Unique contribution of IRF-5-Ikaros axis to the B-cell IgG2a response.

IRF-5 is a transcription factor activated by toll like receptor (TLR)7 and TLR9 during innate immune responses. IRF-5 activates not only Type I IFN, but also inflammatory cytokines. Most importantly, a genetic variation in the IRF-5 gene shows a strong association with autoimmune diseases such as Lupus. Here, we report that IRF5-deficient mice have attenuated IgG2a/c responses to T-cell-dependent and -independent antigens and to polyoma virus infection. This defect is due to the intrinsic deletion of IRF-5 in B cells, as SCID mice reconstituted with Irf5-/- B cells show a decrease in IgG2a/c expression after viral infection compared with mice that received wild-type B cells. Irf5-/-B cells in vitro have diminished TLR and cytokine-induced class switching to IgG2a/c. Addressing the molecular mechanism, we show that IRF-5 regulates IgG2a/c expression by decreasing Ikaros expression; reconstitution of IRF-5 in Irf5-/- B cells downregulates Ikaros levels and increases switching to IgG2a/c. The IRF site in ikzf1 promoter binds IRF-5, IRF-4 and IRF-8. We show that IRF-8 but not IRF-4 activates the ikzf1 promoter, and IRF-5 inhibits the transcriptional activity of IRF-8. Collectively, these results identify the IRF-5-Ikaros axis as a critical modulator of IgG2a/c class switching.

Time-dependent gene expression and immunohistochemical analysis of the injured anterior cruciate ligament.

OBJECTIVES: This study aimed to investigate time-dependent gene expression of injured human anterior cruciate ligament (ACL), and to evaluate the histological changes of the ACL remnant in terms of cellular characterisation. METHODS: Injured human ACL tissues were harvested from 105 patients undergoing primary ACL reconstruction and divided into four phases based on the period from injury to surgery. Phase I was < three weeks, phase II was three to eight weeks, phase III was eight to 20 weeks, and phase IV was ≥ 21 weeks. Gene expressions of these tissues were analysed in each phase by quantitative real-time polymerase chain reaction using selected markers (collagen types 1 and 3, biglycan, decorin, α-smooth muscle actin, IL-6, TGF-ß1, MMP-1, MMP-2 and TIMP-1). Immunohistochemical staining was also performed using primary antibodies against CD68, CD55, Stat3 and phosphorylated-Stat3 (P-Stat3). RESULTS: Expression of IL-6 was mainly seen in phases I, II and III, collagen type 1 in phase II, MMP-1, 2 in phase III, and decorin, TGF-ß1 and α-smooth muscle actin in phase IV. Histologically, degradation and scar formation were seen in the ACL remnant after phase III. The numbers of CD55 and P-Stat3 positive cells were elevated from phase II to phase III. CONCLUSIONS: Elevated cell numbers including P-Stat3 positive cells were not related to collagens but to MMPs' expressions.

Knee osteoarthritis, knee joint pain and aging in relation to increasing serum hyaluronan level in the Japanese population.

OBJECTIVE: To investigate relationship between serum hyaluronan (HA) level and the presence and severity of radiographic knee osteoarthritis (OA) as well as degree of knee pain in Japanese population. DESIGN: A total of 616 volunteers participated in this study. Based on the Kellgren-Lawrence (K-L) grade, participants were radiographically classified into three groups: Normal (K-L grade 0 or 1), Moderate (grade 2) and Severe (grade 3 or 4). The degree of knee pain was quantified by visual analogue scale (VAS) and Knee injury and Osteoarthritis Outcome Score (KOOS) Pain. Serum HA levels were compared among the Normal, Moderate and Severe groups, and the relationship between serum HA level and the severity of knee OA was analyzed after age, sex and body mass index (BMI) were adjusted. In addition, the correlation between serum HA level and the degree of knee pain was analyzed in each group. RESULTS: Regarding relationship between serum HA level and the severity of radiographic knee OA, serum HA levels of the Moderate and Severe groups were significantly higher than in the Normal group (P<0.001). Furthermore, serum HA level correlated with the severity of radiographic knee OA (r=0.289, P<0.001) after adjusting for age, sex and BMI. Serum HA level correlated with VAS of knee pain and/or KOOS Pain in the Normal and Moderate groups. CONCLUSION: Serum HA level has the potential to be useful for the diagnosis of the presence and severity of knee OA.

