Ultrasound efficacy of targeted-synthetic disease-modifying anti-rheumatic drug treatment in rheumatoid arthritis: a multicenter prospective cohort study in Japan.

OBJECTIVE: This study investigated the effectiveness of treatment with Janus kinase (JAK) inhibitors in rheumatoid arthritis (RA) assessed by ultrasonography (US) activity, and the influence of patient characteristics and previous treatments. METHOD: This prospective study assessed 60 treatment initiations among 53 Japanese patients diagnosed with RA who underwent treatment with JAK inhibitors during June 2013 to February 2020. Of the 53 patients, seven patients were enrolled in duplicate because they were treated with two different JAK inhibitors at different periods. For each case, the improvement rate on the power Doppler (PD) score was assessed at 6 month follow-up. Median improvement rate of PD score was used to classify cases as either US responders or non-responders, and patient characteristics were compared between the two groups. RESULTS: All indicators of clinical disease activity and US activity showed a significant improvement at 3 months compared with baseline. Although the JAK inhibitor-cycler group and the interleukin-6 (IL-6) inhibitor inadequate response (IR) group tended to show a later improvement for US activity, all indicators of clinical disease activity and US activity showed a significant improvement at 6 months compared with baseline for both groups. Multivariate analysis showed that concomitant methotrexate use and an IR to the previous biologic or targeted-synthetic disease-modifying anti-rheumatic drug (b/tsDMARD) treatment were independently and significantly associated with US responders. CONCLUSION: Use of a JAK inhibitor in combination with methotrexate and an absence of IR to any previous b/tsDMARDs demonstrated superior effectiveness for patients with RA.

Q-band high-performance notch filters at 56 and 77 GHz notches for versatile fusion plasma diagnostics.

A six-pole Q-band waveguide filter with a notch frequency above the Q-band has been developed for plasma diagnostics. The previous paper [Nishiura et al., J. Instrum. 10, C12014 (2015)] reported that the notch frequency exists within the standard band. In this study, the newly required notch filter extends the function, which prevents a thorny wave from being mixed into an instrument beyond the standard bandwidth of the waveguide. The mode control technique for cavities realizes a deep and sharp filter shape for Q-band notch filters with 56 and 77 GHz notches, respectively. The former filter has an attenuation more than 50 dB at 56.05 GHz and a bandwidth of 1.1 GHz at -3 dB. The latter filter has an attenuation more than 55 dB at 76.95 GHz and a bandwidth of 1.6 GHz at -3 dB. The electron cyclotron emission imaging and the electron cyclotron emission (ECE) diagnostics for the Q-band implemented a pair of the fabricated filters and demonstrated the ECE measurement successfully in the intense stray radiation from a 56 GHz gyrotron.

Discrepancy between clinical and ultrasound remissions in rheumatoid arthritis: a multicentre ultrasound cohort study in Japan.

Objectives: Using multicentre ultrasound (US) cohort data among patients with rheumatoid arthritis (RA), we aimed to identify baseline factors that permit differentiation between two patient cohorts achieving US remission and clinical remission, and to determine the factors contributing to the discrepancy.Method: We reviewed 248 Japanese patients diagnosed with RA who underwent treatment with biological disease-modifying anti-rheumatic drugs at 13 centres. We performed US assessments of the synovia of 22 joints. We assessed the percentages of patients with clinical remission and US remission, defined as total power Doppler scores of 0 at 12 months.Results: The 87 patients who achieved US remission were divided into a group that achieved both clinical and US remission (n = 53) and a group that achieved US remission only (n = 34). Baseline factors that were significantly and independently associated with clinical remission at 12 months among patients who also achieved US remission included short disease duration, the presence of concomitant methotrexate use, and low patient global assessment score (p < 0.05, p < 0.05, and p < 0.005, respectively).Conclusions: RA patients with baseline high patient global assessment scores and long disease duration at baseline were unlikely to achieve clinical remission even after achieving US remission. Objective joint assessments using US provide additional information of potential importance for the management of RA.

Significance of anti-Ro/SSA antibodies in the response and retention of abatacept in patients with rheumatoid arthritis: a multicentre cohort study.

