A novel apoptosis probe, cyclic ApoPep-1, for in vivo imaging with multimodal applications in chronic inflammatory arthritis.

Apoptosis plays an essential role in the pathophysiologic processes of rheumatoid arthritis. A molecular probe that allows spatiotemporal observation of apoptosis in vitro, in vivo, and ex vivo concomitantly would be useful to monitoring or predicting pathophysiologic stages. In this study we investigated whether cyclic apoptosis-targeting peptide-1 (CApoPep-1) can be used as an apoptosis imaging probe in inflammatory arthritis. We tested the utility of CApoPep-1 for detecting apoptotic immune cells in vitro and ex vivo using flow cytometry and immunofluorescence. The feasibility of visualizing and quantifying apoptosis using this probe was evaluated in a murine collagen-induced arthritis (CIA) model, especially after treatment. CApoPep-1 peptide may successfully replace Annexin V for in vitro and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for ex vivo in the measurement of apoptotic cells, thus function as a sensitive probe enough to be used clinically. In vivo imaging in CIA mice revealed that CApoPep-1 had 42.9 times higher fluorescence intensity than Annexin V for apoptosis quantification. Furthermore, it may be used as an imaging probe for early detection of apoptotic response in situ after treatment. The CApoPep-1 signal was mostly co-localized with the TUNEL signal (69.6% of TUNEL+ cells) in defined cell populations in joint tissues of CIA mice. These results demonstrate that CApoPep-1 is sufficiently sensitive to be used as an apoptosis imaging probe for multipurpose applications which could detect the same target across in vitro, in vivo, to ex vivo in inflammatory arthritis.

Robust Therapeutic Efficacy of Matrix Metalloproteinase-2-Cleavable Fas-1-RGD Peptide Complex in Chronic Inflammatory Arthritis.

OBJECTIVE: Therapeutic agents that are transformable via introducing cleavable linkage by locally enriched MMP-2 within inflamed synovium would enhance therapeutic efficacy on chronic inflammatory arthritis. Transforming growth factor-ß-inducible gene-h3 (ßig-h3), which consists of four fas-1 domains and an Arg-Gly-Asp (RGD) motif, intensifies inflammatory processes by facilitating adhesion and migration of fibroblast-like synoviocyte in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to investigate whether a MMP-2-cleavable peptide complex consisting of a fas-1 domain and an RGD peptide blocks the interaction between ßig-h3 and resident cells and leads to the amelioration of inflammatory arthritis. METHODS: We designed ßig-h3-derivatives, including the fourth fas-1 domain truncated for H1 and H2 sequences of mouse (MFK00) and MMP-2-cleavable peptide complex (MFK902). MMP-2 selectivity was examined by treatment with a series of proteases. MFK902 efficacy was determined by the adhesion and migration assay with NIH3T3 cells in vitro and collagen-induced arthritis (CIA) model using male DBA/1J mice in vivo. The mice were treated intraperitoneally with MFK902 at different dosages. RESULTS: MFK902 was specifically cleaved by active MMP-2 in a concentration-dependent manner, and ßig-h3-mediated adhesion and migration were more effectively inhibited by MFK902, compared with RGD or MFK00 peptides. The arthritis activity of murine CIA, measured by clinical arthritis index and incidence of arthritic paws, was significantly ameliorated after treatment with all dosages of MFK902 (1, 10, and 30 mg/kg). MFK902 ameliorated histopathologic deterioration and reduced the expression of inflammatory mediators simultaneously with improvement of clinical features. In addition, a favorable safety profile of MFK902 was demonstrated in vivo. CONCLUSION: The present study revealed that MMP-2-cleavable peptide complex based on ßig-h3 structure is a potent and safe therapeutic agent for chronic inflammatory arthritis, thus providing reliable evidence for a MMP-2-cleavable mechanism as a tissue-targeted strategy for treatment of RA.