Acute coronary artery dilation due to Kawasaki disease and subsequent late calcification as detected by electron beam computed tomography.

We wanted to clarify the relationships between the degree of acute coronary artery dilation caused by Kawasaki disease and subsequent late calcification. Electron beam computed tomography (EBCT) was used to study 79 patients who had previously undergone selective coronary angiograms less than 100 days after the onset of Kawasaki disease. The EBCT was performed using an Imatron C-150 with a 100-ms exposure time and consecutive images at 6-mm intervals. The interval from the onset of Kawasaki disease to EBCT ranged from 2 to 242 months (median, 103 months). The maximum diameters of the right coronary, the left anterior descending, and the left circumflex arteries, as well as the bifurcation of the left coronary artery were measured in the initial coronary angiograms. A total of 250 branches, including 53 left coronary arteries, were measured, and the relationship between the degree of the initial coronary artery dilation and subsequent calcification in the branches and left coronary artery was analyzed. The coronary arterial diameter of all branches that eventually calcified was 6 mm or greater. The incidence of calcification in branches measuring 6 mm or greater on the initial coronary angiogram was 12% at 5 years, 44% at 10 years, and 94% at 20 years (n = 141). Dilation greater than 6 mm is associated with a high probability of late calcification.

Two young adults who had acute coronary syndrome after regression of coronary aneurysms caused by Kawasaki disease in infancy.

We report two young adult patients who had acute coronary syndrome after regression of coronary aneurysms caused by Kawasaki disease (KD). A 26 year-old man had acute anterior myocardial infarction at midnight after drinking alcohol. He had had bilateral coronary aneurysms caused by KD at the age of 8 months. Selective coronary angiograms (CAGs) at the age of 7 years revealed regression of both coronary aneurysms. He had no symptoms until the onset of acute myocardial infarction. The other patient was a 24 year-old man diagnosed as having a subendocardial infarction. He had had bilateral coronary aneurysms caused by KD at the age of 1 year. CAGs at the age of 9 years showed that both had regressed. It should be recognized that young adults with apparently normal coronary arteries angiographically after regression of large coronary aneurysms caused by KD may occasionally have acute coronary syndromes. We suspect intimal involvement of the coronary arterial wall after regression of the large aneurysms underlies the acute coronary syndrome in adults. Risk factors for atherosclerosis must be avoided in this population.

Strategy for localized stenosis caused by Kawasaki disease: midterm results of percutaneous transluminal coronary balloon angioplasty in two infants.

We report the midterm results of percutaneous transluminal coronary balloon angioplasty (PCBA) for localized stenosis (LS) caused by Kawasaki disease (KD). Two 2-year-olds, a girl and a boy, underwent PCBA for severe LS of the left anterior descending artery caused by KD. After 4 years, there was no ischemia and no significant restenosis in the target vessels. PCBA provided a good result for early appearing LS caused by KD in two small infants. The 5-years-old boy was the youngest patient to undergo percutaneous transluminal coronary rotablator. Although the postprocedure result was very effective, 1 year later asymptomatic occlusion occurred.

Dilated coronary arterial lesions in the late period after Kawasaki disease.

OBJECTIVES: There are two types of late coronary dilated lesions after Kawasaki disease: new aneurysms and expanding aneurysms. The development of coronary dilated lesions late after Kawasaki disease was investigated. METHODS: Between 1978 and 2003, 562 patients with coronary arterial lesions underwent selective coronary angiography on at least two occasions. RESULTS: Of the 562 patients studied, 17 new dilated or expanding lesions were found in 15 patients (3%, 11 boys, four girls). The time of detection of new aneurysms after Kawasaki disease ranged from 1.9-19.2 years (median 11.4 years) and their diameters ranged from 2.0-6.5 mm (median 4.4 mm). Thirteen new aneurysms occurred in vessels in which previous aneurysms had regressed and all new aneurysms were associated with localised stenosis. A new aneurysm at the bifurcation or in the branches was seen in 14 (93%) and 13 were eccentric (87%). Of two expanding aneurysms, one involved the right coronary artery in one patient and the other the left anterior descending coronary artery. One expanding aneurysm increased from 4.4 mm to 19.5 mm over 17 years, and the other expanding aneurysm increased from 10 mm to 15 mm in one year. CONCLUSIONS: Neither new nor expanding aneurysms have caused cardiac events. New aneurysms often develop as a pre-stenotic or post-stenotic dilatation secondary to localised stenosis. New and expanding aneurysms may be caused by haemodynamic factors in addition to the abnormality of the coronary arterial wall after severe acute vasculitis. Coronary arterial wall abnormalities were stenosis as well as, rarely, dilatation of the vessels in the late period. It is important to recognise that the changes of the coronary arterial wall persist late after regression of a large aneurysm.

