Immunity to type IX collagen in rodents: a study of type IX collagen for autoimmune and arthritogenic activities

Type IX collagen (CIX), a cartilage-specific glycoprotein, constitutes ≤ 10% of cartilage collagen. To ascertain whether CIX can induce arthritis as shown for type II and XI collagen (CII and CXI), outbred rats were sensitized with bovine, chick and human CIX; inbred rats, mice, and guinea pigs were sensitized with bovine CIX. Mice and guinea pigs proved resistant to arthritis, as did rats sensitized with CIX/Freund's incomplete adjuvant (FIA). Arthritis was seen in rats when 100 μg of Mycobacterium tuberculosis (Mtb) were added to FIA, but seldom with smaller doses of Mtb, suggesting the arthritis was adjuvant-induced. High levels of antibodies to rat CIX, containing complement-fixing subclasses, were detected in rat sera in addition to DTH and lymphocyte proliferation responses to rat CIX. Given the potential for CIX-induced disease, CIX-sensitized rats were injected intraperitoneally with lipopolysaccharide (LPS) to stimulate proinflammatory cytokine release, and intra-articularly with rat CIX to stimulate arthritis. LPS stimulation was ineffective; however, intra-articularly injected CIX produced transient synovitis. When rats with stable adjuvant arthritis were sensitized with CIX/FIA, significant increases in paw volume were measured compared with controls given CI/FIA. Immunohistochemical studies of actively and passively sensitized rats revealed deposits of CIX antibody, but not C3, at the joint margins where proteoglycan staining was weak. Together, these findings suggest that autoimmunity to CIX, in contrast to CII and CXI, is not directly pathogenic but may contribute to joint injury provided arthritis is initiated by an independent disease process.     

Autoreactivity against matrilin-1 in a patient with relapsing polychondritis

Relapsing polychondritis (RP) is a rare inflammatory disease of cartilage. Chondritis of the auricular, nasal, and tracheal cartilages predominates in this disease, suggesting a response to a tissue-specific antigen. One potential antigen is matrilin-1, a cartilage matrix protein found uniquely in the tracheal, auricular, and nasal cartilage of adults. We describe herein a patient with RP who had both a humoral and a cellular immune response directed toward the cartilage matrix protein matrilin-1.     

Studies on iron absorption

Various iron chelates and polymers with molecular weights below 100,000 were prepared in order to study the mechanism of iron absorption by the intestine in vitro and in vivo.It was observed by in vitro experiments using mouse intestine that iron chelates and polymer with molecular weights below 10,000 were similarly transported from mucosal to serosal medium, but iron polymer with a molecular weight of 100,000 was not transported at all. The amount of iron transported from mucosa to serosa proportionally depended on the iron concentration in the mucosal medium, and the iron concentration in serosal and mucosal medium was equilibrated with a concentration gradient which was parallel to the reciprocal of the molecular weight of iron complexes.Furthermore, it was confirmed by in vivo study using ligated rat intestine that iron is transferred from mucosa into the blood stream not only in ionic or chelate forms but also as low molecular weight iron polymers which are unaffected in intestinal cells.     

Characterization and radio-resistant function of manganese superoxide dismutase of Rubrobacter radiotolerans

Rubrobacter radiotolerans is the most radio-resistant eubacterium without spore-formation in the life cycle, and its D(37) is 16,000 Gy against gamma-rays. To understand the molecular mechanism of the high radio-resistance, we purified and characterized superoxide dismutase (SOD) of this organism as enzymatic radical scavenger, and then analyzed its genetic information. The purified SOD protein formed homo-tetramerization of 24,000 Da-monomer, while maintaining its enzymatic activity against potassium cyanide and hydrogen peroxide. We obtained a partial amino acid sequence of the protein and cloned the gene from it. Sequence analysis of the cloned gene indicated that the protein showed a similarity to other bacterial manganese SODs (Mn-SODs). Sequencing for adjacent regions of the gene showed that the gene had promoter elements with an open reading frame for putative PAS/PAC sensor protein at the 5'-adjacent region. Introduction of the gene into Escherichia coli cells lacking intrinsic SOD genes restored the cellular enzymatic activity and resistance to methyl viologen, indicating the gene at work. A mutant cell harboring this gene also became resistant against gamma-rays. The present results suggest that the protein in question is the Mn-SOD of R. radiotolerans, a good candidate as a radio-protection factor for this bacterial radio-resistance.     

Radon inhalation decreases DNA damage induced by oxidative stress in mouse organs via the activation of antioxidative functions

Radon inhalation decreases the level of lipid peroxide (LPO); this is attributed to the activation of antioxidative functions. This activation contributes to the beneficial effects of radon therapy, but there are no studies on the risks of radon therapy, such as DNA damage. We evaluated the effect of radon inhalation on DNA damage caused by oxidative stress and explored the underlying mechanisms. Mice were exposed to radon inhalation at concentrations of 2 or 20 kBq/m³ (for one, three, or 10 days). The 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels decreased in the brains of mice that inhaled 20 kBq/m³ radon for three days and in the kidneys of mice that inhaled 2 or 20 kBq/m³ radon for one, three or 10 days. The 8-OHdG levels in the small intestine decreased by approximately 20–40% (2 kBq/m³ for three days or 20 kBq/m³ for one, three or 10 days), but there were no significant differences in the 8-OHdG levels between mice that inhaled a sham treatment and those that inhaled radon. There was no significant change in the levels of 8-oxoguanine DNA glycosylase, which plays an important role in DNA repair. However, the level of Mn-superoxide dismutase (SOD) increased by 15–60% and 15–45% in the small intestine and kidney, respectively, following radon inhalation. These results suggest that Mn-SOD probably plays an important role in the inhibition of oxidative DNA damage.     