Objective: To determine whether the positivity of baseline anti-Ro/Sjögren's syndrome antigen A (SSA) antibodies influences the response to abatacept, we compared therapeutic responses between anti-Ro/SSA antibody-negative and -positive patients with rheumatoid arthritis (RA) using a multicentre RA ultrasonography prospective cohort. Method: We reviewed Japanese patients with RA who started abatacept as the first biological disease-modifying anti-rheumatic drug between June 2013 and April 2018. We assessed 28-joint Disease Activity Score-erythrocyte sedimentation rate (DAS28-ESR) change between baseline and 6 or 12 months after treatment in RA patients treated with abatacept, and European League Against Rheumatism (EULAR) response at 6 and 12 months. The Global OMERACT-EULAR Synovitis Score (GLOESS) was calculated at baseline and at 6 and 12 months. Results: Overall, 51 patients were enrolled and divided into anti-Ro/SSA antibody-negative and -positive groups of 35 and 16, respectively. Median age at baseline was significantly higher in the anti-Ro/SSA antibody-negative group (p = 0.04). The retention rate and percentage of EULAR good responders at 12 months were significantly higher in the anti-Ro/SSA antibody-negative group (both p = 0.02). Anti-Ro/SSA antibody-negative patients exhibited larger decreases in both DAS28-ESR and DAS28-C-reactive protein at 12 months than anti-Ro/SSA antibody-positive patients (p = 0.02 and 0.04, respectively). GLOESS decreased significantly at 6 months in anti-Ro/SSA antibody-negative patients (p = 0.03). Multivariate analyses showed that anti-Ro/SSA antibody positivity was an independent factor associated with change in the DAS28-ESR at 6 months (p < 0.05). Conclusion: Anti-Ro/SSA antibody positivity predicts a poor response to abatacept and low retention rate.

Fluorescence optical imaging in patients with active rheumatoid arthritis: a comparison with ultrasound and an association with biomarkers.

Objectives: This study compared indocyanine green (ICG)-enhanced fluorescence optical imaging (FOI) and musculoskeletal ultrasound (MSUS), and explored the significance of the FOI findings based on the association between the FOI and MSUS findings and serum biomarkers in patients with rheumatoid arthritis (RA). The study also explored the association between the FOI findings and patients' joint destruction at the joint-area level.Method: We enrolled 50 consecutive patients with active RA from among the patients hospitalized from May 2014 to March 2016 at Nagasaki University Hospital, Japan. FOI images were acquired with the Xiralite® fluorescence imaging system and compared with the patients' clinical examination results and MSUS findings. On the same day, the patients' clinical disease activity and levels of serum biomarkers (including vascular endothelial growth factor) were obtained.Results: Although the FOI detected synovitis with high sensitivity, the frequency of positive findings and the diagnostic performance with MSUS as the reference standard for FOI differed considerably among the phases of FOI as well as among the affected joint regions. The FOI scores were positively correlated with clinical disease activity, MSUS scores, and serum biomarkers. The severity of FOI-proven synovitis was associated with the presence of MSUS-proven bone erosion.Conclusion: FOI is effective for detecting joint inflammation in RA patients, with high accuracy. The severity of the FOI score was closely associated with the joint destruction at the joint-area level. However, the significance of positive FOI findings differed depending on not only the phase of FOI but also the affected joint regions.

IL-15 is a biomarker involved in the development of rapidly progressive interstitial lung disease complicated with polymyositis/dermatomyositis.

BACKGROUND: Polymyositis/dermatomyositis (PM/DM) is an autoimmune disease that is sometimes complicated with rapidly progressive interstitial lung disease (RPILD). However, serum and lung biomarkers that can predict RPILD development remain unclear. OBJECTIVES: To determine potential serum and lung biomarkers that can predict RPILD development in patients with PM/DM-ILD. METHODS: In total, 49 patients with PM/DM-ILD were enrolled. We measured the serum levels of 41 cytokines/chemokines, ferritin and anti-MDA5 antibody, compared them between the RPILD (n = 23) and non-RPILD (n = 26) groups, and ranked them by their importance through random forest analysis. To distinguish the two groups, we determined biomarker combinations by logistic regression analysis. We also measured the bronchoalveolar lavage fluid (BALF) levels of 41 cytokines/chemokines. Using immunohistochemistry, we examined IL-15 expression in lung tissues. The IL-15 production was also investigated using A549 and BEAS-2B cells. RESULTS: The RPILD group had significantly higher IL-15, IL-1RA, IL-6, CXCL10, VCAM-1, anti-MDA5 antibody and ferritin serum levels than the non-RPILD group, but it had a significantly low CCL22 level. Meanwhile, anti-MDA5 antibody, IL-15, CXCL8, CCL22, IL-1RA and ferritin were the best combination to distinguish the two groups. IL-15 and CCL22 were also predictive marker for RPILD development in anti-MDA5 antibody-positive patients. Additionally, the RPILD group had significantly high IL-15 levels in BALF. The lung tissues expressed IL-15, which increased after cytokine stimulation in the A549 cells. CONCLUSION: This study identified a combination of biomarkers predicting PM/DM-RPILD progression, and IL-15 is an important cytokine for predicting RPILD development and reflecting ILD severity.

