have developed a water purification system that enables long-term experiment with aquatic animals for 90 days or more on the space station. We designed the system that combined a biofilter for ammonia removal (nitrification) with another for nitrate removal (denitrification). The experiment with goldfish was for 90 days with an aquatic animals' examination device. The equipment consists of a fish tank, a filter module, pumps, and an artificial lung for gas exchange. The goldfish were kept in the tank without any water replacement throughout the experiment. When a filter module consists of adsorbents without bacteria, the concentration of the nitrite and ammonia begin to increase so that the goldfish die. On the contrary, neither ammonia nor nitrite accumulated throughout the experiment, and the concentration of T-N also maintained 30 ppm or less when the combined biofilter was used. Moreover, no fish died throughout the period. The water purification system with biofilter enabled us to examine the long-term life support testing. We also report a new denitrification (correction of dentrification) method for the life support system.

Separation of bacterial cells by free flow electrophoresis under microgravity: a result of the SpaceLab-Japan project on Space Shuttle flight STS-47.

We demonstrated free flow electrophoresis (FFE) of charged cells under microgravity, where gravitational effects are almost eliminated. Separation of a mixture of three bacterial strains (mutants of Salmonella typhimurium LT2) by FFE was conducted on NASA Space Shuttle flight STS-47 (September 1992). The experiment was designed to differentiate three strains having different lipopolysaccharide core structures in the cell membrane. The results were compared to those of ground experiments, in order to examine whether or not FFE in a weightless environment provides distinct advantages. Smooth strain SL1027 and rough strain SL3749 migrated to two separated fractions. The quality (viability) and the yields of the separated samples were sufficient to show the advantage of microgravity. Another rough strain, SL1102, exhibited unexpected electrophoretic behavior, which prevented the complete resolution of the three strains. All the strains were recovered as viable cells after 8 days of flight. The present study suggests that electrophoretic separation of bacterial cells is much more effective under microgravity conditions with relatively good resolution in comparison with the ground operation.

Electrophoretic free mobility and viability of microbial cells: a preliminary study in preparation for space experiments.

Electrophoretic free mobilities (EFM) of four fungal spores and five bacterial cells were determined in 7 mM triethanolamine/acetate (TEA) buffer by means of microscopic electrophoresis (ME) and free flow electrophoresis (FFE). Spores of Aspergillus terreus, Penicillium citrinum, Gliocladium virens, and Rhizopus oryzae had similar EFM from 2.2 to 3.1 microns sec-1/V cm-1. The resolution of the spore mixture by FFE was therefore determined to be poor. Bacterial cells of Salmonella typhimurium LT2 mutants showed a distinctive EFM from 0 to 4.2 microns sec-1/V cm-1, which is a large enough difference to produce a clear separation of each mutant type from the mixture in FFE. The differences in the EFM of bacteria result from defective structures in the lipopolysaccharide of their outer membranes. The viability of bacteria in TEA buffer at 4 degrees C was investigated, and it was found to be stable for 14 days. This period is long enough to allow the performance of space experiments.

Immunogenicity of a new type of yeast-derived hepatitis B vaccine consisting of M (pre-S2 + S) protein particles.

Genetically modified M (pre-S2+S; P31) protein (M-P31c) particles were formulated into a vaccine (TGP-943) through adsorption on to an alum adjuvant. The immunogenicity of this vaccine was investigated using guinea-pigs and various kinds of mice. In terms of anti-HBs(S) response, TGP-943 was found to be as immunogenic as the control plasma-derived vaccine (PDV) and yeast-derived S vaccine (YDSV) in Balb/c mice. TGP-943 induced anti-S antibodies even in S low-responder mice. In addition to the anti-S antibodies, TGP-943 was shown to elicit anti-pre-S2 antibodies dose-dependently, and the anti-pre-S2 antibodies were maintained at a high level after immunization with a high dose of TGP-943. The antibody response to pre-S2 had a tendency to appear earlier than that to S. M-P31c particles induced in vitro proliferation of TGP-943-primed lymph node cells, indicating that TGP-943 is more immunogenic than conventional vaccines in terms of T-cell levels. These results suggest that TGP-943 would be a promising candidate for a third generation hepatitis B vaccine.

Effect of indomethacin, aspirin, and acetaminophen on in vitro antiviral and antiproliferative activities of recombinant human interferon-alpha 2a.

The effects of indomethacin, aspirin, and acetaminophen on the antiviral and antiproliferative activities of recombinant human interferon-alpha 2a (rIFN-alpha 2a) were studied in vitro. None of the drugs inhibited the antiviral activity of rIFN-alpha 2a in human amnion FL cells against vesicular stomatitis virus, or interfered with its antiproliferative activity against acute lymphoblastic leukemia MOLT-4 cells or renal cell carcinoma NC 65 cells. Although, at high concentrations, aspirin (1 mM) or indomethacin (0.1 mM) alone inhibited the cell growth, rIFN-alpha 2a showed clear additive growth inhibition. It was concluded that neither indomethacin, aspirin, nor acetaminophen directly inhibited the antiviral and antiproliferative activities of rIFN-alpha 2a. The possible use of these three drugs to reduce the adverse effects of rIFN-alpha 2a without spoiling its profitable efficacy in clinical practice is suggested.

Suppression of HBsAg production in PLC/PRF/5 human hepatoma cell line by interferons.

