Myelin Basic Protein as a Novel Genetic Risk Factor in Rheumatoid Arthritis—A Genome-Wide Study Combined with Immunological Analyses

Rheumatoid arthritis (RA) is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2), followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4). SNPs showing p<0.005 in the first two collections and p<10−4 by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP) at chromosome 18q23 showed strong association with RA (p = 2.7×10−8, OR 1.23, 95% CI: 1.14–1.32). The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001). We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001) and patients with other connective tissue disorders (p<0.001). ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease

2019年度 八戸工業大学公開講座

Last updated: 6/15/201

Evaluation of New Cage Lid with Partitioning Barrier Based on Transmission of CAR Bacillus in Mice

We developed a new cage lid made of stainless steel wire mesh and having a screen barrier for partitioning a small laboratory animal cage into compartments. To evaluate the effectiveness of the lid, we tested the transmissibility of Cilia-Associated Respiratory (CAR) bacillus from infected mice to uninfected sentinel mice, which were kept in separate compartments using this lid. Infection from the infected mice to the uninfected mice was confirmed by microbiological, serological, pathological, and molecular diagnostic examinations, as previously observed in an intra-cage contact route. The cage lid that we developed is very useful when uninfected mice are used in quarantine and contagion experiments to prevent fighting among the mice

2010 AALAS National Meeting

Pasteurella pneumotropica, a Gram-negative opportunistic pathogen, is frequently isolated from the respiratory organs and digestive tracts of clinically normal conventional mice. Oral or subcutaneous treatment with enrofloxacin, an antibiotic, at a daily dose of 25.5 mg/kg for 2 weeks is effective in eliminating P. pneumotropica. However, the drawbacks of this treatment include substituted microbism and inhibited growth of indigenous fungi in the mouth. We therefore examined whether reducing the enrofloxacin treatment duration and dosage could eliminate P. pneumotropica from mice and eliminate these drawbacks.Healthy C3H/HeMsNrs (TLR4-competent) and C.B-17/Icr- Prkdcscid/JclNrs mice were intranasally inoculated with 1 X 107 P. pneumotropica. Twenty-two days after inoculation, the mice were subcutaneous injected with enrofloxacin once a day for 3 days at doses ranging from 3 to 300 mg/kg. Day 22 was chosen for treatment initiation because in a preliminary examination P. pneumotropica was detected in nasal samples by PCR assay 21 days after inoculation. Seven days after the completion of treatment, all mice were sacrificed and oral, nasal, and tracheal swabs were collected. Each swab sample was plated directly onto blood agar and incubated. To identify P. pneumotropica among the isolates, PCR assay was used to identify the 16S rRNA gene. The lungs and skin were examined histopathologically. Pasteurella pneumotropica was not detected, even in the minimum-dose group (3 mg/kg), in C3H/HeMsNrs mice. In the 100- and 300-mg/kg dose groups, induration or inflammation was observed in the skin at the injection site. In C.B-17/Icr- Prkdcscid/JclNrs mice, P. pneumotropica was detected at enrofloxacin doses of 3 and 10 mg/kg, and these mice developed pneumonia. Because of skin inflammation, higher doses were not tested. Subcutaneous administration of enrofloxacin at a dose of 3 mg/kg for 3 days can successfully eradicate latent infection of P. pneumotropica in immunocompetent mice. Further investigation is needed to clarify the lack of efficacy of the same treatment in immunodeficient mice.2010 AALAS National Meetin