Complement profile in microscopic polyangiitis and granulomatosis with polyangiitis: analysis using sera from a nationwide prospective cohort study.

OBJECTIVE: The complement cascade, especially the alternative pathway of complement, has been shown in basic research to be associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). We aimed to elucidate relationships between serum complement components and clinical characteristics in AAV. METHOD: In a nationwide prospective cohort study (RemIT-JAV-RPGN), we measured the serum levels of C1q, C2, C3, C3b/iC3b, C4, C4b, C5, C5a, C9, factor B, factor D, factor H, factor I, mannose-binding lectin, and properdin in 52 patients with microscopic polyangiitis (MPA) and 39 patients with granulomatosis with polyangiitis (GPA). RESULTS: The properdin level of MPA and GPA was significantly lower than that of healthy donors. The properdin level was negatively correlated with the Birmingham Vasculitis Activity Score (BVAS) (ρ = -0.2148, p = 0.0409). The factor D level at 6 months was significantly positively correlated with the Vasculitis Damage Index (VDI) at 6, 12, and 24 months (ρ = 0.4207, 0.4132, and 0.3115, respectively). Patients with a higher ratio of C5a to C5 had higher neutrophil percentage and serum immunoglobulin G levels, and significantly lower creatinine levels. Cluster analysis divided the MPA and GPA patients into three subgroups. A principal component (PC) analysis aggregated 15 types of complements into alternative pathway-related PC 1 and complement classical pathway and common pathway-related PC 2. CONCLUSIONS: The serum levels of properdin and factor D were correlated with the BVAS and the VDI in MPA and GPA, respectively. Our analyses suggested the pathological heterogeneity of MPA and GPA from the aspect of complement components.

Anti-citrullinated protein antibody titre as a predictor of abatacept treatment persistence in patients with rheumatoid arthritis: a prospective cohort study in Japan.

Objective: Successful rheumatoid arthritis (RA) outcome depends on treatment efficacy in the early stages of the disease and its sustainability. It is thus critical to identify factors predicting treatment persistence with biological agents, such as abatacept. We compared clinical profiles, including early changes in autoantibody titres at 3 months, between patients with RA demonstrating sustained persistence and those discontinuing abatacept treatment.Method: We prospectively enrolled 71 and 78 active RA patients treated with abatacept and tumour necrosis factor inhibitors (TNF-Is), respectively, who had previous disease-modifying anti-rheumatic drug) failure. Clinical characteristics were compared between non-continuation and continuation groups stratified according to abatacept or TNF-I persistence for at least 12 months from treatment initiation.Results: Significantly larger decreases in rheumatoid factor titre and anti-citrullinated protein autoantibody (ACPA) titre were observed in the continuation group of abatacept therapy at 3 months, and early reduction in ACPA titre remained a significant and independent predictor of sustained persistence with abatacept in multivariate analysis. In addition, we obtained the area under the receiver operator characteristics curve of 0.904 from a model including baseline ACPA titre and reduction of ACPA titre at 3 months. Sustained reduction of RA disease activity score at 12 months was significantly and independently associated with reduced ACPA titre at 3 months.Conclusions: Persistence with abatacept and sustained therapeutic response are associated with an early reduction in ACPA titre. Prediction of abatacept continuation and efficacy will facilitate the optimal design of therapy in the early stages of RA.

Complete renal response at 12 months after induction therapy is associated with renal relapse-free rate in lupus nephritis: a single-center, retrospective cohort study.