Quantitative analysis of isolated and clustered DNA damage induced by gamma-rays, carbon ion beams, and iron ion beams

Ionizing radiation induces multiple damaged sites (clustered damage) together with isolated lesions in DNA. Clustered damage consists of closely spaced lesions within a few helical turns of DNA and is considered to be crucial for understanding the biological consequences of ionizing radiation. In the present study, two types of DNA, supercoiled plasmid DNA and linear lambda DNA, were irradiated with gamma-rays, carbon ion beams, and iron ion beams, and the spectra and yield of isolated DNA damage and bistranded clustered DNA damage were fully analyzed. Despite using different methods for damage analysis, the experiments with plasmid and lambda DNA gave largely consistent results. The spectra of both isolated and clustered damage were essentially independent of the quality of the ionizing radiation used for irradiation. The yields of clustered damage as well as of isolated damage decreased with the different radiation beams in the order gamma> C > Fe, thus exhibiting an inverse correlation with LET [gamma (0.2 keV/microm) < C (13 keV/microm) < Fe (200 keV/microm)]. Consistent with in vitro data, the yield of chromosomal DNA DSBs decreased with increasing LET in Chinese hamster cells irradiated with carbon ion beams with different LETs, suggesting that the decrease in the yield of clustered damage with increasing LET is not peculiar to in vitro irradiation of DNA, but is common for both in vitro and in vivo irradiation. These results suggest that the adverse biological effect of the ionizing radiation is not simply accounted for by the yield of clustered DNA damage, and that the complexity of the clustered damage needs to be considered to understand the biological consequences of ionizing radiation.     

Pharmacological properties of N-methoxy-3-(3,5-ditert-butyl-4-hydroxybenzylidene)-2-pyrrolidone (E-5110), a novel nonsteroidal antiinflammatory agent

The antiinflammatory activity of a novel pyrrolidone derivative E-5110 was investigated using anti-inflammatory, analgesic and antipyretic animal models in comparison to indomethacin (IND) and piroxicam (PIR). The acute antiinflammatory activity of E-5110 on carrageenin paw edema was similar to IND, and half of PIR. E-5110 inhibited the pleural exudate volume and leucocyte infiltration in a reversed passive Arthus reaction more potent than IND. The chronic inflammatory responses in the established adjuvant- and type II collagen-induced arthritis were suppressed by E-5110 similar to IND and PIR. The analgesic potency of E-5110 was similar to IND and PIR, but the antipyretic activity of E-5110 was more potent than that of IND, and slightly more potent than that of PIR. The ulcerogenic effect of E-5110 on rat gastric mucosa was less than that of the reference drugs.     

Sex-linked differences in susceptibility of cynomolgus monkeys to type II collagen-induced arthritis. Evidence that epitope-specific immune suppression is involved in the regulation of type II collagen autoantibody formation

To determine whether sex-linked factors are important in the susceptibility and resistance of cynomolgus monkeys to type II collagen-induced arthritis, we immunized 5 males and 10 females with chick type II collagen (CII) and studied their clinical and immune responses. All 10 females developed overt arthritis and produced antibodies to CII and cross-reactive antibodies to monkey type II collagen (MkII). In contrast, only 1 male monkey developed arthritis, which was transient and mild in severity. Examination of antisera obtained from the male monkeys disclosed high titers of antibody to CII, but little antibody to MkII. The cyanogen bromide peptide recognition patterns varied markedly from animal to animal, implying that monkeys are capable of recognizing multiple arthritogenic determinants on CII.     

Purification and characterization of a novel DNA repair enzyme from the extremely radioresistant bacterium Rubrobacter radiotolerans

Rubrobacter radiotolerans is an extremely radioresistant bacterium. It exhibits higher resistance than the well-known radioresistant bacterium Deinococcus radiodurans, but the molecular mechanisms responsible for the radio-resistance of R. radiotolerans remain unknown. In the present study, we have demonstrated the presence of a novel DNA repair enzyme in R. radiotolerans cells that recognizes radiation-induced DNA damages such as thymine glycol, urea residues, and abasic sites. The enzyme was purified from the crude cell extract by a series of chromatography to an apparent physical homogeneity. The purified enzyme showed a single band with a molecular mass of approximately 40 kDa in SDS-polyacrylamide gel electrophoresis, and was designated as R-endonuclease. R-Endonuclease exhibited repair activity for thymine glycol, urea residues, and abasic sites present in plasmid DNA, but did not act on intact DNA, UV-irradiated DNA and DNA containing reduced abasic sites. The substrate specificity together with the salt and pH optima suggests that R-endonuclease is a functional homolog of endonuclease III of Escherichia coli.