Recombinant human interferon alpha 2a as well as natural human interferons alpha and beta significantly suppressed the production of hepatitis B surface antigen by PLC/PRF/5 cells (which have been established from a human primary hepatocellular carcinoma and proven to carry the hepatitis B virus DNA) and inhibited proliferation of these cells in vitro. However, the production of alpha-fetoprotein by PLC/PRF/5 cells was less significantly affected by any of the interferons. These results suggest that these interferons not only suppress cellular proliferation but also selectively inhibit the action of the HBV gene which is persistently present in these cells.

The activity of synthetic analogs of serum thymic factor (FTS) to convert mouse pre-T cells into Thy-1 positive cells.

Serum thymic factor (FTS) and its 33 analogs were compared with regard to the ability to induce Thy-1 antigen on mouse pre-T cells. The pre-T cells were prepared from spleen cells of athymic nu/nu mice by passing them through a nylon wool column, and the induction of Thy-1 antigen was analysed using a fluorescence-activated cell sorter. Thy-1 negative cells were converted into Thy-1 positive cells by FTS and some of its analogs. Deletion of pGlu at the amino terminus of FTS did not alter the activity. Des ( pGlu1 , Ala2)-FTS and des ( pGlu1 , Ala2, Lys3 )-FTS were inactive and deletion of the carboxyl terminal residue, Asn, also resulted in the loss of activity. Substitution or modification of pGlu1 and Asn9 with an amino acid, an acyl group or an amine caused a marked reduction of the activity. The residue 3, Lys, could be substituted without any loss of the activity by Arg, but not by Leu. These results show that the octapeptide moiety, Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn, is the minimum essential part of the FTS molecule for the expression of activity and they suggest that the positive charge at the residue 3 is also important for its activity.

Radioimmunoassay of serum thymic factor (FTS).

We established a radioimmunoassay (RIA) method which enables a quantitative estimation of serum thymic factor (FTS). This assay is based on the displacement of 125I-(Lys[Tyr] 3)-FTS bound to the anti-FTS antibodies by FTS. Formaldehyde-fixed Staphylococcus aureus Cowan I cells were used to precipitate the antigen-antibody complex. The anti-FTS antiserum was raised in rabbit by repeated injections of FTS-conjugated bovine serum albumin. The antiserum seemed to recognize the carboxy-terminal seven amino acid residues, Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH, of the FTS molecule. As little as 0.5 to 1 pg FTS can be detected by this method. To measure the FTS content in the serum, a serum sample was pretreated with 10% trichloroacetic acid and then with ethanol for removal of serum proteins. The FTS content in pig serum was around 22 pg/ml, and that in BALB/c mouse serum was 1.3 pg/ml. The FTS contents in the sera of DBA/2, C57BL/6 and (C57BL/6 X DBA/2)F1 mice were also less than 5 pg/ml. Since more than 70% of FTS exogenously added to pig or mouse serum was recovered, the mouse serum probably contains only a small amount of FTS.

Identification of an I-J+ antigen-presenting cell required for third order suppressor cell activation.

We have found that an I-J+ I-A- antigen-presenting cell (APC) is required for Ts3 activation in vivo. Together with the I-J restriction previously reported for Ts3 induction (Takaoki, M., M.-S. Sy, A. Tominaga, A. Lowy, M. Tsurifiji, B. Benacerraf, R. Finberg, and M. I. Greene, 1982, J. Exp. Med., 156:1325), it appears that this I-J+ APC is responsible for I-J restriction in the triggering of Ts3. This restriction may be exerted via a pre-Ts3 associative recognition of antigen and I-J encoded determinants, analogous to the T helper recognition of antigen in the context of I-A and I-E determinants.

The role of the epitope density and cross-reactivity between two different purine nucleosides coupled to cells.

Cellular immune responses against nucleic acid antigens were analyzed in BALB/C mice. Delayed-type hypersensitivity (DTH) could be elicited by immunizing and challenging with either guanosine-coupled spleen cells (G-SC) or adenosine-coupled spleen cells (A-SC), and measured by footpad swellings. The epitope density was critical for immunization. This cellular reaction was specific to nucleosides, and cross-immunity was observed between A-SC and G-SC. In addition, cross-unresponsiveness was observed between these two nucleosides. In contrast, soluble carrier proteins coupled with either guanosine or adenosine did not induce cross-reactive immunity or unresponsiveness. The significance of the difference between these two forms of antigens in the ability to induce cross-reactivity is discussed in the context of T versus B-cell recognition in the induction or the expression of the immune response.

I-J-restricted interactions in the generation of azobenzenearsonate-specific suppressor T cells.

The genetic restrictions of the activation of third-order suppressor cells (Ts3) were studied in mice, using two different types of anti-azobenzenearsonate (ABA)-immune responses, namely delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) generation. Ts2 cells were induced in several different strains of mice by injecting monoclonal T hybridoma molecules or first-order suppressor factors (TsF1) originating in A/J (H-2a, Igh-1e) mice and then testing the TsF2 molecules derived from these Ts2 in A/J and A.By (H-2b, Igh-1e) or (A/J X A.By)F1 (H-2a/b, Igh-1e) and (C57Bl/6 X A/J)F1 (H-2b/a, Igh-1e) mice. It was shown that the activity of TsF2 was restricted to the I-J of the strain in which Ts2 was induced. By genetic analysis, restriction was shown to be due to the requirement of H-2 identity between ABA-coupled cells used for Ts3 activation and the strain of the TsF2 origin. Moreover, by using H-2-congenic ABA-coupled cells, we were also able to precisely map and demonstrate that ABA-coupled cells I-J identical to TsF2 induced in various strains were necessary for effective suppression to occur. This selective activation of Ts3 suggested the existence of I-J-related antigen presentation for suppression as the counterpart of I-A or I-A-I-E-restricted antigen presentation for positive immune responses.