BACKGROUND: Lupus nephritis (LN) is a major risk factor for overall morbidity and mortality in systemic lupus erythematosus (SLE). METHODS: We retrospectively analyzed cases of proliferative and membranous LN patients who underwent a renal biopsy at our hospital in 1993-2016. We analyzed the association between complete renal response (CR) rates at 12 months after induction therapy and predictive factors for CR and their association with renal flares. RESULTS: Of the 95 cases analyzed, we were able to track the therapeutic responses of 81 patients at 12 months after their induction therapy. The median follow-up duration after renal biopsy was 51 months (interquartile range: 16.5-154.5 months). The Cox proportional hazards model showed that, compared to not attaining CR at 12 months, the attainment of CR at 12 months was correlated with being free from renal flares. The multivariate logistic analysis revealed that the predictive factors for CR at 12 months were the anti-La/SSB antibodies (U/ml) (odds ratio (OR) 1.22, 95% confidence interval (CI) 1.01-1.63, p = 0.0220), blood urea nitrogen (BUN) (OR 0.68, 95% CI 0.44-0.90, p = 0.00048) and serum ß2 microglobulin (MG) (OR 0.26, 95% CI 0.06-0.74, p = 0.00098) levels. CONCLUSIONS: Among LN patients, being free from renal flares was associated with attaining CR at 12 months after induction therapy. Anti-La/SSB antibodies were a positive predictive factor, and BUN and serum ß2MG levels were negative predictive factors of CR at 12 months.

Factors predictive of long-term mortality in lupus nephritis: a multicenter retrospective study of a Japanese cohort.

BACKGROUND: Lupus nephritis (LN) is a major determinant of mortality in systemic lupus erythematosus (SLE). Here we evaluated the association between complete renal response (CR) and mortality in LN. METHODS: We retrospectively analyzed the cases of 172 of 201 patients with LN for whom data on the therapeutic response at 6 and 12 months after induction therapy were available. The patients underwent a renal biopsy at Nagasaki University Hospital and community hospitals in Nagasaki between the years 1990 and 2016. We determined the CR rates at 6 and 12 months after induction therapy initiation and evaluated the predictive factors for CR and their relationship with mortality. We performed univariate and multivariable competing risks regression analyses to determine the factors predictive of CR. The patients' survival data were analyzed by the Kaplan-Meier method with a log-rank test. RESULTS: The median follow-up duration after renal biopsy was 120 months (interquartile range: 60.3-191.8 months). The 5-, 10-, 15- and 20-year survival rates of our cohort were 99.3, 94.6, 92.0 and 85.4%, respectively. During follow-up, nine patients (5.2%) died from cardiovascular events, infection, malignancy and other causes. The multivariate analysis revealed that the following factors were predictive of CR. At 6 months: male gender (odds ratio (OR) 0.23, 95% confidence interval (CI) 0.08-0.65, p = 0.0028), proteinuria (g/gCr) (OR 0.83, 95% CI 0.71-0.97, p = 0.0098) and index of activity (0-24) (OR 0.84, 95% CI 0.71-0.99, p = 0.0382). At 12 months: male gender (OR 0.25, 95% CI 0.09-0.67, p = 0.0043) and index of activity (0-24) (OR 0.82, 95% CI 0.69-0.98, p = 0.0236). The Kaplan-Meier analysis showed that compared to not achieving CR at 12 months, achieving CR at 12 months was significantly correlated with the survival rate (OR 0.18, 95% CI 0.04-0.92, p = 0.0339). CONCLUSIONS: Our results suggest that the survival rate of patients with LN is associated with the achievement of CR at 12 months after induction therapy, and that male gender and a higher index of activity (0-24) are the common predictive factors for failure to achieve CR at 6 and 12 months.

