Kazunobu Ouchi

ITPKC functional polymorphism associated with Kawasaki disease susceptibility and formation of coronary artery aneurysms

Kawasaki disease is a pediatric systemic vasculitis of unknown etiology for which a genetic influence is suspected. We identified a functional SNP (itpkc_3) in the inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) gene on chromosome 19q13.2 that is significantly associated with Kawasaki disease susceptibility and also with an increased risk of coronary artery lesions in both Japanese and US children. Transfection experiments showed that the C allele of itpkc_3 reduces splicing efficiency of the ITPKC mRNA. ITPKC acts as a negative regulator of T-cell activation through the Ca2+/NFAT signaling pathway, and the C allele may contribute to immune hyper-reactivity in Kawasaki disease. This finding provides new insights into the mechanisms of immune activation in Kawasaki disease and emphasizes the importance of activated T cells in the pathogenesis of this vasculitis.

Characterization and Molecular Analysis of Macrolide-Resistant Mycoplasma pneumoniae Clinical Isolates Obtained in Japan

ABSTRACT In recent years, Mycoplasma pneumoniae strains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of M. pneumoniae isolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, ≥256 μg/ml) and 1 was weakly resistant (MIC, 8 μg/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in Escherichia coli numbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in E. coli numbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical M. pneumoniae strains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of M. pneumoniae, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant M. pneumoniae infection is not unusual in Japan.

A genome-wide association study identifies three new risk loci for Kawasaki disease

We performed a genome-wide association study (GWAS) of Kawasaki disease in Japanese subjects using data from 428 individuals with Kawasaki disease (cases) and 3,379 controls genotyped at 473,803 SNPs. We validated the association results in two independent replication panels totaling 754 cases and 947 controls. We observed significant associations in the FAM167A-BLK region at 8p22-23 (rs2254546, P = 8.2 × 10−21), in the human leukocyte antigen (HLA) region at 6p21.3 (rs2857151, P = 4.6 × 10−11) and in the CD40 region at 20q13 (rs4813003, P = 4.8 × 10−8). We also replicated the association of a functional SNP of FCGR2A (rs1801274, P = 1.6 × 10−6) identified in a recently reported GWAS of Kawasaki disease. Our findings provide new insights into the pathogenesis and pathophysiology of Kawasaki disease.

Intravenous immunoglobulin therapy in acute disseminated encephalomyelitis

Three children ranging in age from 2 to 5 years with acute disseminated encephalomyelitis (ADEM) were successfully treated with high-dose intravenous immunoglobulin (IVIG). Their symptoms were somnolence, fever, headache, vomiting, and resting tremor. In all of these patients, it was difficult to distinguish the condition from viral encephalitis before analyzing the myelin basic protein. ADEM was diagnosed because of increased levels of myelin basic protein in their cerebrospinal fluid and abnormal high-signal intensity on T2-weighted magnetic resonance imaging. All patients were given IVIG at a dose of 400 mg/kg/day for 5 consecutive days. The patients rapidly regained consciousness in 14 hours, 2 days, and 4 days and demonstrated a complete clinical improvement within 18 days, 10 days, and 7 days of the initiation of the treatment, respectively. IVIG may prove useful as an alternative treatment to corticosteroids for ADEM.