Incidence of stenotic lesions predicted by acute phase changes in coronary arterial diameter during Kawasaki disease.

To clarify the incidence of stenotic lesions according to the coronary arterial diameter in the acute phase. we investigated 190 patients with coronary arterial lesions who underwent an initial coronary angiogram (CAG) less than 100 days after the onset of Kawasaki disease. The largest diameters of the major branches were measured in the initial CAGs. The diameter of the large group was > or = 8.0 mm, that of the medium group was > or = 6.0 mm but < 8.0 mm, and that of the small group was > or = 4.0 mm but < 6.0 mm. There were 121 patients in the large group, 85 in the medium group, 77 in the small group. We investigated the stenotic lesions in the follow-up CAGs and evaluated the incidence of stenotic lesions in each group by the Kaplan-Meier method. The mean interval from the initial CAGs to the latest CAG was 97 months. The incidence of stenosis at 5, 10, and 15 years in the large group was 44, 62, and 74%, respectively. In the medium group the corresponding values were 6, 20, 58%, respectively. None of the patients in the small group developed stenotic lesions. Dilatation of more than 6.0 mm produces a high probability of irreversible change in the coronary arterial wall, leading to subsequent stenotic lesions.

Osteoclastogenesis inhibitory factor/osteoprotegerin reduced bone loss induced by mechanical unloading.

Skeletal unloading resulting from space flight and prolonged immobilization causes bone loss. Such bone loss ostensibly results from a rapid increase in bone resorption and subsequent sustained reduction in bone formation, but this mechanism remains unclear. Osteoclastogenesis inhibitory factor/osteoprotegerin (OCIF/OPG) is a recently identified potent inhibitor of osteoclast formation. We studied effects of OPG administration on tail-suspended growing rats to explore the therapeutic potential of OPG in the treatment and prevention of bone loss during mechanical unloading, such as that which occurs during space flight. Treatment with OPG in tail suspension increased the total bone mineral content (BMC g) of the tibia and femur and the total bone mineral density (BMD g/cm2) of the tibia. Moreover, treatment with OPG prevented reduction not only of BMC and BMD, but also of bone strength occurring through femoral diaphysis. Treatment with OPG in tail-suspended rats improved BMC, BMD and bone strength to levels of normally loaded rats treated with vehicle. Treatment with OPG in normally loaded rats significantly decreased urinary excretion of deoxypyridinoline, but the effect of OPG in tail suspension was unclear. These results indicate that OPG may be useful in inhibiting bone loss-engendered mechanical unloading.

Mycoplasma infection induces a scleroderma-like centrosome autoantibody response in mice.

Development of autoantibodies to intracellular molecules is a universal feature of autoimmune diseases and parallels onset of chronic inflammatory pathology. Initiating antigens of disease-specific autoantibody responses are unknown. We previously showed that the major targets of autoantibodies in scleroderma are centrosomes, organelles involved in mitotic spindle organization. Here we show that centrosome autoantibodies are induced in mice by mycoplasma infection. The centrosome-specific antibody response involves class switching of preexisting IgM to IgG isotypes, suggesting a T cell-dependent mechanism. The antibody response spreads to include additional intracellular targets, with newly recruited autoantibody specificities arising as IgM isotypes. Antibiotic treatment of mice prevents autoantibody development. Centrosome autoantibodies may provide an aetiological link between infection and human autoimmunity and suggest novel therapeutic strategies in these disorders.

Changes in osteoprotegerin and markers of bone metabolism during glucocorticoid treatment in patients with chronic glomerulonephritis.