The efficacy of adjunct tacrolimus treatment in pregnancy outcomes in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) involves multiple organ systems and primarily affects women during their reproductive years. Pregnancy in a woman with SLE may lead to higher rates of disease flares. Little is known regarding which medications are safe to maintain remission and/or treat flares throughout such pregnancies. Here we retrospectively analyzed the efficacy of tacrolimus (TAC) in the pregnancy outcomes of SLE patients. We studied the 54 deliveries of 40 SLE patients over an eight-year period from 2008 to 2016. We used analyses of covariance with adjustments for the propensity score and inverse probability of treatment weights to compare the patient backgrounds between the TAC users and non-TAC users. TAC was administered to the patient in 15 of the 54 (27.8%) pregnancies, and these patients had a significantly higher dose of prednisolone, hypocomplementemia, lower estimated glomerular filtration rate, past history of lupus nephritis, and complication with antiphospholipid syndrome. In the adjusted background of the TAC deliveries, the risks of decreased fetal body weight, low birth weight infant, non-reassuring fetal status (NRFS), and preterm birth were not increased compared to the non-TAC deliveries. Thrombocytopenia and hypertension during the pregnancy were extracted as independent predictive risk factors for decreased fetal body weight and NRFS, respectively. We had anticipated that the maternal and fetal outcomes in the TAC-use deliveries would be poor before the analysis; however, the TAC-use group showed no significant difference in risks contributing to outcomes compared to the non-TAC group, suggesting that adjunct TAC treatment corrected various risk factors during the lupus pregnancies.

Sweet potato resistance to Euscepes postfasciatus (Coleoptera: Curculionidae): larval performance adversely effected by adult's preference to tuber for food and oviposition.

The preferences of the West Indian sweet potato weevil, Euscepes postfasciatus (Fairmaire), to tubers of sweet potato, Ipomoea batatas (L.), for food and for oviposition were evaluated, and correlated to sweet potato's resistance to immatures. Adults (parent) were released in a plastic box containing tubers of sweet potato cultivars and maintained for 5 d, after which the adults on each tuber were counted. All adults were then removed and each tuber was maintained separately. New adults that emerged from the tubers were counted. Cultivars were grouped by cluster analyses using the number of parent adults on the tubers and the number of new adults emerging from the tubers, adjusted for the weight of each tuber. Cultivars were divided into five groups: average level of preference, preferred, preferred for oviposition but not for food, preferred for food but not for oviposition, and not preferred. New adults from the first two groups took less time to eclose than those from the other groups, and their body size was smaller. In a second experiment, one to five cultivars were selected from each group and inoculated each tuber with 10 weevil eggs on each cultivar. Although the proportion of eclosed adults was not significantly different between cultivars, the time to eclosion was shorter and body size was smaller on preferred cultivars. The selection of tubers by parent adults was not linearly related with larval development, and did not reduce the survival of the immatures.

Association of distinct clinical subsets with myositis-specific autoantibodies towards anti-155/140-kDa polypeptides, anti-140-kDa polypeptides, and anti-aminoacyl tRNA synthetases in Japanese patients with dermatomyositis: a single-centre, cross-sectional study.

OBJECTIVE: To determine the association of distinct clinical subsets with myositis-specific autoantibodies (MSAs) towards anti-155/140-kDa polypeptides [anti-155/140 antibodies (Abs)], anti-140-kDa polypeptides (anti-140 Abs), and anti-aminoacyl tRNA synthetases (ARS Abs) in Japanese patients with dermatomyositis (DM). METHODS: We compared the clinical features and short-term prognoses of 30 DM patients whose serological status included these MSAs. The MSAs were determined by immunoprecipitation. RESULTS: Anti-155/140 Abs (n = 5), anti-140 Abs (n = 8), and anti-ARS Abs (n = 7) did not overlap each other. All of the anti-155/140 Ab-positive patients (n = 5) were complicated by malignancies, as were all of the anti-140 Ab-positive patients (n = 8), who showed rapidly progressive interstitial lung disease (ILD). The survival rate at 6 months from the diagnosis of DM was significantly lower in the anti-140 Ab-positive patients than in the other patients. CONCLUSION: This is the first study to report, in a single cohort of DM patients, that distinct clinical subsets are distributed in an anti-155/140 Ab-positive group, an anti-140 Ab-positive group, or an anti-ARS Ab-positive group. Our data also confirm previous evidence that anti-155/140 Abs are involved in malignancies and that anti-140 Abs are involved in rapidly progressive ILD.

First Report of Root-Knot Nematode Meloidogyne enterolobii on Guava in Vietnam.