Absence of Chlamydia psittaci in ocular adnexal lymphoma from Japanese patients

Low-grade malignant lymphomas arising from mucosa-associated lymphoid tissue (MALT) represent a distinct clinicopathological entity of B-cell non-Hodgkin lymphoma (Isaacson & Wright, 1984). MALT lymphoma tends to remain localised and the extranodal sites of involvement include the gastrointestinal tract, salivary gland, thyroid and ocular region. MALT lymphoma often occurs in relation to autoimmune disorders or chronic antigen stimulation, but the pathogenetic mechanisms of this type of lymphoma have not been well defined. Recently, Ferreri et al (2004) reported that 32 (80%) of 40 Italian patients with ocular adnexal lymphoma harboured Chlamydia psittaci DNA. The findings suggested that C. psittaci infection might contribute to the development of these lymphomas. Soon after this, two other groups independently investigated ocular adnexal lymphoma for the presence of C. psittaci DNA in 57 patients from south Florida (Rosado et al, 2005) and 11 patients from the north-eastern USA (Vargas et al, 2005) and found that none of them carried C. psittaci DNA. One explanation for this discrepancy concerns geographic differences in the incidence of C. psittaci infection. However, all of these reports are based on findings from patients in the USA and Europe, and therefore additional surveys evaluating the association between C. psittaci and ocular adnexal lymphomas including MALT lymphoma in other geographical regions of the world would be warranted. There has been no report from Asia thus far. In this study, we looked for C. psittaci DNA in tumour specimens from Japanese patients with primary ocular adnexal lymphoma. Tissues obtained by an incisional biopsy from 24 Japanese patients (12 males and 12 females, mean age 56 years; range 34–73 years) with localised lymphoproliferative conditions of the ocular adnexa, including 18 MALT lymphomas, three nonMALT lymphomas and three reactive ocular lymphoid hyperplasias, were studied. The localities of the tumours were the orbit in 14 cases, lacrimal glands in four cases, and conjunctiva in six cases. DNA was obtained from frozen or paraffinembedded tissues using the phenol/chroroform extraction technique or DNA extraction system according to the manufacturer’s instructions (Takara, Tokyo, Japan). All samples were successfully amplified by polymerase chain reaction (PCR) for human b-globin sequences, yielding a 123-bp amplicon, which indicated that amplifiable DNAs were present. Three independent PCRs were used to detect C. psittaci DNA in this study. First, we used the same method employed in the previous studies (Ferreri et al, 2004; Rosado et al, 2005; Vargas et al, 2005). The touchdown enzyme time release PCR with CPS100 and CPS101 primers produces a 111-bp amplicon (Madico et al, 2000). The primers amplify a highly conserved region encoding the 16S ribosomal RNA gene. The second method was a nested PCR to detect the same DNA region with the first-step primers (5¢-ACGGAATAATGACTTCGG-3¢ and 5¢-TACCTGGTACGCTCAATT-3¢) and the second-step primers (5¢-ATAATGACTTCGGTTGTTATT-3¢ and 5¢-TGTTTTA GATGCCTAAACAT-3¢), generating a 127-bp amplicon. The PCR primers for the third C. psittaci PCR were derived from another DNA region encoding the ompA gene, and the primer sequences were 5¢-GCCTTAAACATCTGGGATCG-3¢ and 5¢-GCACAACCACATTCCCATAAAG-3¢, giving a 248-bp fragment. All three PCRs failed to detect C. psittaci DNA in any of our samples, while positive control reactions using DNA prepared from C. psittaci strain Cal-10 yielded amplicons of expected size. In addition, our samples were negative for Chlamydia pneumoniae and Chlamydia trachomatis DNA sequences. Our results showed no correlation between C. psittaci infection and ocular adnexal lymphoma in Japan, supporting the observation from the USA (Rosado et al, 2005; Vargas et al, 2005). All these studies were based on testing C. psittaci DNA sequences. Given the serological evidence of an association between chronic chlamydia infections (C. pneumoniae and C. trachomatis) and lymphomas (Anttila et al, 1998), serological surveys for C. psittaci may also help to better understand the relationship between C. psittaci and ocular adnexal lymphoma. Although the ‘hit-and-run’ mechanism cannot be fully excluded, the present findings indicate that C. psittaci is unlikely to have played a pathogenetic role in ocular adnexal lymphoma in the Japanese population.

Nationwide Surveillance of Macrolide-Resistant Mycoplasma pneumoniae Infection in Pediatric Patients

ABSTRACT We conducted nationwide surveillance to investigate regional differences in macrolide-resistant (MR) Mycoplasma pneumoniae strains in Japan. The prevalence of MR M. pneumoniae in pediatric patients gradually increased between 2008 and 2012. Although regional differences were observed, high levels of MR genes were detected in all seven surveillance areas throughout Japan and ranged in prevalence from 50% to 93%. These regional differences were closely related to the previous administration of macrolides.

CD40 ligand gene and Kawasaki disease.