It is well known that long-term glucocorticoid treatment causes osteoporosis, but the precise mechanism remains unclear. Recently, osteoprotegerin (OPG) has been identified as a cytokine that inhibits osteoclast differentiation. We have previously demonstrated that serum OPG is suppressed by glucocorticoids. Therefore, the present study was carried out to clarify the interrelationships between OPG and other markers of bone metabolism during glucocorticoid treatment. Thirteen patients (7 men, 6 women; 44.1 +/- 5.9 years old) with chronic glomerulonephritis who were to be treated with glucocorticoids for the first time were chosen for this study. Markers of bone metabolism, including serum OPG, osteocalcin (OC), bone-specific alkaline phosphatase activity (bAP), parathyroid hormone (PTH), tartrate-resistant acid phosphatase (TRAP), and bone mineral density (BMD), were measured before and during the treatment period. Glucocorticoids significantly reduced BMD of the lumbar spine in the 6 month treatment period (p < 0.01). Serum OPG was decreased significantly by glucocorticoids within 2 weeks (p < 0.001), and serum TRAP, a marker of bone resorption, was markedly increased (p < 0.001). On the other hand, there were no remarkable changes in serum PTH. Serum OC and bAP, markers of bone formation, were transiently reduced during the treatment period (p < 0.01). Furthermore, only serum OPG was positively and independently correlated with percentage BMD of age-matched reference (%AMR). These findings imply that glucocorticoid-induced bone loss develops rapidly via enhanced bone resorption and suppressed bone formation. Moreover, the increased bone resorption caused by glucocorticoids may be, at least in part, mediated by inhibition of OPG, not increment of PTH.

Coronary artery dilatation exceeding 4.0 mm during acute Kawasaki disease predicts a high probability of subsequent late intima-medial thickening.

We used intravascular ultrasound (IVUS) to compare the degree of coronary artery dilatation during the acute phase of Kawasaki disease with the extent of intima-medial thickening more than 10 years later. We wanted to determine if there was a threshold degree of dilatation that was highly predictive of later thickening. Twenty-eight patients with a mean age of 17.3 +/- 1.7 years were studied; the mean interval from the initial selective coronary angiography to the IVUS study was 15.0 +/- 1.6 years. We measured the maximum intima medial thickness of selected coronary arterial segments in IVUS images and measured the largest diameters of the corresponding coronary arterial segments in the initial coronary angiograms. A significant correlation was found between the initial diameters of the coronary arteries and the intima medial thickness more than 10 years later in the right coronary, the left anterior descending coronary, and the left circumflex arteries. The coefficient of correlation was 0.77 (n = 120, p < 0.0001), and for the bifurcation of the left coronary artery it was 0.50 (n = 26, p < 0.01). For this study, abnormal intima medial thickness was defined as more than 0.40 mm. When the initial coronary arterial dilatation exceeded 4.0 mm, the sensitivity was 28/31 (90%) and the specificity was 87/89 (98%) in the right coronary, the left anterior descending coronary, and the left circumflex arteries. For the bifurcation of the left coronary artery, the sensitivity was 14/21 (67%) and the specificity was 5/5 (100%).

Guidelines for catheter intervention in coronary artery lesion in Kawasaki disease.

The Research Committee of Ministry of Health, Labour and Welfare 'Study of treatment and long-term management in Kawasaki disease' reported the guidelines for catheter intervention in coronary artery lesion in Kawasaki disease in this paper. The contents include: (i) background and natural history of coronary artery lesion in Kawasaki disease; (ii) indication of catheter intervention; (iii) types of procedure, and their indication and care; (iv) institute and backup system; (v) the management after procedure, evaluation and follow up; and (vi) prospects, especially in relation to bypass surgery.

Reversal of multidrug resistance by novel nitrophenyl pyrones, SNF4435C and D.

SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis, were examined for their reversing effects in vitro on various multidrug-resistant (MDR) tumor cells overexpressing P-glycoprotein. These two compounds in the range of 3-10 microM completely reversed the resistance of MDR variant cells, mouse leukemia P388 cells [vincristine (VCR)-resistant P388/VCR and adriamycin (ADM)-resistant P388/ADM], human myelogenous leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM) and human ovarian cancer A2780 cells (ADM-resistant AD(10)), against VCR. Both compounds moderately potentiated the sensitivity of the MDR cells to ADM but the reversal was not complete. SNF4435C and D significantly increased the intracellular accumulation of VCR in AD(10) cells as potently as verapamil, cyclosporin A (CysA) and FK506, whereas the compounds exerted no effect on the accumulation of VCR in the drug-sensitive parent cells. Moreover, SNF4435C improved the chemotherapeutic efficacy of VCR in the treatment of P388/VCR-bearing mice. When 10 mg/kg SNF4435C was administered intraperitoneally to the mice concurrently with 0.2 mg/kg VCR for every 5 days, a treated/control (T/C) value of 143% was obtained. These results suggest that the compounds are useful candidates or tools for MDR modification in cancer chemotherapy.