In southern Vietnam, citrus is interplanted with guava (Psidium guajava L.) for the management of citrus greening disease. Guava seedlings exhibited decline symptoms including leaf browning, growth inhibition, leaf drop, and death. These symptomatic seedlings were accompanied by severely galled roots, possibly attributable to root-knot nematodes. We collected second-stage juveniles from the soil and picked adult females from roots to perform morphological observations. The perineal patterns of most specimens were similar to those in the original description of Meloidogyne enterolobii (3). However, some were similar to that of M. incognita. Such variability among perineal patterns precluded unequivocal identification. Therefore, DNA was extracted from these nematodes and their sequences were compared with those in the DNA sequence database. For the comparison, we employed the primer sets of Powers and Harris (1) to amplify a region between cytochrome oxidase II and the 16S ribosomal DNA gene of mitochondria by PCR. An approximate 700-bp fragment was obtained and analyzed, revealing more than 99.6% homology to M. mayaguensis, a synonym of M. enterolobii (2), when aligned with sequence data of isolates from France (GenBank Accession No. AJ421396), the United States (GenBank Accession No. AY446978), and China (GenBank Accession No. AY831967). These results indicate that the nematode species responsible for guava damage in southern Vietnam is M. enterolobii. To our knowledge, this is the first report of M. enterolobii in Vietnam. References: (1) T. O. Powers and T. S. Harris. J. Nematol. 25:1, 1993. (2) J. Xu et al. Eur. J. Plant Pathol. 110:309, 2004. (3) B. Yang and J. D. Eisenback. J. Nematol. 15:381, 1983.

Crystal structures of the Streptomyces coelicolor TetR-like protein ActR alone and in complex with actinorhodin or the actinorhodin biosynthetic precursor (S)-DNPA.

Actinorhodin, an antibiotic produced by Streptomyces coelicolor, is exported from the cell by the ActA efflux pump. actA is divergently transcribed from actR, which encodes a TetR-like transcriptional repressor. We showed previously that ActR represses transcription by binding to an operator from the actA/actR intergenic region. Importantly, actinorhodin itself or various actinorhodin biosynthetic intermediates can cause ActR to dissociate from its operator, leading to derepression. This suggests that ActR may mediate timely self-resistance to an endogenously produced antibiotic by responding to one of its biosynthetic precursors. Here, we report the structural basis for this precursor-mediated derepression with crystal structures of homodimeric ActR by itself and in complex with either actinorhodin or the actinorhodin biosynthetic intermediate (S)-DNPA [4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho-[2,3-c]-pyran-3-(S)-acetic acid]. The ligand-binding tunnel in each ActR monomer has a striking hydrophilic/hydrophobic/hydrophilic arrangement of surface residues that accommodate either one hexacyclic actinorhodin molecule or two back-to-back tricyclic (S)-DNPA molecules. Moreover, our work also reveals the strongest structural evidence to date that TetR-mediated antibiotic resistance may have been acquired from an antibiotic-producer organism.

Endostatin peptide, an inhibitor of angiogenesis, prevents the progression of peritoneal sclerosis in a mouse experimental model.

Peritoneal sclerosis is a major and serious complication in patients on long-term continuous ambulatory peritoneal dialysis (PD). The involvement of angiogenesis and proangiogenic factors such as vascular endothelial growth factor (VEGF)-A in progressing peritoneal sclerosis has been reported. We previously reported the therapeutic efficacy of endostatin peptide, a potent inhibitor of angiogenesis derived from type XVIII collagen, in a mouse diabetic nephropathy model. Here, we examined the therapeutic effect of endostatin peptide in preventing progression in a mouse peritoneal sclerosis model. Male ICR mice received intraperitoneal injections of chlorhexidine gluconate (CG) every other day to induce peritoneal sclerosis. Endostatin peptide (1 or 4 mg/kg/day) was administered via subcutaneously implanted osmotic minipumps. Peritoneal sclerosis (day 24) was significantly suppressed by endostatin peptide in a dose-dependent manner. Peritoneal accumulation of type III collagen was significantly suppressed by endostatin peptide. Increase in the number of CD31(+) blood vessels, F4/80(+) monocyte/macrophage accumulation, and 5-bromodeoxyuridine(+) proliferating cells was significantly inhibited by endostatin peptide. Increase in peritoneal expression of VEGF-A, profibrotic transforming growth factor-beta1, and alpha-smooth muscle actin was suppressed by endostatin peptide. Immunoreactivity for endogenous endostatin (whole molecule) and endostatin receptor alpha5beta1-integrin was increased and colocalized to CD31(+) blood vessels in the thickened peritonea of CG-injected mice. These results demonstrate the potential use of antiangiogenic endostatin peptide as a novel therapeutic agent in preventing peritoneal sclerosis, a severe complication in patients undergoing long-term PD.