Anthocyanin Profile and Antioxidant Activity of Various Berries Cultivated in Korea.

This study examined the anthocyanin composition and antioxidant activity of various berries cultivated in Korea: blueberry, crowberry, Korean black raspberry, mulberry, and strawberry. The anthocyanins in berries were identified by high-performance liquid chromatography (HPLC) analysis, and each component was quantitatively analyzed. Furthermore, the antioxidant activity of berries was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization, oxygen radical absorbance capacity (ORAC), and ferric ion reducing antioxidant (FRAP) assays. The results revealed that the total content of anthocyanins in crowberry was 35.1 mg/g of extract, which was higher than that in the other four major berry species (1.9-27.7 mg/g of extract). Nineteen anthocyanins were identified in the various berries. The major anthocyanins of crowberry were cyanidin-3-galactoside and delphinidin-3-galactoside, and those from Korean black raspberry were cyanidin-3-rutinoside and cyanidin-3-sambubioside-5-rhamnoside. These two berries also had relatively strong antioxidant activity accompanied by high total polyphenol contents. Thus, consumption of crowberry and Korean black raspberry may be beneficial in reducing the risk of developing lifestyle-related chronic diseases because of their strong antioxidant activity.

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma arising in a patient with hypersensitivity to mosquito bites.

Hypersensitivity to mosquito bites is defined as the appearance of intense skin reactive lesions and systemic symptoms subsequent to mosquito bites. Most cases of hypersensitivity to mosquito bites reported thus far have been associated with chronic Epstein-Barr virus infection or natural killer cell leukemia/lymphoma. In this study, we describe the case of an 18-year-old Korean boy who had hypersensitivity to mosquito bites associated with primary systemic anaplastic lymphoma kinase-positive anaplastic large cell lymphoma. After a mosquito bite, the patient developed a progressive cutaneous nodule on his left lower leg and regional lymphadenopathy in the left inguinal area. The histopathological and immunohistochemical findings suggested anaplastic lymphoma kinase-positive anaplastic large cell lymphoma. Positron emission tomography-computed tomography revealed increased fluorodeoxyglucose uptake in the left T4 vertebrae, left external iliac lymph nodes, left inguinal lymph nodes, and lateral subcutaneous region of the left lower leg. According to the clinical, histopathological, and immunohistochemical findings, as well as the imaging data, the patient was diagnosed with primary systemic anaplastic lymphoma kinase-positive anaplastic large cell lymphoma. Consequently, the patient received a total of 6 cycles of cyclophosphamide + doxorubicin + vincristine + prednisolone chemotherapy at 3-week intervals, after which the lesions regressed.

Comparison of single-port, two-port and four-port laparoscopic surgery for cyst enucleation in benign ovarian cysts.

OBJECTIVE: To compare the perioperative outcomes of three laparoscopic approaches for performing ovarian cyst enucleation. METHODS: A total of 148 patients underwent laparoscopic cyst enucleation at the CHA Gangnam Medical Center between September 2010 and May 2011. We reviewed retrospectively the medical records including patient demographics, operative outcomes and complications. RESULTS: We assigned the 148 patients into three groups: single-port (group A: 40), 2-port (group B: 30) and 4-port (group C: 78). There were no statistically significant differences in patient characteristics. The operation times were 90.4 ± 43.6, 74.7 ± 22.0 and 63.8 ± 30.5 min, and the estimated blood loss was 179.3 ± 253.9, 73 ± 75.2 and 89.9 ± 106.7 ml, respectively. Mean operation time was longer (p < 0.001) and estimated blood loss was higher (p = 0.005) in group A than in the other groups. There was no statistical difference in perioperative complications among the three groups. In group A, additional port insertion rate was higher than in groups B and C (p < 0.001). CONCLUSION: Single-port surgery required longer operation time, had a higher estimated blood loss and used additional ports more frequently during the operation than the other groups. However, 2-port surgery had no significant differences from 4-port surgery in the surgical outcomes. Therefore, 2-port surgery can be an alternative surgical option for 4-port surgery in ovarian cyst enucleation.