Lid with barrier for partitioning cage for laboratory animals

Genetically modified laboratory mice are frequently transferred among research institutions. When these mice are quarantined, sentinel animals are used to indirectly monitor the health of the mice in order to avoid having to sacrifice the valuable genetically modified mice. Recent data on the health monitoring of laboratory mice indicate that opportunistic infections are more common than severe contagious infections. During quarantine, test animals and sentinel animals should be placed in the same cage because opportunistic infections often exhibit low levels of transmissibility. However, it is important to prevent fighting among the animals. Thus, we developed a new cage lid, made of a stainless steel wire screen, having a screen barrier for partitioning a cage into compartments. To evaluate the effectiveness of the cage lid with the partitioning barrier, the transmission of Cilia-Associated Respiratory (CAR) bacillus was tested. A total of 40 specific-pathogen-free(SPF) mice were used. Sixteen mice were inoculated intranasally with the SMR strain of the CAR bacillus under light ether anesthesia. Two inoculated mice were placed in one compartment and 3 uninfected sentinel mice were placed in the other compartment. Each cage was covered with the lid we developed. At weekly intervals, the five mice were sacrificed by exsanguination under anesthesia and were examined. Using a PCR assay, the 16S rRNA gene from the CAR bacillus was detected from swab samples of the larynx and the respiratory organs of both inoculated and sentinel mice at 4 weeks. Histopathologically, CAR bacilli were shown on the surface of the epithelium of the respiratory tracts of both inoculated and sentinel mice at 4 weeks. Typical bronchopneumonia and peribronchitis were observed in both inoculated and sentinel mice at 5 weeks. Serologically, IFA antibodies against the CAR bacillus were detected in both inoculated and sentinel mice at 6 weeks.The cage lid with the partitioning barrier we developed is very useful when sentinel animals are used in the quarantine of laboratory mice, such as genetically modified or immunodeficient mice, because sacrifice of valuable genetically modified mice should be avoided, and because immunodeficient mice cannot produce serum antibodies.59th AALAS National Meetin

Histopathological Studies on Cases of Chronic Mouse Hepatitis by Natural Helicobacter Infections

It has been known that Helicobacter hepaticus or Helicobacter bilis infection cause chronic inflammation of the colon and liver. Chronic active hepatitis was found in radiation exposure experiments using male C3H/HeNrs mice at our institute. Histopathologically, 103 cases among 978 mice (64-91 weeks of age at autopsy) had hepatic lesions regardless of irradiation exposure. Mild lesions showed only focal necrosis and focal inflammation in the liver. Severe cases were accompanied by hepatocytomegaly, bile duct hyperplasia, hypertrophy and activation of Kupffer cells, cholangitis, pleomorphic hepatocytes and/or tumor. Helical-shaped bacteria were detected between hepatocytes by Warthin-Starry silver stain and immunohistochemistry (IHC) with an antibody against Helicobacter pylori. It was suggested that these chronic hepatitis caused by Helicobacter spp. Although chronic hepatitis occurred frequently in mice exposed high-dose irradiation compared to non-irradiated mice in one lot, it was not concluded whether radiation might influence in incidence or degree of hepatitis or not. Our report suggested that natural Helicobacter spp. infection in mice can occur in an experimental animal facility. Therefore, it is suggested that monitor of the Helicobacter infection is very important for quality control of animal experiments

Differences in the Transmissibility of Cilia-Associated Respiratory Bacillus between Mouse Strains

We have developed a new type of cage lid which divide in two compartments of a cage. In the present study, we placed at infected BALB/c-nu/+ mice or A/J mice with the Cilia-Associated Respiratory(CAR) bacillus of one side compartment, in the other side, placed at non-infected mice. We researched whether they would be transmitted to non-infected healthy mice.Method:6-week-old female BALB/c-nu/+ mice and A/J mice were nasally inoculated with the SMR strain of CAR bacillus. Two infected mice and three healthy mice were kept in separate compartments of a cage using the new lid. PCR tests were performed using oral and nasal swabs, the trachea and the lungs of the mice at 7, 14, 21, 28, 35, 42, 49, and 56 days after inoculation. The histopathological features of the respiratory organs and the serum antibody to CAR bacillus were assessed by the indirect fluorescent antibody method.Results and Conclusions : Apparent changes, including histopathologic changes and increases in serum antibodies, were observed in the infected BALB/c-nu/+ mice and A/J mice, but no obvious changes indicating transmission of the bacillus were found in the healthy A/J mice. The reasons for this may be that (1) the bacillus had not yet reached the stage of excretion from the infected mice, (2) transmission of CAR bacillus failed because defense mechanisms of the healthy A/J mice were more active than that of BALB/c-nu/+ mice or (3) the lesions of respiratory organs did not appeared because CAR bacillus did not proliferated at respiratory organs.第２回アジア実験動物学会連合(AFLAS)学術大