A new mode of stereochemical control revealed by analysis of the biosynthesis of dihydrogranaticin in Streptomyces violaceoruber Tü22.

A class of Streptomyces aromatic polyketide antibiotics, the benzoisochromanequinones, all shows trans stereochemistry at C-3 and C-15 in the pyran ring. The opposite stereochemical control found in actinorhodin (3S, 15R, ACT) from S. coelicolor A3(2) and dihydrogranaticin (3R, 15S, DHGRA) from S. violaceoruber Tü22 was studied by functional expression of the potentially relevant ketoreductase genes, actIII, actVI-ORF1, gra-ORF5, and gra-ORF6. A common bicyclic intermediate was postulated to undergo stereospecific reduction to provide either the 3-(S) or the 3-(R) configuration of an advanced intermediate, 4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho[2,3-c]pyran-3-acetic acid (DNPA). Combinations of the four ketoreductase genes were coexpressed with the early biosynthetic genes encoding a type II minimal polyketide synthase, aromatase, and cyclase. gra-ORF6 was essential to produce (R)-DNPA in DHGRA biosynthesis. Out of the various recombinants carrying the relevant ketoreductases, the set of gra-ORF5 and -ORF6 under translational coupling (on pIK191) led to the most efficient production of (R)-DNPA as a single product, implying a possible unique cooperative function whereby gra-ORF6 might encode a "guiding" protein to control the regio- and stereochemical course of reduction at C-3 catalyzed by the gra-ORF5 protein. Updated BLAST-based database analysis suggested that the gra-ORF6 product, a putative short-chain dehydrogenase, has virtually no sequence homology with the actVI-ORF1 protein, which was previously shown to determine the 3-(S) configuration of DNPA in ACT biosynthesis. This demonstrates an example of opposite stereochemical control in antibiotic biosynthesis, providing a key branch point to afford diverse chiral metabolic pools.

Resistance of CD4-positive T lymphocytes to etoposide-induced apoptosis mediated by upregulation of Bcl-xL expression in patients with HTLV-I-associated myelopathy.

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM) is characterized by chronic inflammation of the spinal cord. The exact mechanisms that enhance the development of chronic myelopathy remain to be determined. One such mechanism could be an altered response of peripheral blood CD4(+) T lymphocytes to apoptotic stimuli. We examined the sensitivity of these cells to apoptosis in HAM patients and control. Apoptosis was induced by etoposide, which induces mitochondria-dependent apoptosis through the release of cytochrome c from the mitochondria. The percentage of apoptotic cells that expressed hypodiploid DNA among etoposide-treated CD4(+) T lymphocytes was significantly lower in HAM patients than in the control. Western blot analysis of cell lysates derived from CD4(+) T lymphocytes demonstrated that the expression level of Bcl-xL protein was significantly higher in HAM patients than in the control. Our results indicate that peripheral blood CD4(+) T lymphocytes of HAM patients are resistant to apoptosis triggered through mitochondrial death pathway through upregulation of expression of anti-apoptotic protein, Bcl-xL. This phenomenon might contribute to the prolongation and perpetuation of the chronic inflammatory process in the spinal cord of HAM patients.

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity.

BACKGROUND: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Generally, it is proposed that GTs are two-domain proteins with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aimed at finding out the region responsible for determination of substrate specificities of these two urdamycin GTs. RESULTS: A series of 10 chimeric GT genes were constructed consisting of differently sized and positioned portions of urdGT1b and urdGT1c. Gene expression experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT1c-like activity. A 31 amino acid region (aa 52-82) located close to the N-terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chimeric gene product of the 10 was not functional. Targeted stepwise alterations of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues conserved among streptomycete GTs. Alterations G226S and G226R restored a weak activity, whereas G226SR showed an activity comparable with other functional chimeras. CONCLUSIONS: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it.