A matrix metalloproteinase 1-cleavable composite peptide derived from transforming growth factor ß-inducible gene h3 potently inhibits collagen-induced arthritis.

OBJECTIVE: Transforming growth factor ß-inducible gene h3 (ßIG-H3), which is abundantly expressed in rheumatoid synovium, and matrix metalloproteinases (MMP) play important roles in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the therapeutic efficacy of ßIG-H3-derived peptides using MMP-1-dependent target tissue delivery in chronic inflammatory arthritis. METHODS: Peptides developed from ßIG-H3 derivatives, including the second and fourth YH peptides, the fourth fas-1 domain, the fourth fas-1 domain truncated for H1 and H2 sequences (dhfas-1), and an MMP-1- cleavable composite peptide (MFK24), were cloned. We confirmed the specificity of MFK24 cleavage by immunoblot analysis after treatment with different proteases. RESULTS: The YH18 peptide in the fourth fas-1 domain of ßIG-H3 was weakly effective in suppressing arthritis severity in mice with collagen-induced arthritis (CIA). Treatment with higher-dose dhfas-1 (30 mg/kg) showed remarkable efficacy, whereas treatment with a lower dose (10 mg/kg) resulted in only partial improvement. MFK24, a composite peptide consisting of dhfas-1 and RGD peptide linked by MMP-1 substrate, was cleaved specifically by MMP-1. The adhesion and migration of NIH3T3 cells mediated by ßIG-H3 were inhibited by MFK24 at a low concentration. MFK24 suppressed the adhesion of NIH3T3 cells more efficiently compared with murine dhfas-1 (MFK00) or RGD, either alone or in combination. The therapeutic efficacy of MFK24 in mice with CIA was remarkably enhanced, with consistently reduced expression of inflammatory mediators within joint tissue. CONCLUSION: This proof-of-concept study showed that an MMP-cleavable composite peptide, based on ßIG-H3 derivatives, had markedly improved therapeutic efficacy in chronic inflammatory arthritis, implicating a new expandable strategy for enhancement of the efficacy of 2 different active molecules in RA.

Orally active desulfated low molecular weight heparin and deoxycholic acid conjugate, 6ODS-LHbD, suppresses neovascularization and bone destruction in arthritis.

The regulation of angiogenesis is an interesting area to consider for novel therapeutic approaches to rheumatoid arthritis (RA). Chemically modified heparins have been developed as possible candidates for angiogenesis inhibitor; however, they have a major clinical drawback in exhibiting poor oral bioavailability. Here, orally absorbable O-desulfated low molecular weight heparin (ODS-LMWH) derivatives were newly synthesized by conjugating 2-O- or 6-O-desulfated LMWH with deoxycholic acid (DOCA) or bisDOCA (a dimer of DOCA), and their physicochemical properties, antiangiogenic potency and pharmacokinetic profiles were assessed. After selecting the best candidate among those derivatives, its therapeutic efficacy on arthritis was investigated in a murine collagen antibody-induced arthritis (CAIA) model. ODS-LMWH derivatives significantly inhibited the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs) and basic fibroblast growth factor (bFGF)-induced angiogenesis in the Matrigel plug assay. Among all the compounds, 6ODS-LHbD showed the highest oral bioavailability in rats (19.3%). In the CAIA mouse model, 6ODS-LHbD (10 mg/kg, p.o., S.I.D.) significantly inhibited neovascularization in the joint, the increase of hind-paw thickness, and the structural damage in the bone. Therefore, 6ODS-LHbD would be a promising candidate for an orally active drug for the treatment of RA.

In vivo quantitative measurement of arthritis activity based on hydrophobically modified glycol chitosan in inflammatory arthritis: more active than passive accumulation.