Synovial cells from a patient with rheumatoid arthritis produce osteoclastogenesis inhibitory factor/osteoprotegerin: reciprocal regulation of the production by inflammatory cytokines and basic fibroblast growth factor.

To understand the involvement of osteoclastogenesis inhibitory factor (OCIF), also called osteoprotegerin (OPG), in the pathogenesis of bone destruction in rheumatoid arthritis (RA), we investigated the cytokine network involved in the production of OCIF by human fibroblast-like synovial (HFLS) cells from a patient with RA. Inflammatory cytokines, such as interleukin (IL)-1beta, IL-6 plus soluble IL-6 receptor (sIL-6R), IL-17, and tumor necrosis factor (TNF)-alpha, which are elevated in synovial fluid in RA, upregulated the production of OCIF to a level (5-20 ng/ml) sufficient to inhibit osteoclastogenesis in vitro. These inflammatory cytokines (except for IL-6 plus sIL-6R) stimulate OCIF production directly or indirectly through stimulation of prostaglandin E2 (PGE2) synthesis. In contrast to the findings with inflammatory cytokines, basic fibroblast growth factor (bFGF) inhibited the production of OCIF by the cells in a dose-dependent manner. While bFGF enhanced both the inflammatory cytokine-mediated release of PGE2 and the PGE2-mediated OCIF production, it significantly suppressed OCIF production by negating the direct stimulatory effect of the inflammatory cytokines. These findings suggest that bFGF in the synovial fluid of patients with RA may lead to severe joint destruction by suppressing the production of OCIF by HFLS cells.

Immunological study on circulating murine osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF): possible role of OPG/OCIF in the prevention of osteoporosis in pregnancy.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a soluble member of the tumor necrosis factor receptor family and plays a crucial role in the negative regulation of osteoclastic bone resorption. We have immunized OPG/OCIF knockout mice with murine rOPG/rOCIF and established a panel of hybridomas producing monoclonal antibodies (mAbs) to murine rOPG/rOCIF. Utilizing the mAbs, we developed enzyme-linked immunosorbent assay (ELISA) systems: one detecting both homodimeric and monomeric forms of murine OPG/OCIF and the other detecting only dimeric form of murine OPG/OCIF. With the aid of these ELISA systems we showed that OPG/OCIF is present mainly as a monomer in murine blood. The concentration of OPG/OCIF in normal mouse sera was approximately 500 pg/ml and there was no statistical difference in the serum concentration of OPG/OCIF among genders, age, and strains. Interestingly, the concentration of circulating OPG/OCIF in mouse markedly increased during pregnancy. The result indicated that circulating OPG/OCIF plays an important role in the protection of bone from excess resorption during pregnancy in mammals.

SW-163C and E, novel antitumor depsipeptides produced by Streptomyces sp. I. Taxonomy, fermentation, isolation and biological activities.

Novel depsipeptides, SW-163C and E were isolated from the culture broth of an actinomycete strain. The producing organism, designated as SNA15896, was identified as a member of Streptomyces from its morphological and cultural characteristics. SW-163C and E exhibited potent antitumor activities against various tumor cell lines in vitro and against murine leukemia P388 in vivo. The compounds also showed antimicrobial activities.

SW-163C and E, novel antitumor depsipeptides produced by Streptomyces sp. II. Structure elucidation.

SW-163C and E are novel antitumor antibiotics, which belong to quinomycin family, isolated from the culture broth of Streptomyces sp. SNA15896. These compounds were determined to be cyclic depsipeptides having 3-hydroxyquinaldic acid as a chromophore and a sulfur-containing intramolecular cross linkage through various spectroscopic analyses.

SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis. I. Taxonomy, fermentation, isolation and biological activities.

SNF4435C and D, novel nitrophenyl pyrones, have been isolated from the culture broth of an actinomycete strain SNF4435. The strain was identified as Streptomyces spectabilis from its morphological and cultural characteristics. SNF4435C and D showed a potent immunosuppressive activity in vitro and selectively suppressed B-cell proliferation induced by LPS versus T-cell proliferation induced by Con A.