Kawasaki disease (KD) is an acute systemic vasculitis syndrome of infants and young children. Although its etiology is largely unknown, epidemiological findings suggest that genetic factors play a role in the pathogenesis of KD. To identify genetic factors, affected sib-pair analysis has been performed. One of the identified peaks was located on the Xq26 region. A recent report of elevated expression of CD40 ligand (CD40L), which maps to Xq26, during the acute-phase KD, and its relationship to the development of coronary artery lesions (CAL) prompted us to screen for polymorphism of CD40L and to study the association of the gene to KD. A newly identified SNP in intron 4 (IVS4+121 A>G) is marginally over-represented in KD patients as compared to controls (109/602, 18.1 vs 111/737, 15.1%). When male KD patients with CAL were analyzed as a patient group, the SNP was significantly more frequent than in controls (15/58, 25.9%, vs 111/737, 15.1%, OR=2.0, 95% CI=1.07–3.66; P=0.030). Interestingly, this variation was extremely rare in a control Caucasian population (1/145, 0.7%). Our results suggest a role of CD40L in the pathogenesis of CAL and might explain the excess of males affected with KD.

Chlamydia pneumoniae in coronary and iliac arteries of Japanese patients with atherosclerotic cardiovascular diseases.

Recent studies suggest the association of atherosclerotic cardiovascular diseases with Chlamydia pneumoniae infection in western populations. It is of great interest whether such an association exists in Asians with their distinct genetic background. Symptomatic patients with coronary heart disease (29) or arteriosclerosis obliterans (10) who underwent directional endo-atherectomy were studied. Atherectomy specimens of coronary and iliac arteries were examined for C. pneumoniae by culture, nested PCR and immunohistochemical stain (IHC) with one Chlamydia genus-specific, two C. pneumoniae species-specific, and two C. trachomatis species-specific monoclonal antibodies. Among the 29 patients with coronary artery disease, C. pneumoniae was detected in the coronary arteries of 13 by IHC, 16 by PCR and 20 by IHC or PCR, or both. C. pneumoniae was also found in the iliac arteries of four patients by IHC, three by PCR and five by IHC or PCR, or both, of the 10 patients with arteriosclerosis obliterans. Attempts to isolate C. pneumoniae by culture were unsuccessful. The re-stenotic rate after atherectomy was higher in the C. pneumoniae-positive group than in the negative group, but not significantly so. These findings support the high incidence of C. pneumoniae in atherosclerotic lesions of symptomatic patients with coronary heart disease and arteriosclerosis obliterans in Asians.

Therapeutic Efficacy of Macrolides, Minocycline, and Tosufloxacin against Macrolide-Resistant Mycoplasma pneumoniae Pneumonia in Pediatric Patients

ABSTRACT The importance of macrolide-resistant (MR) Mycoplasma pneumoniae has become much more apparent in the past decade. We investigated differences in the therapeutic efficacies of macrolides, minocycline, and tosufloxacin against MR M. pneumoniae. A total of 188 children with M. pneumoniae pneumonia confirmed by culture and PCR were analyzed. Of these, 150 patients had a strain with an MR gene and 134 had one with an A-to-G mutation at position 2063 of M. pneumoniae 23S rRNA domain V. Azithromycin (n = 27), clarithromycin (n = 23), tosufloxacin (n = 62), or minocycline (n = 38) was used for definitive treatment of patients with MR M. pneumoniae. Defervescence within 48 h after the initiation of antibiotic therapy was observed in 41% of the patients in the azithromycin group, 48% of those in the clarithromycin group, 69% of those in the tosufloxacin group, and 87% of those in the minocycline group. The average number of days of fever after the administration of antibiotic treatment was lower in the minocycline and tosufloxacin groups than in the macrolide groups. The decrease in the M. pneumoniae burden, as estimated by the number of DNA copies, after 48 to 96 h of treatment was more rapid in patients receiving minocycline (P = 0.016) than in those receiving tosufloxacin (P = 0.049), azithromycin (P = 0.273), or clarithromycin (P = 0.107). We found that the clinical and bacteriological efficacies of macrolides against MR M. pneumoniae pneumonia was low. Our results indicated that minocycline rather than tosufloxacin can be considered the first-choice drug for the treatment of M. pneumoniae pneumonia in children aged ≥8 years.

Clinical efficacy of macrolide antibiotics against genetically determined macrolide-resistant Mycoplasma pneumoniae pneumonia in paediatric patients.

Background and objective:  Since 2000, the prevalence of macrolide‐resistant (MR) Mycoplasma pneumoniae has increased among paediatric patients in Japan. To determine the efficacy of macrolides against MR M. pneumoniae pneumonia, microbiological and clinical efficacies were compared during the antibiotic treatment.