We demonstrated that arthritis could be visualized noninvasively using hydrophobically modified glycol chitosan nanoparticles labeled with Cy5.5 (HGC-Cy5.5) and an optical imaging system. Activated macrophages expressing Mac-1 molecules effectively phagocytosed HGC-Cy5.5, which formed spherical nanoparticles under physiologic conditions. We estimated the applicability of HGC-Cy5.5 to quantitative analysis of arthritis development and progression. Near-infrared fluorescence images, captured after HGC-Cy5.5 injection in mice with collagen-induced arthritis, showed stronger fluorescence intensity in the active arthritis group than in the nonarthritis group. According to the progression of arthritis in both collagen-induced arthritis and collagen antibody-induced arthritis models, total photon counts (TPCs) increased in parallel with the clinical arthritis index. Quantitative analysis of fluorescence after treatment with methotrexate showed a significant decrease in TPC in a dose-dependent manner. Histologic evaluation confirmed that the mechanism underlying selective accumulation of HGC-Cy5.5 within synovitis tissues included enhanced phagocytosis of the probe by Mac-1-expressing macrophages as well as enhanced permeability through leaky vessels. These results suggest that optical imaging of arthritis using HGC-Cy5.5 can provide an objective measurement of disease activity and, at the same time, therapeutic responses in rheumatoid arthritis.

The effectiveness of levonorgestrel releasing intrauterine system in the treatment of endometrial hyperplasia in Korean women.

OBJECTIVE: Levonorgestrel releasing intrauterine system (LNG-IUS) has been shown to treat patients with non-atypical & atypical endometrial hyperplasia (EH) successfully in many western studies. Our purpose was to examine the effectiveness of LNG-IUS in the treatment of Korean women with EH. METHODS: We conducted a prospective observational study of 12 women diagnosed with EH and treated with LNG-IUS insertion between February 2007 and August 2009 at the Department of Gynecology of Gangnam CHA Hospital, CHA University School of Medicine. Baseline endometrial biopsies were done before insertion of LNG-IUS, and outpatient follow-up endometrial biopsies were undertaken at 3-month intervals after insertion of LNG-IUS. We investigated the regression rate and the time to regression. RESULTS: Four patients had simple hyperplasia without atypia, 7 patients complex hyperplasia without atypia, and just 1 patient complex atypical hyperplasia. Complete regression of EH was achieved in all cases (100%, 12/12), with the significant proportion (66%, 8/12) achieving it within 3 months. The mean duration to regression was 4.5 months. All cases had regression within 9 months. In the case of complex atypical hyperplasia, the regression was attained at the 9th month after insertion of LNG-IUS. The mean follow-up duration was 12 months (range, 3 to 27 months). As long as LNG-IUS was maintained, the EH did not recur. CONCLUSION: LNG-IUS appears to be as highly effective in treating Korean women with EH.

Gene expression in chorionic villous samples at 11 weeks' gestation from women destined to develop preeclampsia.

OBJECTIVE: To evaluate the direct alterations in mRNA expression among chorionic villous samples from 11 weeks' pregnant women who would develop preeclampsia (PE) later in the pregnancy. METHOD: Case-control study encompassing five women destined to develop PE [cases matched 1:5 for gestational age (GA) with 25 controls]. We quantified mRNA expression on tissue samples from chorionic villous sampling (CVS) of normal and PE patients. We then assessed mRNA expressions of vascular endothelial growth factor (VEGFA), VEGFA receptor 1 (Flt-1), endoglin (Eng), placental growth factor (PlGF), transforming growth factor-beta1 (TGF-beta1), heme oxygenase-1 (HO-1) and superoxide dismutase (SOD). Data were analyzed by nonparametric rank analysis. RESULTS: For all the mRNA species considered in this study, all the mean observed ranks in the PE group were significantly altered compared to the rank expectation among controls. mRNA for Eng and TGF-beta1 were the markers with the highest degree of aberration in PE, in respect to controls. The results are consistent with those already reported for the corresponding circulating proteins. mRNA for HO-1 and SOD were instead associated with the lowest aberration. CONCLUSION: It is assumed that the pathogenesis of PE is associated with pathophysiological alterations to trophoblasts in early gestation. Our study has directly proved that gene expressions relating to angiogenesis or oxidative stress are altered in the first trimester trophoblasts that go on to develop PE later. These results would put the basis for a possible screening method for PE by using residual CVS.

Down syndrome biochemical markers and screening for preeclampsia at first and second trimester: correlation with the week of onset and the severity.

OBJECTIVES: To estimate the combined screening performance of first and early second trimester prenatal serum markers for Down syndrome, in screening for the development of preeclampsia, and analyze the correlation among marker levels, week of onset, and severity of the disease. METHODS: A retrospective cohort study was carried out on 32 women with preeclampsia and 3044 controls. Serum samples from these pregnancies were assayed for pregnancy-associated plasma protein-A (PAPP-A), alpha-fetoprotein (AFP), unconjugated estriol (uE3), human chorionic gonadotrophin (hCG), and inhibin-A. A likelihood ratio and the odds of being affected given a positive result (OAPR) of various combinations of markers were calculated and receiver operating characteristic (ROC) curves analysis was performed. RESULTS: In the pregnancies that subsequently developed preeclampsia, first trimester PAPP-A concentration was significantly lower and concentrations of early second trimester inhibin-A and hCG significantly elevated. Levels of early second trimester uE3 and AFP were not significantly altered. We also found that inhibin-A correlates with both onset of the disease and the severity. CONCLUSION: Down syndrome biochemical markers levels are altered in those patients who subsequently developed preeclampsia and may be a useful screening test for preeclampsia. Inhibin-A is the most predictive marker and correlates with the severity of subsequent preeclampsia and inversely with the week of occurrence of preeclampsia.

LIGHT up-regulated on B lymphocytes and monocytes in rheumatoid arthritis mediates cellular adhesion and metalloproteinase production by synoviocytes.

OBJECTIVE: To study the expression of LIGHT (tumor necrosis factor superfamily 14) and herpesvirus entry mediator (HVEM; tumor necrosis factor receptor superfamily 14) in rheumatoid arthritis (RA) and to determine the regulatory role of LIGHT on the effector functions of fibroblast-like synoviocytes (FLS). METHODS: The expression of LIGHT and HVEM was assessed by immunohistochemical staining of synovial tissue and by flow cytometric analysis of mononuclear cells. The presence of HVEM and lymphotoxin beta receptor was measured by reverse transcriptase-polymerase chain reaction and by flow cytometry. The regulation of effector molecules, including matrix metalloproteinases (MMPs) and adhesion molecules, was evaluated. The adhesiveness of FLS was determined by adhesion assay. RESULTS: HVEM was detected in most cell types within rheumatoid synovial tissue, while only a few cells were positive for LIGHT. In RA patients, LIGHT expression was significantly up-regulated only in CD20+ B cells and monocytes, whereas the mean fluorescence intensity of HVEM was down-regulated in mononuclear cells. The stimulation of FLS with LIGHT resulted in the production of MMPs and the expression of adhesion molecules, which were efficiently inhibited by dexamethasone. LIGHT-mediated up-regulation of MMPs and intercellular adhesion molecule 1 was blocked by inhibitors of NF-kappaB and JNK, whereas up-regulation of vascular cell adhesion molecule 1 was blocked by inhibitors of phosphatidylinositol 3-kinase, as well as NF-kappaB. CONCLUSION: These data suggest that binding of LIGHT with its receptors may play a role in the progression of inflammation within rheumatoid synovium, especially by mediating the interactions between infiltrating inflammatory cells and stromal cells. These findings thus emphasize the relevance of LIGHT as a potential therapeutic target in RA.